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Sample records for specific adapter protein

  1. The T-cell-specific adapter protein family: TSAd, ALX, and SH2D4A/SH2D4B.

    Science.gov (United States)

    Lapinski, Philip E; Oliver, Jennifer A; Bodie, Jennifer N; Marti, Francesc; King, Philip D

    2009-11-01

    Adapter proteins play key roles in intracellular signal transduction through complex formation with catalytically active signaling molecules. In T lymphocytes, the role of several different types of adapter proteins in T-cell antigen receptor signal transduction is well established. An exception to this is the family of T-cell-specific adapter (TSAd) proteins comprising of TSAd, adapter protein of unknown function (ALX), SH2D4A, and SH2D4B. Only recently has the function of these adapters in T-cell signal transduction been explored. Here, we discuss advances in our understanding of the role of this family of adapter proteins in T cells. Their function as regulators of signal transduction in other cell types is also discussed.

  2. Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Daniil M Prigozhin

    Full Text Available Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.

  3. Pervasive Adaptive Evolution in Primate Seminal Proteins.

    Directory of Open Access Journals (Sweden)

    2005-09-01

    Full Text Available Seminal fluid proteins show striking effects on reproduction, involving manipulation of female behavior and physiology, mechanisms of sperm competition, and pathogen defense. Strong adaptive pressures are expected for such manifestations of sexual selection and host defense, but the extent of positive selection in seminal fluid proteins from divergent taxa is unknown. We identified adaptive evolution in primate seminal proteins using genomic resources in a tissue-specific study. We found extensive signatures of positive selection when comparing 161 human seminal fluid proteins and 2,858 prostate-expressed genes to those in chimpanzee. Seven of eight outstanding genes yielded statistically significant evidence of positive selection when analyzed in divergent primates. Functional clues were gained through divergent analysis, including several cases of species-specific loss of function in copulatory plug genes, and statistically significant spatial clustering of positively selected sites near the active site of kallikrein 2. This study reveals previously unidentified positive selection in seven primate seminal proteins, and when considered with findings in Drosophila, indicates that extensive positive selection is found in seminal fluid across divergent taxonomic groups.

  4. Protein Adaptations in Archaeal Extremophiles

    Directory of Open Access Journals (Sweden)

    Christopher J. Reed

    2013-01-01

    Full Text Available Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity.

  5. Protein Adaptations in Archaeal Extremophiles

    Science.gov (United States)

    Reed, Christopher J.; Lewis, Hunter; Trejo, Eric; Winston, Vern; Evilia, Caryn

    2013-01-01

    Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity. PMID:24151449

  6. The Mitochondrial Lon Protease Is Required for Age-Specific and Sex-Specific Adaptation to Oxidative Stress.

    Science.gov (United States)

    Pomatto, Laura C D; Carney, Caroline; Shen, Brenda; Wong, Sarah; Halaszynski, Kelly; Salomon, Matthew P; Davies, Kelvin J A; Tower, John

    2017-01-09

    Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. However, a possible molecular mechanism for the sex bias in physiological adaptation to oxidative stress remains unclear. Here, we report that Drosophila melanogaster females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H 2 O 2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H 2 O 2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Identification and characterization of insect-specific proteins by genome data analysis

    Directory of Open Access Journals (Sweden)

    Clark Terry

    2007-04-01

    Full Text Available Abstract Background Insects constitute the vast majority of known species with their importance including biodiversity, agricultural, and human health concerns. It is likely that the successful adaptation of the Insecta clade depends on specific components in its proteome that give rise to specialized features. However, proteome determination is an intensive undertaking. Here we present results from a computational method that uses genome analysis to characterize insect and eukaryote proteomes as an approximation complementary to experimental approaches. Results Homologs in common to Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Tribolium castaneum, and Apis mellifera were compared to the complete genomes of three non-insect eukaryotes (opisthokonts Homo sapiens, Caenorhabditis elegans and Saccharomyces cerevisiae. This operation yielded 154 groups of orthologous proteins in Drosophila to be insect-specific homologs; 466 groups were determined to be common to eukaryotes (represented by three opisthokonts. ESTs from the hemimetabolous insect Locust migratoria were also considered in order to approximate their corresponding genes in the insect-specific homologs. Stress and stimulus response proteins were found to constitute a higher fraction in the insect-specific homologs than in the homologs common to eukaryotes. Conclusion The significant representation of stress response and stimulus response proteins in proteins determined to be insect-specific, along with specific cuticle and pheromone/odorant binding proteins, suggest that communication and adaptation to environments may distinguish insect evolution relative to other eukaryotes. The tendency for low Ka/Ks ratios in the insect-specific protein set suggests purifying selection pressure. The generally larger number of paralogs in the insect-specific proteins may indicate adaptation to environment changes. Instances in our insect-specific protein set have been arrived at through

  8. High rate of adaptation of mammalian proteins that interact with Plasmodium and related parasites

    Science.gov (United States)

    Telis, Natalie; Petrov, Dmitri A.

    2017-01-01

    Plasmodium parasites, along with their Piroplasm relatives, have caused malaria-like illnesses in terrestrial mammals for millions of years. Several Plasmodium-protective alleles have recently evolved in human populations, but little is known about host adaptation to blood parasites over deeper evolutionary timescales. In this work, we analyze mammalian adaptation in ~500 Plasmodium- or Piroplasm- interacting proteins (PPIPs) manually curated from the scientific literature. We show that (i) PPIPs are enriched for both immune functions and pleiotropy with other pathogens, and (ii) the rate of adaptation across mammals is significantly elevated in PPIPs, compared to carefully matched control proteins. PPIPs with high pathogen pleiotropy show the strongest signatures of adaptation, but this pattern is fully explained by their immune enrichment. Several pieces of evidence suggest that blood parasites specifically have imposed selection on PPIPs. First, even non-immune PPIPs that lack interactions with other pathogens have adapted at twice the rate of matched controls. Second, PPIP adaptation is linked to high expression in the liver, a critical organ in the parasite life cycle. Finally, our detailed investigation of alpha-spectrin, a major red blood cell membrane protein, shows that domains with particularly high rates of adaptation are those known to interact specifically with P. falciparum. Overall, we show that host proteins that interact with Plasmodium and Piroplasm parasites have experienced elevated rates of adaptation across mammals, and provide evidence that some of this adaptation has likely been driven by blood parasites. PMID:28957326

  9. Social-psychological specific of individual adaptation

    OpenAIRE

    Ovsyanik, Olga

    2012-01-01

    There is analyzing of specific of social-psychological adaptation person by model of adaptation. Structure model of adaptation of women of our age group, which was named “adaptation complex” was made by theoretic analyzes of problem of adaptation adult.

  10. Design Specifications for Adaptive Real-Time Systems

    Science.gov (United States)

    1991-12-01

    TICfl \\ E CT E Design Specifications for JAN’\\ 1992 Adaptive Real - Time Systems fl Randall W. Lichota U, Alice H. Muntz - December 1991 \\ \\\\/ 0 / r...268-2056 Technical Report CMU/SEI-91-TR-20 ESD-91-TR-20 December 1991 Design Specifications for Adaptive Real - Time Systems Randall W. Lichota Hughes...Design Specifications for Adaptive Real - Time Systems Abstract: The design specification method described in this report treats a software

  11. Viruses are a dominant driver of protein adaptation in mammals.

    Science.gov (United States)

    Enard, David; Cai, Le; Gwennap, Carina; Petrov, Dmitri A

    2016-05-17

    Viruses interact with hundreds to thousands of proteins in mammals, yet adaptation against viruses has only been studied in a few proteins specialized in antiviral defense. Whether adaptation to viruses typically involves only specialized antiviral proteins or affects a broad array of virus-interacting proteins is unknown. Here, we analyze adaptation in ~1300 virus-interacting proteins manually curated from a set of 9900 proteins conserved in all sequenced mammalian genomes. We show that viruses (i) use the more evolutionarily constrained proteins within the cellular functions they interact with and that (ii) despite this high constraint, virus-interacting proteins account for a high proportion of all protein adaptation in humans and other mammals. Adaptation is elevated in virus-interacting proteins across all functional categories, including both immune and non-immune functions. We conservatively estimate that viruses have driven close to 30% of all adaptive amino acid changes in the part of the human proteome conserved within mammals. Our results suggest that viruses are one of the most dominant drivers of evolutionary change across mammalian and human proteomes.

  12. Role of X-ray-inducible genes and proteins in adaptive survival responses

    International Nuclear Information System (INIS)

    Meyers, M.; Schea, R.A.; Petrowski, A.E.; Seabury, H.; McLaughlin, P.W.; Lee, I.; Lee, S.W.; Boothman, D.A.

    1992-01-01

    Certain X-ray-inducible genes and their corresponding protein products, appearing following low priming doses of ionizing radiation may subsequently give rise to an adaptive survival response, ultimately leading to increased radioresistance. Further, this adaptive radioresistance may be due to increased DNA repair (or misrepair) processes. Ultimately, the function of low-dose-induced cDNA clones within the cell is hoped to elucidate to follow the effects of specific gene turn-off on adaptive responses. Future research must determine the various functions of adaptive response gene products so that the beneficial or deleterious consequences of adaptive responses, which increases resistance to ionizing radiation, can be determined. (author). 19 refs., 1 fig

  13. Trends in the Design and Development of Specific Aptamers Against Peptides and Proteins.

    Science.gov (United States)

    Tabarzad, Maryam; Jafari, Marzieh

    2016-04-01

    Aptamers are single stranded oligonucleotides, comparable to monoclonal antibodies (mAbs) in selectivity and affinity and have significant strategic properties in design, development and applications more than mAbs. Ease of design and development, simple chemical modification and the attachment of functional groups, easily handling and more adaptability with analytical methods, small size and adaptation with nanostructures are the valuable characteristics of aptamers in comparison to large protein based ligands. Among a broad range of targets that their specific aptamers developed, proteins and peptides have significant position according to the number of related studies performed so far. Since proteins control many of important physiological and pathological incidents in the living organisms, particularly human beings and because of the benefits of aptamers in clinical and analytical applications, aptamer related technologies in the field of proteins and peptides are under progress, exclusively. Currently, there is only one FDA approved therapeutic aptamer in the pharmaceutical market, which is specific to vascular endothelial growth factor and is prescribed for age related macular degenerative disease. Additionally, there are several aptamers in the different phases of clinical trials. Almost all of these aptamers are specific to clinically important peptide or protein targets. In addition, the application of protein specific aptamers in the design and development of targeted drug delivery systems and diagnostic biosensors is another interesting field of aptamer technology. In this review, significant efforts related to development and applications of aptamer technologies in proteins and peptides sciences were considered to emphasis on the importance of aptamers in medicinal and clinical applications.

  14. Human Skeletal Muscle Stem Cells in Adaptations to Exercise; Effects of Resistance Exercise Contraction Mode and Protein Supplementation

    DEFF Research Database (Denmark)

    Farup, Jean

    2014-01-01

    the effect of contraction mode specific resistance training and protein supplementation on whole muscle and tendon hypertrophy. Quadriceps muscle and patellar tendon cross-sectional area (CSA) was quantified using magnetic resonance imaging pre and post 12 weeks of eccentric (Ecc) or concentric (Conc...... concentric resistance training and ingestion of protein influence myocellular adaptations, with special emphasis on muscle stem cell adaptations, during both acute and prolonged resistance exercise in human skeletal muscle. Paper I. Whey protein supplementation accelerates satellite cell proliferation during...... recovery from eccentric exercise In paper I, we evaluated the effect of a single bout of unaccustomed eccentric exercise on fiber type specific SC content by immunohistochemistry. Subjects received either hydrolysed whey protein (Whey) or iso-caloric carbohydrate (Placebo) in the days post eccentric...

  15. Identification of variant-specific surface proteins in Giardia muris trophozoites.

    Science.gov (United States)

    Ropolo, Andrea S; Saura, Alicia; Carranza, Pedro G; Lujan, Hugo D

    2005-08-01

    Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

  16. Identification of Variant-Specific Surface Proteins in Giardia muris Trophozoites

    OpenAIRE

    Ropolo, Andrea S.; Saura, Alicia; Carranza, Pedro G.; Lujan, Hugo D.

    2005-01-01

    Giardia lamblia undergoes antigenic variation, a process that might allow the parasite to evade the host's immune response and adapt to different environments. Here we show that Giardia muris, a related species that naturally infects rodents, possesses multiple variant-specific surface proteins (VSPs) and expresses VSPs on its surface, suggesting that it undergoes antigenic variation similar to that of G. lamblia.

  17. Protein domain evolution is associated with reproductive diversification and adaptive radiation in the genus Eucalyptus.

    Science.gov (United States)

    Kersting, Anna R; Mizrachi, Eshchar; Bornberg-Bauer, Erich; Myburg, Alexander A

    2015-06-01

    Eucalyptus is a pivotal genus within the rosid order Myrtales with distinct geographic history and adaptations. Comparative analysis of protein domain evolution in the newly sequenced Eucalyptus grandis genome and other rosid lineages sheds light on the adaptive mechanisms integral to the success of this genus of woody perennials. We reconstructed the ancestral domain content to elucidate the gain, loss and expansion of protein domains and domain arrangements in Eucalyptus in the context of rosid phylogeny. We used functional gene ontology (GO) annotation of genes to investigate the possible biological and evolutionary consequences of protein domain expansion. We found that protein modulation within the angiosperms occurred primarily on the level of expansion of certain domains and arrangements. Using RNA-Seq data from E. grandis, we showed that domain expansions have contributed to tissue-specific expression of tandemly duplicated genes. Our results indicate that tandem duplication of genes, a key feature of the Eucalyptus genome, has played an important role in the expansion of domains, particularly in proteins related to the specialization of reproduction and biotic and abiotic interactions affecting root and floral biology, and that tissue-specific expression of proteins with expanded domains has facilitated subfunctionalization in domain families. © 2014 University of Pretoria New Phytologist © 2014 New Phytologist Trust.

  18. Specificity and affinity quantification of protein-protein interactions.

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    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  19. Automated sequence-specific protein NMR assignment using the memetic algorithm MATCH

    International Nuclear Information System (INIS)

    Volk, Jochen; Herrmann, Torsten; Wuethrich, Kurt

    2008-01-01

    MATCH (Memetic Algorithm and Combinatorial Optimization Heuristics) is a new memetic algorithm for automated sequence-specific polypeptide backbone NMR assignment of proteins. MATCH employs local optimization for tracing partial sequence-specific assignments within a global, population-based search environment, where the simultaneous application of local and global optimization heuristics guarantees high efficiency and robustness. MATCH thus makes combined use of the two predominant concepts in use for automated NMR assignment of proteins. Dynamic transition and inherent mutation are new techniques that enable automatic adaptation to variable quality of the experimental input data. The concept of dynamic transition is incorporated in all major building blocks of the algorithm, where it enables switching between local and global optimization heuristics at any time during the assignment process. Inherent mutation restricts the intrinsically required randomness of the evolutionary algorithm to those regions of the conformation space that are compatible with the experimental input data. Using intact and artificially deteriorated APSY-NMR input data of proteins, MATCH performed sequence-specific resonance assignment with high efficiency and robustness

  20. Rampant adaptive evolution in regions of proteins with unknown function in Drosophila simulans.

    Directory of Open Access Journals (Sweden)

    Alisha K Holloway

    2007-10-01

    Full Text Available Adaptive protein evolution is pervasive in Drosophila. Genomic studies, thus far, have analyzed each protein as a single entity. However, the targets of adaptive events may be localized to particular parts of proteins, such as protein domains or regions involved in protein folding. We compared the population genetic mechanisms driving sequence polymorphism and divergence in defined protein domains and non-domain regions. Interestingly, we find that non-domain regions of proteins are more frequent targets of directional selection. Protein domains are also evolving under directional selection, but appear to be under stronger purifying selection than non-domain regions. Non-domain regions of proteins clearly play a major role in adaptive protein evolution on a genomic scale and merit future investigations of their functional properties.

  1. Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein.

    OpenAIRE

    Begun, D J; Whitley, P

    2000-01-01

    NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appear...

  2. Cold-water immersion after training sessions: Effects on fiber type-specific adaptations in muscle K+ transport proteins to sprint-interval training in men.

    Science.gov (United States)

    Christiansen, Danny; Bishop, David John; Broatch, James R; Bangsbo, Jens; McKenna, Michael John; Murphy, Robyn M

    2018-05-10

    Effects of regular use of cold-water immersion (CWI) on fiber type-specific adaptations in muscle K + transport proteins to intense training, along with their relationship to changes in mRNA levels after the first training session, were investigated in humans. Nineteen recreationally-active men (24{plus minus}6 y, 79.5{plus minus}10.8 kg, 44.6{plus minus}5.8 mL∙kg -1 ∙min -1 ) completed six weeks of sprint-interval cycling either without (passive rest; CON) or with training sessions followed by CWI (15 min at 10{degree sign}C; COLD). Muscle biopsies were obtained before and after training to determine abundance of Na + ,K + -ATPase isoforms (α 1-3 , β 1-3 ) and FXYD1, and after recovery treatments (+0h and +3h) on the first day of training to measure mRNA content. Training increased (ptraining (p>0.05). CWI after each session did not influence responses to training (p>0.05). However, α 2 mRNA increased after the first session in COLD (+0h, p0.05). In both conditions, α 1 and β 3 mRNA increased (+3h; p 0.05) after the first session. In summary, Na + ,K + -ATPase isoforms are differently regulated in type I and II muscle fibers by sprint-interval training in humans, which for most isoforms do not associate with changes in mRNA levels after the first training session. CWI neither impairs nor improves protein adaptations to intense training of importance for muscle K + regulation.

  3. Protein scissors: Photocleavage of proteins at specific locations

    Indian Academy of Sciences (India)

    Unknown

    Binding of ligands to globular proteins at hydrophobic cavities while making specific ... ched to a PTI model A1010 monochromator. UV cut-off filter ..... >1:1 stoichiometry (protein to ligand), the binding equilibrium favors the thermo- dynamically ...

  4. Disease specific protein corona

    Science.gov (United States)

    Rahman, M.; Mahmoudi, M.

    2015-03-01

    It is now well accepted that upon their entrance into the biological environments, the surface of nanomaterials would be covered by various biomacromolecules (e.g., proteins and lipids). The absorption of these biomolecules, so called `protein corona', onto the surface of (nano)biomaterials confers them a new `biological identity'. Although the formation of protein coronas on the surface of nanoparticles has been widely investigated, there are few reports on the effect of various diseases on the biological identity of nanoparticles. As the type of diseases may tremendously changes the composition of the protein source (e.g., human plasma/serum), one can expect that amount and composition of associated proteins in the corona composition may be varied, in disease type manner. Here, we show that corona coated silica and polystyrene nanoparticles (after interaction with in the plasma of the healthy individuals) could induce unfolding of fibrinogen, which promotes release of the inflammatory cytokines. However, no considerable releases of inflammatory cytokines were observed for corona coated graphene sheets. In contrast, the obtained corona coated silica and polystyrene nanoparticles from the hypofibrinogenemia patients could not induce inflammatory cytokine release where graphene sheets do. Therefore, one can expect that disease-specific protein coronas can provide a novel approach for applying nanomedicine to personalized medicine, improving diagnosis and treatment of different diseases tailored to the specific conditions and circumstances.

  5. The geography of sex-specific selection, local adaptation, and sexual dimorphism.

    Science.gov (United States)

    Connallon, Tim

    2015-09-01

    Local adaptation and sexual dimorphism are iconic evolutionary scenarios of intraspecific adaptive differentiation in the face of gene flow. Although theory has traditionally considered local adaptation and sexual dimorphism as conceptually distinct processes, emerging data suggest that they often act concurrently during evolutionary diversification. Here, I merge theories of local adaptation in space and sex-specific adaptation over time, and show that their confluence yields several new predictions about the roles of context-specific selection, migration, and genetic correlations, in adaptive diversification. I specifically revisit two influential predictions from classical studies of clinal adaptation and sexual dimorphism: (1) that local adaptation should decrease with distance from the species' range center and (2) that opposing directional selection between the sexes (sexual antagonism) should inevitably accompany the evolution of sexual dimorphism. I show that both predictions can break down under clinally varying selection. First, the geography of local adaptation can be sexually dimorphic, with locations of relatively high local adaptation differing profoundly between the sexes. Second, the intensity of sexual antagonism varies across the species' range, with subpopulations near the range center representing hotspots for antagonistic selection. The results highlight the context-dependent roles of migration versus sexual conflict as primary constraints to adaptive diversification. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

  6. Nck adapter proteins: functional versatility in T cells

    Directory of Open Access Journals (Sweden)

    Janssen Ottmar

    2009-02-01

    Full Text Available Abstract Nck is a ubiquitously expressed adapter protein that is almost exclusively built of one SH2 domain and three SH3 domains. The two isoproteins of Nck are functionally redundant in many aspects and differ in only few amino acids that are mostly located in the linker regions between the interaction modules. Nck proteins connect receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. Thereby, Nck regulates activation-dependent processes during cell polarisation and migration and plays a crucial role in the signal transduction of a variety of receptors including for instance PDGF-, HGF-, VEGF- and Ephrin receptors. In most cases, the SH2 domain mediates binding to the phosphorylated receptor or associated phosphoproteins, while SH3 domain interactions lead to the formation of larger protein complexes. In T lymphocytes, Nck plays a pivotal role in the T cell receptor (TCR-induced reorganisation of the actin cytoskeleton and the formation of the immunological synapse. However, in this context, two different mechanisms and adapter complexes are discussed. In the first scenario, dependent on an activation-induced conformational change in the CD3ε subunits, a direct binding of Nck to components of the TCR/CD3 complex was shown. In the second scenario, Nck is recruited to the TCR complex via phosphorylated Slp76, another central constituent of the membrane proximal activation complex. Over the past years, a large number of putative Nck interactors have been identified in different cellular systems that point to diverse additional functions of the adapter protein, e.g. in the control of gene expression and proliferation.

  7. Heat shock protein 70 and heat shock protein 90 expression in light- and dark-adapted adult octopus retinas.

    Science.gov (United States)

    Ochoa, Gina H; Clark, Ying Mei; Matsumoto, Brian; Torres-Ruiz, Jose A; Robles, Laura J

    2002-02-01

    Light- and dark-adaptation leads to changes in rhabdom morphology and photopigment distribution in the octopus retina. Molecular chaperones, including heat shock proteins (Hsps), may be involved in specific signaling pathways that cause changes in photoreceptor actin- and tubulin-based cytoskeletons and movement of the photopigments, rhodopsin and retinochrome. In this study, we used immunoblotting, in situ RT-PCR, immunofluorescence and confocal microscopy to localize the inducible form of Hsp70 and the larger Hsp90 in light- and dark-adapted and dorsal and ventral halves of adult octopus retinas. The Hsps showed differences in distribution between the light and dark and in dorsal vs. ventral position in the retina. Double labeling confocal microscopy co-localized Hsp70 with actin and tubulin, and Hsp90 with the photopigment, retinochrome. Our results demonstrate the presence of Hsp70 and Hsp90 in otherwise non-stressed light- and dark-adapted octopus retinas. These Hsps may help stabilize the cytoskeleton, important for rhabdom structure, and are perhaps involved in the redistribution of retinochrome in conditions of light and dark.

  8. Proteomic Analysis of Rhizoctonia solani Identifies Infection-specific, Redox Associated Proteins and Insight into Adaptation to Different Plant Hosts*

    Science.gov (United States)

    Anderson, Jonathan P.; Hane, James K.; Stoll, Thomas; Pain, Nicholas; Hastie, Marcus L.; Kaur, Parwinder; Hoogland, Christine; Gorman, Jeffrey J.; Singh, Karam B.

    2016-01-01

    Rhizoctonia solani is an important root infecting pathogen of a range of food staples worldwide including wheat, rice, maize, soybean, potato and others. Conventional resistance breeding strategies are hindered by the absence of tractable genetic resistance in any crop host. Understanding the biology and pathogenicity mechanisms of this fungus is important for addressing these disease issues, however, little is known about how R. solani causes disease. This study capitalizes on recent genomic studies by applying mass spectrometry based proteomics to identify soluble, membrane-bound and culture filtrate proteins produced under wheat infection and vegetative growth conditions. Many of the proteins found in the culture filtrate had predicted functions relating to modification of the plant cell wall, a major activity required for pathogenesis on the plant host, including a number found only under infection conditions. Other infection related proteins included a high proportion of proteins with redox associated functions and many novel proteins without functional classification. The majority of infection only proteins tested were confirmed to show transcript up-regulation during infection including a thaumatin which increased susceptibility to R. solani when expressed in Nicotiana benthamiana. In addition, analysis of expression during infection of different plant hosts highlighted how the infection strategy of this broad host range pathogen can be adapted to the particular host being encountered. Data are available via ProteomeXchange with identifier PXD002806. PMID:26811357

  9. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

    Science.gov (United States)

    Bell, Thomas J; Eberwine, James

    2015-01-01

    RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data.

  10. Report on Adaptive Force, a specific neuromuscular function

    Directory of Open Access Journals (Sweden)

    Marko Hoff

    2015-08-01

    Full Text Available In real life motions, as well as in sports, the adaptation of the neuromuscular systems to externally applied forces plays an important role. The term Adaptive Force (AF shall characterize the ability of the nerve-muscle-system to adapt to impacting external forces during isometric and eccentric muscle action. The focus in this paper is on the concept of this neuromuscular action, which is not yet described in this way. A measuring system was constructed and evaluated for this specific neuromuscular function, but only the main information of the evaluation of the measuring system and the preliminary reference values are mentioned here, while an article with detailed description will be published separately. This paper concentrates on the three following points: 1 What is the peculiarity of this neuromuscular function, introduced as AF? 2 Is the measuring system able to capture its specific characteristics and which phases of measurement occur? 3 It seems reasonable to discuss if AF can be distinguished and classified among the known force concepts. The article describes the measuring system and how it is able to capture special features of real life motions like submaximal intensities and the subjects’ option to react adequately on external varying forces. Furthermore, within one measurement the system records three different force qualities: the isometric submaximal Adaptive Force (AFiso, the maximal isometric Adaptive Force (AFisomax and the maximal eccentric Adaptive Force (AFeccmax. Each of these phases provide different and unique information on the nerve-muscle-system that are discussed in detail. Important, in terms of the Adaptive Force, seems to be the combination of conditional and coordinative abilities.

  11. The E-Id Protein Axis Specifies Adaptive Lymphoid Cell Identity and Suppresses Thymic Innate Lymphoid Cell Development.

    Science.gov (United States)

    Miyazaki, Masaki; Miyazaki, Kazuko; Chen, Kenian; Jin, Yi; Turner, Jacob; Moore, Amanda J; Saito, Rintaro; Yoshida, Kenichi; Ogawa, Seishi; Rodewald, Hans-Reimer; Lin, Yin C; Kawamoto, Hiroshi; Murre, Cornelis

    2017-05-16

    Innate and adaptive lymphoid development is orchestrated by the activities of E proteins and their antagonist Id proteins, but how these factors regulate early T cell progenitor (ETP) and innate lymphoid cell (ILC) development remains unclear. Using multiple genetic strategies, we demonstrated that E proteins E2A and HEB acted in synergy in the thymus to establish T cell identity and to suppress the aberrant development of ILCs, including ILC2s and lymphoid-tissue-inducer-like cells. E2A and HEB orchestrated T cell fate and suppressed the ILC transcription signature by activating the expression of genes associated with Notch receptors, T cell receptor (TCR) assembly, and TCR-mediated signaling. E2A and HEB acted in ETPs to establish and maintain a T-cell-lineage-specific enhancer repertoire, including regulatory elements associated with the Notch1, Rag1, and Rag2 loci. On the basis of these and previous observations, we propose that the E-Id protein axis specifies innate and adaptive lymphoid cell fate. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition.

    Science.gov (United States)

    De Vendittis, Emmanuele; Castellano, Immacolata; Cotugno, Roberta; Ruocco, Maria Rosaria; Raimo, Gennaro; Masullo, Mariorosario

    2008-01-07

    The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was

  13. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men.

    Science.gov (United States)

    McGinley, Cian; Bishop, David J

    2016-12-01

    McGinley C, Bishop DJ. Influence of training intensity on adaptations in acid/base transport proteins, muscle buffer capacity, and repeated-sprint ability in active men. J Appl Physiol 121: 1290-1305, 2016. First published October 14, 2016; doi:10.1152/japplphysiol.00630.2016-This study measured the adaptive response to exercise training for each of the acid-base transport protein families, including providing isoform-specific evidence for the monocarboxylate transporter (MCT)1/4 chaperone protein basigin and for the electrogenic sodium-bicarbonate cotransporter (NBCe)1. We investigated whether 4 wk of work-matched, high-intensity interval training (HIIT), performed either just above the lactate threshold (HIITΔ20; n = 8), or close to peak aerobic power (HIITΔ90; n = 8), influenced adaptations in acid-base transport protein abundance, nonbicarbonate muscle buffer capacity (βm in vitro ), and exercise capacity in active men. Training intensity did not discriminate between adaptations for most proteins measured, with abundance of MCT1, sodium/hydrogen exchanger (NHE) 1, NBCe1, carbonic anhydrase (CA) II, and CAXIV increasing after 4 wk, whereas there was little change in CAIII and CAIV abundance. βm in vitro also did not change. However, MCT4 protein content only increased for HIITΔ20 [effect size (ES): 1.06, 90% confidence limits × / ÷ 0.77], whereas basigin protein content only increased for HIITΔ90 (ES: 1.49, × / ÷ 1.42). Repeated-sprint ability (5 × 6-s sprints; 24 s passive rest) improved similarly for both groups. Power at the lactate threshold only improved for HIITΔ20 (ES: 0.49; 90% confidence limits ± 0.38), whereas peak O 2 uptake did not change for either group. Detraining was characterized by the loss of adaptations for all of the proteins measured and for repeated-sprint ability 6 wk after removing the stimulus of HIIT. In conclusion, 4 wk of HIIT induced improvements in each of the acid-base transport protein families, but, remarkably, a 40

  14. Cell-specific monitoring of protein synthesis in vivo.

    Directory of Open Access Journals (Sweden)

    Nikos Kourtis

    Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

  15. Context-specific protein network miner - an online system for exploring context-specific protein interaction networks from the literature

    KAUST Repository

    Chowdhary, Rajesh

    2012-04-06

    Background: Protein interaction networks (PINs) specific within a particular context contain crucial information regarding many cellular biological processes. For example, PINs may include information on the type and directionality of interaction (e.g. phosphorylation), location of interaction (i.e. tissues, cells), and related diseases. Currently, very few tools are capable of deriving context-specific PINs for conducting exploratory analysis. Results: We developed a literature-based online system, Context-specific Protein Network Miner (CPNM), which derives context-specific PINs in real-time from the PubMed database based on a set of user-input keywords and enhanced PubMed query system. CPNM reports enriched information on protein interactions (with type and directionality), their network topology with summary statistics (e.g. most densely connected proteins in the network; most densely connected protein-pairs; and proteins connected by most inbound/outbound links) that can be explored via a user-friendly interface. Some of the novel features of the CPNM system include PIN generation, ontology-based PubMed query enhancement, real-time, user-queried, up-to-date PubMed document processing, and prediction of PIN directionality. Conclusions: CPNM provides a tool for biologists to explore PINs. It is freely accessible at http://www.biotextminer.com/CPNM/. © 2012 Chowdhary et al.

  16. Context-specific protein network miner - an online system for exploring context-specific protein interaction networks from the literature

    KAUST Repository

    Chowdhary, Rajesh; Tan, Sin Lam; Zhang, Jinfeng; Karnik, Shreyas; Bajic, Vladimir B.; Liu, Jun S.

    2012-01-01

    Background: Protein interaction networks (PINs) specific within a particular context contain crucial information regarding many cellular biological processes. For example, PINs may include information on the type and directionality of interaction (e.g. phosphorylation), location of interaction (i.e. tissues, cells), and related diseases. Currently, very few tools are capable of deriving context-specific PINs for conducting exploratory analysis. Results: We developed a literature-based online system, Context-specific Protein Network Miner (CPNM), which derives context-specific PINs in real-time from the PubMed database based on a set of user-input keywords and enhanced PubMed query system. CPNM reports enriched information on protein interactions (with type and directionality), their network topology with summary statistics (e.g. most densely connected proteins in the network; most densely connected protein-pairs; and proteins connected by most inbound/outbound links) that can be explored via a user-friendly interface. Some of the novel features of the CPNM system include PIN generation, ontology-based PubMed query enhancement, real-time, user-queried, up-to-date PubMed document processing, and prediction of PIN directionality. Conclusions: CPNM provides a tool for biologists to explore PINs. It is freely accessible at http://www.biotextminer.com/CPNM/. © 2012 Chowdhary et al.

  17. Adaptive resolution simulation of an atomistic protein in MARTINI water

    International Nuclear Information System (INIS)

    Zavadlav, Julija; Melo, Manuel Nuno; Marrink, Siewert J.; Praprotnik, Matej

    2014-01-01

    We present an adaptive resolution simulation of protein G in multiscale water. We couple atomistic water around the protein with mesoscopic water, where four water molecules are represented with one coarse-grained bead, farther away. We circumvent the difficulties that arise from coupling to the coarse-grained model via a 4-to-1 molecule coarse-grain mapping by using bundled water models, i.e., we restrict the relative movement of water molecules that are mapped to the same coarse-grained bead employing harmonic springs. The water molecules change their resolution from four molecules to one coarse-grained particle and vice versa adaptively on-the-fly. Having performed 15 ns long molecular dynamics simulations, we observe within our error bars no differences between structural (e.g., root-mean-squared deviation and fluctuations of backbone atoms, radius of gyration, the stability of native contacts and secondary structure, and the solvent accessible surface area) and dynamical properties of the protein in the adaptive resolution approach compared to the fully atomistically solvated model. Our multiscale model is compatible with the widely used MARTINI force field and will therefore significantly enhance the scope of biomolecular simulations

  18. Biomimetic conformation-specific assembly of proteins at artificial binding sites nano-patterned on silicon

    Science.gov (United States)

    de la Rica, Roberto; Matsui, Hiroshi

    2009-01-01

    Biomolecules such as enzymes and antibodies possess binding sites where the molecular architecture and the physicochemical properties are optimum for their interaction with a particular target, in some cases even differentiating between stereoisomers. Here, we mimic this exquisite specificity via the creation of a suitable chemical environment by fabricating artificial binding sites for the protein calmodulin (CaM). By downscaling well-known surface chemical modification methodologies to the nanometer scale via silicon nanopatterning, the Ca2+-CaM conformer was found to selectively bind the biomimetic binding sites. The methodology could be adapted to mimic other protein-receptor interactions for sensing and catalysis. PMID:19757782

  19. Site-Specific PEGylation of Therapeutic Proteins

    Directory of Open Access Journals (Sweden)

    Jonathan K. Dozier

    2015-10-01

    Full Text Available The use of proteins as therapeutics has a long history and is becoming ever more common in modern medicine. While the number of protein-based drugs is growing every year, significant problems still remain with their use. Among these problems are rapid degradation and excretion from patients, thus requiring frequent dosing, which in turn increases the chances for an immunological response as well as increasing the cost of therapy. One of the main strategies to alleviate these problems is to link a polyethylene glycol (PEG group to the protein of interest. This process, called PEGylation, has grown dramatically in recent years resulting in several approved drugs. Installing a single PEG chain at a defined site in a protein is challenging. Recently, there is has been considerable research into various methods for the site-specific PEGylation of proteins. This review seeks to summarize that work and provide background and context for how site-specific PEGylation is performed. After introducing the topic of site-specific PEGylation, recent developments using chemical methods are described. That is followed by a more extensive discussion of bioorthogonal reactions and enzymatic labeling.

  20. Radio-adaptive response

    International Nuclear Information System (INIS)

    Ikushima, Takaji

    1991-01-01

    An adaptive response to radiation stress was found in cultured Chinese hamster V79 cells, as a suppressed induction of micronuclei (MNs) and sister chromatid exchanges (SCEs) in the cells conditioned by very low doses. The important characteristics of the novel chromosomal response, called radio-adaptive response (RAR), that have newly emerged in this study are: 1) Low doses of beta-rays from tritiated water (HTO) as well as tritiated thymidine can cause the RAR. 2) Thermal neutrons, a high LET radiation, can not act as tritium beta-rays or gamma-rays. 3) The RAR expression is suppressed by an inhibition of protein synthesis. 4) Several proteins are newly synthesized concurrently with the RAR expression after adapting doses, viewed by two-dimensional electrophoresis of cellular proteins. These results suggest that the RAR is an adaptive chromosomal DNA repair induced by very low doses of low LET radiations under restricted conditions, accompanying the inducible specific gene expression. (author)

  1. Cross cultural adaptation of the menopause specific questionnaire ...

    African Journals Online (AJOL)

    Cross cultural adaptation of the menopause specific questionnaire into the Persian language. ... Annals of Medical and Health Sciences Research ... good internal consistency in vasomotor, physical and psychosocial domains, but not sexual.

  2. Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

    Directory of Open Access Journals (Sweden)

    Martin Flück

    2014-01-01

    Full Text Available We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL and soleus (SOL muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS and subjected to fibre typing and measures for costameric (FAK and FRNK, mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1, and MHCI protein and RNA content. Mean cross-sectional area (MCSA of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05≤P<0.10. FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P=0.029. SOL muscle demonstrated a specific reduction in UCP3 content (-23%; P = 0.012. Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading.

  3. Early Changes in Costameric and Mitochondrial Protein Expression with Unloading Are Muscle Specific

    Science.gov (United States)

    Li, Ruowei; Linnehan, Richard M.; Castells, Josiane; Tesch, Per; Gustafsson, Thomas

    2014-01-01

    We hypothesised that load-sensitive expression of costameric proteins, which hold the sarcomere in place and position the mitochondria, contributes to the early adaptations of antigravity muscle to unloading and would depend on muscle fibre composition and chymotrypsin activity of the proteasome. Biopsies were obtained from vastus lateralis (VL) and soleus (SOL) muscles of eight men before and after 3 days of unilateral lower limb suspension (ULLS) and subjected to fibre typing and measures for costameric (FAK and FRNK), mitochondrial (NDUFA9, SDHA, UQCRC1, UCP3, and ATP5A1), and MHCI protein and RNA content. Mean cross-sectional area (MCSA) of types I and II muscle fibres in VL and type I fibres in SOL demonstrated a trend for a reduction after ULLS (0.05 ≤ P < 0.10). FAK phosphorylation at tyrosine 397 showed a 20% reduction in VL muscle (P = 0.029). SOL muscle demonstrated a specific reduction in UCP3 content (−23%; P = 0.012). Muscle-specific effects of ULLS were identified for linear relationships between measured proteins, chymotrypsin activity and fibre MCSA. The molecular modifications in costamere turnover and energy homoeostasis identify that aspects of atrophy and fibre transformation are detectable at the protein level in weight-bearing muscles within 3 days of unloading. PMID:25313365

  4. Human-specific protein isoforms produced by novel splice sites in the human genome after the human-chimpanzee divergence

    Directory of Open Access Journals (Sweden)

    Kim Dong Seon

    2012-11-01

    Full Text Available Abstract Background Evolution of splice sites is a well-known phenomenon that results in transcript diversity during human evolution. Many novel splice sites are derived from repetitive elements and may not contribute to protein products. Here, we analyzed annotated human protein-coding exons and identified human-specific splice sites that arose after the human-chimpanzee divergence. Results We analyzed multiple alignments of the annotated human protein-coding exons and their respective orthologous mammalian genome sequences to identify 85 novel splice sites (50 splice acceptors and 35 donors in the human genome. The novel protein-coding exons, which are expressed either constitutively or alternatively, produce novel protein isoforms by insertion, deletion, or frameshift. We found three cases in which the human-specific isoform conferred novel molecular function in the human cells: the human-specific IMUP protein isoform induces apoptosis of the trophoblast and is implicated in pre-eclampsia; the intronization of a part of SMOX gene exon produces inactive spermine oxidase; the human-specific NUB1 isoform shows reduced interaction with ubiquitin-like proteins, possibly affecting ubiquitin pathways. Conclusions Although the generation of novel protein isoforms does not equate to adaptive evolution, we propose that these cases are useful candidates for a molecular functional study to identify proteomic changes that might bring about novel phenotypes during human evolution.

  5. p53 Protein interacts specifically with the meiosis-specific mammalian RecA-like protein DMC1 in meiosis.

    Science.gov (United States)

    Habu, Toshiyuki; Wakabayashi, Nobunao; Yoshida, Kayo; Yomogida, Kenntaro; Nishimune, Yoshitake; Morita, Takashi

    2004-06-01

    The tumor suppressor protein p53 is specifically expressed during meiosis in spermatocytes. Subsets of p53 knockout mice exhibit testicular giant cell degenerative syndrome, which suggests p53 may be associated with meiotic cell cycle and/or DNA metabolism. Here, we show that p53 binds to the mouse meiosis-specific RecA-like protein Mus musculus DMC1 (MmDMC1). The C-terminal domain (amino acid 234-340) of MmDMC1 binds to DNA-binding domain of p53 protein. p53 might be involved in homologous recombination and/or checkpoint function by directly binding to DMC1 protein to repress genomic instability in meiotic germ cells.

  6. Design of multi-specificity in protein interfaces.

    Directory of Open Access Journals (Sweden)

    Elisabeth L Humphris

    2007-08-01

    Full Text Available Interactions in protein networks may place constraints on protein interface sequences to maintain correct and avoid unwanted interactions. Here we describe a "multi-constraint" protein design protocol to predict sequences optimized for multiple criteria, such as maintaining sets of interactions, and apply it to characterize the mechanism and extent to which 20 multi-specific proteins are constrained by binding to multiple partners. We find that multi-specific binding is accommodated by at least two distinct patterns. In the simplest case, all partners share key interactions, and sequences optimized for binding to either single or multiple partners recover only a subset of native amino acid residues as optimal. More interestingly, for signaling interfaces functioning as network "hubs," we identify a different, "multi-faceted" mode, where each binding partner prefers its own subset of wild-type residues within the promiscuous binding site. Here, integration of preferences across all partners results in sequences much more "native-like" than seen in optimization for any single binding partner alone, suggesting these interfaces are substantially optimized for multi-specificity. The two strategies make distinct predictions for interface evolution and design. Shared interfaces may be better small molecule targets, whereas multi-faceted interactions may be more "designable" for altered specificity patterns. The computational methodology presented here is generalizable for examining how naturally occurring protein sequences have been selected to satisfy a variety of positive and negative constraints, as well as for rationally designing proteins to have desired patterns of altered specificity.

  7. Gender specific pattern of left ventricular cardiac adaptation to ...

    African Journals Online (AJOL)

    EB

    2013-09-03

    Sep 3, 2013 ... Some gender specific associations with left ventricular structure and function have been described ... used for analysis. ... risk due to LVH and that cardiac adaptation to ... had history taking, physical examination and ..... between the gender specific differences in cardiac ... A metaanalysis of individual.

  8. Gender specific pattern of left ventricular cardiac adaptation to ...

    African Journals Online (AJOL)

    Background: Cardiac adaptation to hypertension and obesity may be related to many factors such as race, gender and haemodynamic status. Some gender specific associations with left ventricular structure and function have been described among Caucasians. Objectives: To describe the sex specific pattern of left ...

  9. The limits of adaptation of functional protein synthesis to severe undernutrition

    International Nuclear Information System (INIS)

    Forrester, T.; Jahoor, F.; Reeds, P.

    1996-01-01

    This project was designed to investigate the limits of adaptation of protein metabolism in the stree of severe childhood malnutrition, representing as it does chronic dietary insufficiency of macronutrients and superimposed infection. The tasks included measurement of concentrations and rates of synthesis of nutrient transport proteins and hepatic acute phase proteins inseverely malnourished children during their acute illness and a recovery

  10. Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.

    Directory of Open Access Journals (Sweden)

    Thierry Gauthier

    Full Text Available Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous in vivo protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.

  11. Sequence-specific capture of protein-DNA complexes for mass spectrometric protein identification.

    Directory of Open Access Journals (Sweden)

    Cheng-Hsien Wu

    Full Text Available The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1 promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

  12. Specificity of molecular interactions in transient protein-protein interaction interfaces.

    Science.gov (United States)

    Cho, Kyu-il; Lee, KiYoung; Lee, Kwang H; Kim, Dongsup; Lee, Doheon

    2006-11-15

    In this study, we investigate what types of interactions are specific to their biological function, and what types of interactions are persistent regardless of their functional category in transient protein-protein heterocomplexes. This is the first approach to analyze protein-protein interfaces systematically at the molecular interaction level in the context of protein functions. We perform systematic analysis at the molecular interaction level using classification and feature subset selection technique prevalent in the field of pattern recognition. To represent the physicochemical properties of protein-protein interfaces, we design 18 molecular interaction types using canonical and noncanonical interactions. Then, we construct input vector using the frequency of each interaction type in protein-protein interface. We analyze the 131 interfaces of transient protein-protein heterocomplexes in PDB: 33 protease-inhibitors, 52 antibody-antigens, 46 signaling proteins including 4 cyclin dependent kinase and 26 G-protein. Using kNN classification and feature subset selection technique, we show that there are specific interaction types based on their functional category, and such interaction types are conserved through the common binding mechanism, rather than through the sequence or structure conservation. The extracted interaction types are C(alpha)-- H...O==C interaction, cation...anion interaction, amine...amine interaction, and amine...cation interaction. With these four interaction types, we achieve the classification success rate up to 83.2% with leave-one-out cross-validation at k = 15. Of these four interaction types, C(alpha)--H...O==C shows binding specificity for protease-inhibitor complexes, while cation-anion interaction is predominant in signaling complexes. The amine ... amine and amine...cation interaction give a minor contribution to the classification accuracy. When combined with these two interactions, they increase the accuracy by 3.8%. In the case of

  13. Adaptive GDDA-BLAST: fast and efficient algorithm for protein sequence embedding.

    Directory of Open Access Journals (Sweden)

    Yoojin Hong

    2010-10-01

    Full Text Available A major computational challenge in the genomic era is annotating structure/function to the vast quantities of sequence information that is now available. This problem is illustrated by the fact that most proteins lack comprehensive annotations, even when experimental evidence exists. We previously theorized that embedded-alignment profiles (simply "alignment profiles" hereafter provide a quantitative method that is capable of relating the structural and functional properties of proteins, as well as their evolutionary relationships. A key feature of alignment profiles lies in the interoperability of data format (e.g., alignment information, physio-chemical information, genomic information, etc.. Indeed, we have demonstrated that the Position Specific Scoring Matrices (PSSMs are an informative M-dimension that is scored by quantitatively measuring the embedded or unmodified sequence alignments. Moreover, the information obtained from these alignments is informative, and remains so even in the "twilight zone" of sequence similarity (<25% identity. Although our previous embedding strategy was powerful, it suffered from contaminating alignments (embedded AND unmodified and high computational costs. Herein, we describe the logic and algorithmic process for a heuristic embedding strategy named "Adaptive GDDA-BLAST." Adaptive GDDA-BLAST is, on average, up to 19 times faster than, but has similar sensitivity to our previous method. Further, data are provided to demonstrate the benefits of embedded-alignment measurements in terms of detecting structural homology in highly divergent protein sequences and isolating secondary structural elements of transmembrane and ankyrin-repeat domains. Together, these advances allow further exploration of the embedded alignment data space within sufficiently large data sets to eventually induce relevant statistical inferences. We show that sequence embedding could serve as one of the vehicles for measurement of low

  14. Milk whey protein modification by coffee-specific phenolics: effect on structural and functional properties.

    Science.gov (United States)

    Ali, Mostafa; Homann, Thomas; Khalil, Mahmoud; Kruse, Hans-Peter; Rawel, Harshadrai

    2013-07-17

    A suitable vehicle for integration of bioactive plant constituents is proposed. It involves modification of proteins using phenolics and applying these for protection of labile constituents. It dissects the noncovalent and covalent interactions of β-lactoglobulin with coffee-specific phenolics. Alkaline and polyphenol oxidase modulated covalent reactions were compared. Tryptic digestion combined with MALDI-TOF-MS provided tentative allocation of the modification type and site in the protein, and an in silico modeling of modified β-lactoglobulin is proposed. The modification delivers proteins with enhanced antioxidative properties. Changed structural properties and differences in solubility, surface hydrophobicity, and emulsification were observed. The polyphenol oxidase modulated reaction provides a modified β-lactoglobulin with a high antioxidative power, is thermally more stable, requires less energy to unfold, and, when emulsified with lutein esters, exhibits their higher stability against UV light. Thus, adaptation of this modification provides an innovative approach for functionalizing proteins and their uses in the food industry.

  15. Proteomic analysis of the intestinal adaptation response reveals altered expression of fatty acid binding proteins following massive small bowel resection.

    Science.gov (United States)

    Stephens, Andrew N; Pereira-Fantini, Prue M; Wilson, Guineva; Taylor, Russell G; Rainczuk, Adam; Meehan, Katie L; Sourial, Magdy; Fuller, Peter J; Stanton, Peter G; Robertson, David M; Bines, Julie E

    2010-03-05

    Intestinal adaptation in response to the loss of the small intestine is essential to restore enteral autonomy in patients who have undergone massive small bowel resection (MSBR). In a proportion of patients, intestinal function is not restored, resulting in chronic intestinal failure (IF). Early referral of such patients for transplant provides the best prognosis; however, the molecular mechanisms underlying intestinal adaptation remain elusive and there is currently no convenient marker to predict whether patients will develop IF. We have investigated the adaptation response in a well-characterized porcine model of intestinal adaptation. 2D DIGE analysis of ileal epithelium from piglets recovering from massive small bowel resection (MSBR) identified over 60 proteins that changed specifically in MSBR animals relative to nonoperational or sham-operated controls. Three fatty acid binding proteins (L-FABP, FABP-6, and I-FABP) showed changes in MSBR animals. The expression changes and localization of each FABP were validated by immunoblotting and immunohistochemical analysis. FABP expression changes in MSBR animals occurred concurrently with altered triglyceride and bile acid metabolism as well as weight gain. The observed FABP expression changes in the ileal epithelium occur as part of the intestinal adaptation response and could provide a clinically useful marker to evaluate adaptation following MSBR.

  16. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    Science.gov (United States)

    Hursel, Rick; Martens, Eveline A P; Gonnissen, Hanne K J; Hamer, Henrike M; Senden, Joan M G; van Loon, Luc J C; Westerterp-Plantenga, Margriet S

    2015-01-01

    Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates. To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake. A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d) or low protein (0.4 g protein/kg/d) energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat) for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y) were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans. After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;Pprotein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;Psynthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;Pprotein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042±0.01 vs 0.045±0.01%/h;P = 0.620). In the overnight fasted state, adaptation to a low-protein intake (0.4 g/kg/d) does not result in a more negative whole-body protein balance and

  17. Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells

    Directory of Open Access Journals (Sweden)

    Kristin eSurmann

    2014-08-01

    Full Text Available Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549, and human embryonic kidney cells (HEK 293. Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen´s proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2x106 bacteria, roughly 1,450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreases in levels of ribosomal proteins and metabolic enzymes or increases in amounts of proteins involved in arginine and lysine biosynthesis, coding for terminal oxidases and stress responsive genes or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and

  18. Mucin-like protein, a saliva component involved in brown planthopper virulence and host adaptation.

    Science.gov (United States)

    Huang, Hai-Jian; Liu, Cheng-Wen; Xu, Hai-Jun; Bao, Yan-Yuan; Zhang, Chuan-Xi

    2017-04-01

    The rice brown planthopper (BPH), Nilaparvata lugens, can rapidly adapt to new resistant rice varieties within several generations, rendering its management burdensome. However, the molecular mechanism underlying its adaptability remains unclear. In this study, we investigated the potential role of mucin-like protein (NlMul) in N. lugens virulence and adaptation to host resistance. NlMul is an important glycoprotein that constitutes both gelling and watery saliva, and specifically expressed in the salivary glands at all developmental stages except the egg period. Knocking down the expression of NlMul resulted in the secretion of short and single-branched salivary sheaths. NlMul might help BPH deal with plant resistance, and altered gene expression was observed when BPHs were transferred from a susceptible rice variety to a resistant one. The NlMul-deficient BPHs showed disordered developmental duration and a portion of these insects reared on resistant rice exhibited lethal effects. Our results uncover a saliva-mediated interaction between insect and host plant, and provide useful information in rice breeding and planthopper management. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. The role of stimulus-specific adaptation in songbird syntax generation

    Science.gov (United States)

    Wittenbach, Jason D.

    Sequential behaviors are an important part of the behavioral repertoire of many animals and understanding how neural circuits encode and generate such sequences is a long-standing question in neuroscience. The Bengalese finch is a useful model system for studying variable action sequences. The songs of these birds consist of well-defined vocal elements (syllables) that are strung together to form sequences. The ordering of the syllables within the sequence is variable but not random - it shows complex statistical patterns (syntax). While often thought to be first-order, the syntax of the Bengalese finch song shows a distinct form of history dependence where the probability of repeating a syllable decreases as a function of the number of repetitions that have already occurred. Current models of the Bengalese finch song control circuitry offer no explanation for this repetition adaptation. The Bengalese finch also uses real-time auditory feedback to control the song syntax. Considering these facts, we hypothesize that repetition adaptation in the Bengalese finch syntax may be caused by stimulus-specific adaptation - a wide-spread phenomenon where neural responses to a specific stimulus become weaker with repeated presentations of the same stimulus. We begin by proposing a computational model for the song-control circuit where an auditory feedback signal that undergoes stimulus-specific adaptation helps drive repeated syllables. We show that this model does indeed capture the repetition adaptation observed in Bengalese finch syntax; along the way, we derive a new probabilistic model for repetition adaptation. Key predictions of our model are analyzed in light of experiments performed by collaborators. Next we extend the model in order to predict how the syntax will change as a function of brain temperature. These predictions are compared to experimental results from collaborators where portions of the Bengalese finch song circuit are cooled in awake and behaving birds

  20. Distinct distribution of specific members of protein 4.1 genefamily in the mouse nephron

    Energy Technology Data Exchange (ETDEWEB)

    Ramez, Mohamed; Blot-Chabaud, Marcel; Cluzeaud, Francoise; Chanan, Sumita; Patterson, Michael; Walensky, Loren D.; Marfatia, Shirin; Baines, Anthony J.; Chasis, Joel A.; Conboy, John G.; Mohandas, Narla; Gascard, Philippe

    2002-12-11

    Background: Protein 4.1 is an adapter protein which linksthe actin cytoskeleton to various transmembrane proteins. 4.1 proteinsare encoded by four homologous genes, 4.1R, 4.1G, 4.1N, and 4.1B, whichundergo complex alternative splicing. Here we performed a detailedcharacterization of the expression of specific 4.1 proteins in the mousenephron. Methods: Distribution of renal 4.1 proteins was investigated bystaining of paraformaldehyde fixed mouse kidney sections with antibodieshighly specific for each 4.1 protein. Major 4.1 splice forms, amplifiedfrom mouse kidney marathon cDNA, were expressed in transfected COS-7cells in order to assign species of known exon composition to proteinsdetected in kidney. Results: A 105kDa4.1R splice form, initiating atATG-2 translation initiation site and lacking exon 16, but including exon17B, was restricted to thick ascending limb of Henle's loop. A 95kDa 4.1Nspliceform,lacking exons 15 and 17D, was expressed in either descendingor ascending thin limb of Henle'sloop, distal convoluted tubule and allregions of the collecting duct system. A major 108kDa 4.1B spliceform,initiating at a newly characterized ATG translation initiation site, andlacking exons 15, 17B, and 21, was present only in Bowman's capsule andproximal convoluted tubule (PCT). There was no expression of 4.1G inkidney. Conclusion: Distinct distribution of 4.1 proteins along thenephron suggests their involvement in targeting of selected transmembraneproteins in kidney epithelium andtherefore in regulation of specifickidney functions.

  1. Cross Cultural Adaptation of the Menopause Specific Questionnaire ...

    African Journals Online (AJOL)

    Cross Cultural Adaptation of the Menopause Specific. Questionnaire into the Persian Language. Ghazanfarpour M, Kaviani M1, Rezaiee M2, Ghaderi E3, Zandvakili F2. Department of Midwifery, School of Nursing and Midwifery, Mashhad University of Medical Sciences, Mashhad, 1Nursing and Midwifery College, Shiraz ...

  2. Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

    Directory of Open Access Journals (Sweden)

    Lauren A. Cooper

    2018-04-01

    Full Text Available In clustered regularly interspaced short palindromic repeat (CRISPR-Cas (CRISPR-associated immunity systems, short CRISPR RNAs (crRNAs are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing.

  3. Staphylococcal Immune Evasion Proteins: Structure, Function, and Host Adaptation.

    Science.gov (United States)

    Koymans, Kirsten J; Vrieling, Manouk; Gorham, Ronald D; van Strijp, Jos A G

    2017-01-01

    Staphylococcus aureus is a successful human and animal pathogen. Its pathogenicity is linked to its ability to secrete a large amount of virulence factors. These secreted proteins interfere with many critical components of the immune system, both innate and adaptive, and hamper proper immune functioning. In recent years, numerous studies have been conducted in order to understand the molecular mechanism underlying the interaction of evasion molecules with the host immune system. Structural studies have fundamentally contributed to our understanding of the mechanisms of action of the individual factors. Furthermore, such studies revealed one of the most striking characteristics of the secreted immune evasion molecules: their conserved structure. Despite high-sequence variability, most immune evasion molecules belong to a small number of structural categories. Another remarkable characteristic is that S. aureus carries most of these virulence factors on mobile genetic elements (MGE) or ex-MGE in its accessory genome. Coevolution of pathogen and host has resulted in immune evasion molecules with a highly host-specific function and prevalence. In this review, we explore how these shared structures and genomic locations relate to function and host specificity. This is discussed in the context of therapeutic options for these immune evasion molecules in infectious as well as in inflammatory diseases.

  4. Computational identification of strain-, species- and genus-specific proteins

    Directory of Open Access Journals (Sweden)

    Thiagarajan Rathi

    2005-11-01

    Full Text Available Abstract Background The identification of unique proteins at different taxonomic levels has both scientific and practical value. Strain-, species- and genus-specific proteins can provide insight into the criteria that define an organism and its relationship with close relatives. Such proteins can also serve as taxon-specific diagnostic targets. Description A pipeline using a combination of computational and manual analyses of BLAST results was developed to identify strain-, species-, and genus-specific proteins and to catalog the closest sequenced relative for each protein in a proteome. Proteins encoded by a given strain are preliminarily considered to be unique if BLAST, using a comprehensive protein database, fails to retrieve (with an e-value better than 0.001 any protein not encoded by the query strain, species or genus (for strain-, species- and genus-specific proteins respectively, or if BLAST, using the best hit as the query (reverse BLAST, does not retrieve the initial query protein. Results are manually inspected for homology if the initial query is retrieved in the reverse BLAST but is not the best hit. Sequences unlikely to retrieve homologs using the default BLOSUM62 matrix (usually short sequences are re-tested using the PAM30 matrix, thereby increasing the number of retrieved homologs and increasing the stringency of the search for unique proteins. The above protocol was used to examine several food- and water-borne pathogens. We find that the reverse BLAST step filters out about 22% of proteins with homologs that would otherwise be considered unique at the genus and species levels. Analysis of the annotations of unique proteins reveals that many are remnants of prophage proteins, or may be involved in virulence. The data generated from this study can be accessed and further evaluated from the CUPID (Core and Unique Protein Identification system web site (updated semi-annually at http://pir.georgetown.edu/cupid. Conclusion CUPID

  5. mPLR-Loc: an adaptive decision multi-label classifier based on penalized logistic regression for protein subcellular localization prediction.

    Science.gov (United States)

    Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

    2015-03-15

    Proteins located in appropriate cellular compartments are of paramount importance to exert their biological functions. Prediction of protein subcellular localization by computational methods is required in the post-genomic era. Recent studies have been focusing on predicting not only single-location proteins but also multi-location proteins. However, most of the existing predictors are far from effective for tackling the challenges of multi-label proteins. This article proposes an efficient multi-label predictor, namely mPLR-Loc, based on penalized logistic regression and adaptive decisions for predicting both single- and multi-location proteins. Specifically, for each query protein, mPLR-Loc exploits the information from the Gene Ontology (GO) database by using its accession number (AC) or the ACs of its homologs obtained via BLAST. The frequencies of GO occurrences are used to construct feature vectors, which are then classified by an adaptive decision-based multi-label penalized logistic regression classifier. Experimental results based on two recent stringent benchmark datasets (virus and plant) show that mPLR-Loc remarkably outperforms existing state-of-the-art multi-label predictors. In addition to being able to rapidly and accurately predict subcellular localization of single- and multi-label proteins, mPLR-Loc can also provide probabilistic confidence scores for the prediction decisions. For readers' convenience, the mPLR-Loc server is available online (http://bioinfo.eie.polyu.edu.hk/mPLRLocServer). Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Adaptive response in Drosophila melanogaster heat shock proteins mutant strains

    International Nuclear Information System (INIS)

    Shaposhnikov, M.V.; Moskalev, A.A.; Turysheva, E.V.

    2007-01-01

    Complete text of publication follows. The members of the heat shock proteins (Hsp) family function as molecular chaperones and assist intracellular folding of newly synthesized proteins. Also it is possible that molecular chaperones are induced during adaptive response to oxidative stress and radiation. The aim of our research was to exam the role of heat shock proteins in adaptive response to oxidative stress after low dose rate gamma-irradiation in Drosophila melanogaster. Drosophilamelanogaster strains were kindly provided by Bloomington Drosophila Stock Center (University of state of Indiana, Bloomington, USA). We used wild type strain (CS), heat shock protein mutant strains (Hsp22, Hsp70, Hsp83), and heat shock factor mutant strain (Hsf). Strains were chronically exposured to adaptive dose of gamma-irradiation in dose rate of 0.17 cGy/h during all stages of life history (from the embrional stage to the stage of matured imago). The rate of absorbed dose was 60 cGy. For oxidative-stress challenge twodays old flies were starved in empty vials for 6 h and then transferred to vials containing only filter paper soaked with 20 mM paraquat in 5% sucrose solution. Survival data were collected after 26 h of treatment. Dead flies were counted daily. The obtained data were subjected to survival analysis by Kaplan and Meier method and presented as survival curves. Statistical analysis was held by non-parametric methods. To test the significance of the difference between the two age distributions Kolmogorov-Smirnov test was applied. Gehan-Braslow- Wilcoxon and Cox-Mantel tests were used for estimation of median life span differences. In addition the minimal and maximal life span, time of 90% death, and mortality rate doubling time (MRDT) were estimated. The obtained results will be discussed in presentation.

  7. Specificity of transmembrane protein palmitoylation in yeast.

    Directory of Open Access Journals (Sweden)

    Ayelén González Montoro

    Full Text Available Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs, characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD. There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as

  8. Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

    Science.gov (United States)

    Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

    2015-01-01

    ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can

  9. Naturally occurring, tumor-specific, therapeutic proteins.

    Science.gov (United States)

    Argiris, Konstantinos; Panethymitaki, Chrysoula; Tavassoli, Mahvash

    2011-05-01

    The emerging approach to cancer treatment known as targeted therapies offers hope in improving the treatment of therapy-resistant cancers. Recent understanding of the molecular pathogenesis of cancer has led to the development of targeted novel drugs such as monoclonal antibodies, small molecule inhibitors, mimetics, antisense and small interference RNA-based strategies, among others. These compounds act on specific targets that are believed to contribute to the development and progression of cancers and resistance of tumors to conventional therapies. Delivered individually or combined with chemo- and/or radiotherapy, such novel drugs have produced significant responses in certain types of cancer. Among the most successful novel compounds are those which target tyrosine kinases (imatinib, trastuzumab, sinutinib, cetuximab). However, these compounds can cause severe side-effects as they inhibit pathways such as epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor, which are also important for normal functions in non-transformed cells. Recently, a number of proteins have been identified which show a remarkable tumor-specific cytotoxic activity. This toxicity is independent of tumor type or specific genetic changes such as p53, pRB or EGFR aberrations. These tumor-specific killer proteins are either derived from common human and animal viruses such as E1A, E4ORF4 and VP3 (apoptin) or of cellular origin, such as TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) and MDA-7 (melanoma differentiation associated-7). This review aims to present a current overview of a selection of these proteins with preferential toxicity among cancer cells and will provide an insight into the possible mechanism of action, tumor specificity and their potential as novel tumor-specific cancer therapeutics.

  10. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...... the relationship between the immunogenic proteins involved in this nondeleterious antibody response and the pathological response associated with food allergy. The objective of the present study was to characterize the antigenic specificity of the soy protein-specific antibody response generated in healthy mice...... ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...

  11. Prolonged Adaptation to a Low or High Protein Diet Does Not Modulate Basal Muscle Protein Synthesis Rates - A Substudy.

    Directory of Open Access Journals (Sweden)

    Rick Hursel

    Full Text Available Based on controlled 36 h experiments a higher dietary protein intake causes a positive protein balance and a negative fat balance. A positive net protein balance may support fat free mass accrual. However, few data are available on the impact of more prolonged changes in habitual protein intake on whole-body protein metabolism and basal muscle protein synthesis rates.To assess changes in whole-body protein turnover and basal muscle protein synthesis rates following 12 weeks of adaptation to a low versus high dietary protein intake.A randomized parallel study was performed in 40 subjects who followed either a high protein (2.4 g protein/kg/d or low protein (0.4 g protein/kg/d energy-balanced diet (30/35/35% or 5/60/35% energy from protein/carbohydrate/fat for a period of 12 weeks. A subgroup of 7 men and 8 women (body mass index: 22.8±2.3 kg/m2, age: 24.3±4.9 y were selected to evaluate the impact of prolonged adaptation to either a high or low protein intake on whole body protein metabolism and basal muscle protein synthesis rates. After the diet, subjects received continuous infusions with L-[ring-2H5]phenylalanine and L-[ring-2H2]tyrosine in an overnight fasted state, with blood samples and muscle biopsies being collected to assess post-absorptive whole-body protein turnover and muscle protein synthesis rates in vivo in humans.After 12 weeks of intervention, whole-body protein balance in the fasted state was more negative in the high protein treatment when compared with the low protein treatment (-4.1±0.5 vs -2.7±0.6 μmol phenylalanine/kg/h;P<0.001. Whole-body protein breakdown (43.0±4.4 vs 37.8±3.8 μmol phenylalanine/kg/h;P<0.03, synthesis (38.9±4.2 vs 35.1±3.6 μmol phenylalanine/kg/h;P<0.01 and phenylalanine hydroxylation rates (4.1±0.6 vs 2.7±0.6 μmol phenylalanine/kg/h;P<0.001 were significantly higher in the high vs low protein group. Basal muscle protein synthesis rates were maintained on a low vs high protein diet (0.042

  12. Domain requirements for the Dock adapter protein in growth- cone signaling

    OpenAIRE

    Rao, Yong; Zipursky, S. Lawrence

    1998-01-01

    Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly speci...

  13. Sequence motifs in MADS transcription factors responsible for specificity and diversification of protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Aalt D J van Dijk

    Full Text Available Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and

  14. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H.; Burk, Robert D.

    2015-01-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution. PMID:26086730

  15. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Directory of Open Access Journals (Sweden)

    Koenraad Van Doorslaer

    2015-06-01

    Full Text Available In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  16. Degradation of Human PDZ-Proteins by Human Alphapapillomaviruses Represents an Evolutionary Adaptation to a Novel Cellular Niche.

    Science.gov (United States)

    Van Doorslaer, Koenraad; DeSalle, Rob; Einstein, Mark H; Burk, Robert D

    2015-06-01

    In order to complete their life cycle, papillomaviruses have evolved to manipulate a plethora of cellular pathways. The products of the human Alphapapillomavirus E6 proteins specifically interact with and target PDZ containing proteins for degradation. This viral phenotype has been suggested to play a role in viral oncogenesis. To analyze the association of HPV E6 mediated PDZ-protein degradation with cervical oncogenesis, a high-throughput cell culture assay was developed. Degradation of an epitope tagged human MAGI1 isoform was visualized by immunoblot. The correlation between HPV E6-induced degradation of hMAGI1 and epidemiologically determined HPV oncogenicity was evaluated using a Bayesian approach within a phylogenetic context. All tested oncogenic types degraded the PDZ-containing protein hMAGI1d; however, E6 proteins isolated from several related albeit non-oncogenic viral types were equally efficient at degrading hMAGI1. The relationship between both traits (oncogenicity and PDZ degradation potential) is best explained by a model in which the potential to degrade PDZ proteins was acquired prior to the oncogenic phenotype. This analysis provides evidence that the ancestor of both oncogenic and non-oncogenic HPVs acquired the potential to degrade human PDZ-containing proteins. This suggests that HPV E6 directed degradation of PDZ-proteins represents an ancient ecological niche adaptation. Phylogenetic modeling indicates that this phenotype is not specifically correlated with oncogenic risk, but may act as an enabling phenotype. The role of PDZ protein degradation in HPV fitness and oncogenesis needs to be interpreted in the context of Alphapapillomavirus evolution.

  17. Radio-adaptive response

    International Nuclear Information System (INIS)

    Ikushima, T.

    1992-01-01

    An adaptive response to radiation stress was found as a suppressed induction of chromosomal damage including micronuclei and sister chromatid exchanges in cultured Chinese hamster V79 cells pre-exposed to very low doses of ionizing radiations. The mechanism underlying this novel chromosomal response, called 'radio-adaptive response (RAR)' has been studied progressively. The following results were obtained in recent experiments. 1. Low doses of β-rays from tritiated water (HTO) as well as tritium-thymidine can cause RAR. 2. Thermal neutrons, a high LET radiation, can not act as tritium β-rays or γ-rays. 3. The RAR expression is suppressed not only by the treatment with an inhibitor of protein synthesis but also by RNA synthesis inhibition. 4. Several proteins are newly synthesized concurrently with the RAR expression after the adapting doses, viewed by two-dimensional electrophoresis of cellular proteins. These results suggests that the RAR might be a cellular stress response to a signal produced preferentially by very low doses of low LET radiation under restricted conditions, accompany the inducible specific gene expression. (author)

  18. Adaptive evolution of the symbiotic gene NORK is not correlated with shifts of rhizobial specificity in the genus Medicago

    Directory of Open Access Journals (Sweden)

    Ronfort Joëlle

    2007-11-01

    Full Text Available Abstract Background The NODULATION RECEPTOR KINASE (NORK gene encodes a Leucine-Rich Repeat (LRR-containing receptor-like protein and controls the infection by symbiotic rhizobia and endomycorrhizal fungi in Legumes. The occurrence of numerous amino acid changes driven by directional selection has been reported in this gene, using a limited number of messenger RNA sequences, but the functional reason of these changes remains obscure. The Medicago genus, where changes in rhizobial associations have been previously examined, is a good model to test whether the evolution of NORK is influenced by rhizobial interactions. Results We sequenced a region of 3610 nucleotides (encoding a 392 amino acid-long region of the NORK protein in 32 Medicago species. We confirm that positive selection in NORK has occurred within the Medicago genus and find that the amino acid positions targeted by selection occur in sites outside of solvent-exposed regions in LRRs, and other sites in the N-terminal region of the protein. We tested if branches of the Medicago phylogeny where changes of rhizobial symbionts occurred displayed accelerated rates of amino acid substitutions. Only one branch out of five tested, leading to M. noeana, displays such a pattern. Among other branches, the most likely for having undergone positive selection is not associated with documented shift of rhizobial specificity. Conclusion Adaptive changes in the sequence of the NORK receptor have involved the LRRs, but targeted different sites than in most previous studies of LRR proteins evolution. The fact that positive selection in NORK tends not to be associated to changes in rhizobial specificity indicates that this gene was probably not involved in evolving rhizobial preferences. Other explanations (e.g. coevolutionary arms race must be tested to explain the adaptive evolution of NORK.

  19. Neuron specific metabolic adaptations following multi-day exposures to oxygen glucose deprivation.

    Science.gov (United States)

    Zeiger, Stephanie L H; McKenzie, Jennifer R; Stankowski, Jeannette N; Martin, Jacob A; Cliffel, David E; McLaughlin, BethAnn

    2010-11-01

    Prior exposure to sub toxic insults can induce a powerful endogenous neuroprotective program known as ischemic preconditioning. Current models typically rely on a single stress episode to induce neuroprotection whereas the clinical reality is that patients may experience multiple transient ischemic attacks (TIAs) prior to suffering a stroke. We sought to develop a neuron-enriched preconditioning model using multiple oxygen glucose deprivation (OGD) episodes to assess the endogenous protective mechanisms neurons implement at the metabolic and cellular level. We found that neurons exposed to a five minute period of glucose deprivation recovered oxygen utilization and lactate production using novel microphysiometry techniques. Using the non-toxic and energetically favorable five minute exposure, we developed a preconditioning paradigm where neurons are exposed to this brief OGD for three consecutive days. These cells experienced a 45% greater survival following an otherwise lethal event and exhibited a longer lasting window of protection in comparison to our previous in vitro preconditioning model using a single stress. As in other models, preconditioned cells exhibited mild caspase activation, an increase in oxidized proteins and a requirement for reactive oxygen species for neuroprotection. Heat shock protein 70 was upregulated during preconditioning, yet the majority of this protein was released extracellularly. We believe coupling this neuron-enriched multi-day model with microphysiometry will allow us to assess neuronal specific real-time metabolic adaptations necessary for preconditioning. Copyright © 2010 Elsevier B.V. All rights reserved.

  20. Improved innate and adaptive immunostimulation by genetically modified HIV-1 protein expressing NYVAC vectors.

    Directory of Open Access Journals (Sweden)

    Esther D Quakkelaar

    Full Text Available Attenuated poxviruses are safe and capable of expressing foreign antigens. Poxviruses are applied in veterinary vaccination and explored as candidate vaccines for humans. However, poxviruses express multiple genes encoding proteins that interfere with components of the innate and adaptive immune response. This manuscript describes two strategies aimed to improve the immunogenicity of the highly attenuated, host-range restricted poxvirus NYVAC: deletion of the viral gene encoding type-I interferon-binding protein and development of attenuated replication-competent NYVAC. We evaluated these newly generated NYVAC mutants, encoding HIV-1 env, gag, pol and nef, for their ability to stimulate HIV-specific CD8 T-cell responses in vitro from blood mononuclear cells of HIV-infected subjects. The new vectors were evaluated and compared to the parental NYVAC vector in dendritic cells (DCs, RNA expression arrays, HIV gag expression and cross-presentation assays in vitro. Deletion of type-I interferon-binding protein enhanced expression of interferon and interferon-induced genes in DCs, and increased maturation of infected DCs. Restoration of replication competence induced activation of pathways involving antigen processing and presentation. Also, replication-competent NYVAC showed increased Gag expression in infected cells, permitting enhanced cross-presentation to HIV-specific CD8 T cells and proliferation of HIV-specific memory CD8 T-cells in vitro. The recombinant NYVAC combining both modifications induced interferon-induced genes and genes involved in antigen processing and presentation, as well as increased Gag expression. This combined replication-competent NYVAC is a promising candidate for the next generation of HIV vaccines.

  1. The adapter protein, Grb10, is a positive regulator of vascular endothelial growth factor signaling.

    Science.gov (United States)

    Giorgetti-Peraldi, S; Murdaca, J; Mas, J C; Van Obberghen, E

    2001-07-05

    Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and angiogenesis. Activation of VEGF receptors leads to the recruitment of SH2 containing proteins which link the receptors to the activation of signaling pathways. Here we report that Grb10, an adapter protein of which the biological role remains unknown, is tyrosine phosphorylated in response to VEGF in endothelial cells (HUVEC) and in 293 cells expressing the VEGF receptor KDR. An intact SH2 domain is required for Grb10 tyrosine phosphorylation in response to VEGF, and this phosphorylation is mediated in part through the activation of Src. In HUVEC, VEGF increases Grb10 mRNA level. Expression of Grb10 in HUVEC or in KDR expressing 293 cells results in an increase in the amount and in the tyrosine phosphorylation of KDR. In 293 cells, this is correlated with the activation of signaling molecules, such as MAP kinase. By expressing mutants of Grb10, we found that the positive action of Grb10 is independent of its SH2 domain. Moreover, these Grb10 effects on KDR seem to be specific since Grb10 has no effect on the insulin receptor, and Grb2, another adapter protein, does not mimic the effect of Grb10 on KDR. In conclusion, we propose that VEGF up-regulates Grb10 level, which in turn increases KDR molecules, suggesting that Grb10 could be involved in a positive feedback loop in VEGF signaling.

  2. Neuromuscular adaptations associated with knee joint angle-specific force change.

    Science.gov (United States)

    Noorkõiv, Marika; Nosaka, Kazunori; Blazevich, Anthony J

    2014-08-01

    Neuromuscular adaptations to joint angle-specific force increases after isometric training have not yet been fully elucidated. This study examined angle-specific neuromuscular adaptations in response to isometric knee extension training at short (SL, joint angle 38.1° ± 3.7°) versus long (LL, 87.5° ± 6.0°) muscle lengths. Sixteen men trained three times a week for 6 wk either at SL (n = 8) or LL (n = 8). Voluntary maximal isometric knee extensor (MVC) force, doublet twitch force, EMG amplitudes (EMG/Mmax), and voluntary activation during MVC force (VA%) were measured at eight knee joint angles (30°-100°) at weeks 0, 3, and 6. Muscle volume and cross-sectional area (CSA) were measured from magnetic resonance imaging scans, and fascicle length (Lf) was assessed using ultrasonography before and after training. Clear joint angle specificity of force increase was seen in SL but not in LL. The 13.4% ± 9.7% (P = 0.01) force increase around the training angle in SL was related to changes in vastus lateralis and vastus medialis EMG/Mmax around the training angle (r = 0.84-0.88, P < 0.05), without changes in the doublet twitch force-angle relation or muscle size. In LL, muscle volume and CSA increased and the changes in CSA at specific muscle regions were correlated with changes in MVC force. A 5.4% ± 4.9% (P = 0.001) increase in Lf found in both groups was not associated with angle-specific force changes. There were no angle-specific changes in VA%. The EMG/Mmax, although not VA%, results suggest that neural adaptations underpinned training-related changes at short quadriceps lengths, but hypertrophic changes predominated after training at long lengths. The findings of this study should contribute to the development of more effective and evidence-based rehabilitation and strength training protocols.

  3. Absence of direction-specific cross-modal visual-auditory adaptation in motion-onset event-related potentials.

    Science.gov (United States)

    Grzeschik, Ramona; Lewald, Jörg; Verhey, Jesko L; Hoffmann, Michael B; Getzmann, Stephan

    2016-01-01

    Adaptation to visual or auditory motion affects within-modality motion processing as reflected by visual or auditory free-field motion-onset evoked potentials (VEPs, AEPs). Here, a visual-auditory motion adaptation paradigm was used to investigate the effect of visual motion adaptation on VEPs and AEPs to leftward motion-onset test stimuli. Effects of visual adaptation to (i) scattered light flashes, and motion in the (ii) same or in the (iii) opposite direction of the test stimulus were compared. For the motion-onset VEPs, i.e. the intra-modal adaptation conditions, direction-specific adaptation was observed--the change-N2 (cN2) and change-P2 (cP2) amplitudes were significantly smaller after motion adaptation in the same than in the opposite direction. For the motion-onset AEPs, i.e. the cross-modal adaptation condition, there was an effect of motion history only in the change-P1 (cP1), and this effect was not direction-specific--cP1 was smaller after scatter than after motion adaptation to either direction. No effects were found for later components of motion-onset AEPs. While the VEP results provided clear evidence for the existence of a direction-specific effect of motion adaptation within the visual modality, the AEP findings suggested merely a motion-related, but not a direction-specific effect. In conclusion, the adaptation of veridical auditory motion detectors by visual motion is not reflected by the AEPs of the present study. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  4. Specific Proteins in Nontuberculous Mycobacteria: New Potential Tools

    Directory of Open Access Journals (Sweden)

    Patricia Orduña

    2015-01-01

    Full Text Available Nontuberculous mycobacteria (NTM have been isolated from water, soil, air, food, protozoa, plants, animals, and humans. Although most NTM are saprophytes, approximately one-third of NTM have been associated with human diseases. In this study, we did a comparative proteomic analysis among five NTM strains isolated from several sources. There were different numbers of protein spots from M. gordonae (1,264, M. nonchromogenicum type I (894, M. nonchromogenicum type II (935, M. peregrinum (806, and M. scrofulaceum/Mycobacterium mantenii (1,486 strains, respectively. We identified 141 proteins common to all strains and specific proteins to each NTM strain. A total of 23 proteins were selected for its identification. Two of the common proteins identified (short-chain dehydrogenase/reductase SDR and diguanylate cyclase did not align with M. tuberculosis complex protein sequences, which suggest that these proteins are found only in the NTM strains. Some of the proteins identified as common to all strains can be used as markers of NTM exposure and for the development of new diagnostic tools. Additionally, the specific proteins to NTM strains identified may represent potential candidates for the diagnosis of diseases caused by these mycobacteria.

  5. Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

    Science.gov (United States)

    Qiao, Liang; Xiong, Gao; Wang, Ri-xin; He, Song-zhen; Chen, Jie; Tong, Xiao-ling; Hu, Hai; Li, Chun-lin; Gai, Ting-ting; Xin, Ya-qun; Liu, Xiao-fan; Chen, Bin; Xiang, Zhong-huai; Lu, Cheng; Dai, Fang-yin

    2014-04-01

    Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.

  6. Homeoviscous adaptation and the regulation of membrane lipids

    DEFF Research Database (Denmark)

    Ernst, Robert; Ejsing, Christer S; Antonny, Bruno

    2016-01-01

    Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold. This ada......Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold....... This adaptive response was termed homeoviscous adaptation and has been frequently studied with a specific focus on the acyl chain composition of membrane lipids. Massspectrometry-based lipidomics can nowadays provide more comprehensive insights into the complexity of lipid remodeling during adaptive responses...... such as neurons maintain unique lipid compositions with specific physicochemical properties. To date little is known about the sensory mechanisms regulating the acyl chain profile in such specialized cells or during adaptive responses. Here we summarize our current understanding of lipid metabolic networks...

  7. Neandertals' large lower thorax may represent adaptation to high protein diet.

    Science.gov (United States)

    Ben-Dor, Miki; Gopher, Avi; Barkai, Ran

    2016-07-01

    Humans are limited in their capacity to convert protein into energy. We present a hypothesis that a "bell" shaped thorax and a wide pelvis evolved in Neandertals, at least in part, as an adaptation to a high protein diet. A high protein diet created a need to house an enlarged liver and urinary system in a wider lower trunk. To test the hypothesis, we applied a model developed to identify points of nutritional stress. A ratio of obligatory dietary fat to total animal fat and protein sourced calories is calculated based on various known and estimated parameters. Stress is identified when the obligatory dietary fat ratio is higher than fat content ratios in available prey. The model predicts that during glacial winters, when carbohydrates weren't available, 74%-85% of Neandertals' caloric intake would have had to come from animal fat. Large animals contain around 50% fat calories, and their fat content is diminished during winter, so a significant stressful dietary fat deficit was identified by the model. This deficit could potentially be ameliorated by an increased capability to convert protein into energy. Given that high protein consumption is associated with larger liver and kidneys in animal models, it appears likely that the enlarged inferior section of the Neandertals thorax and possibly, in part, also his wide pelvis, represented an adaptation to provide encasement for those enlarged organs. Behavioral and evolutionary implications of the hypothesis are also discussed. Am J Phys Anthropol 160:367-378, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Quantitative Proteomics Reveals Membrane Protein-Mediated Hypersaline Sensitivity and Adaptation in Halophilic Nocardiopsis xinjiangensis.

    Science.gov (United States)

    Zhang, Yao; Li, Yanchang; Zhang, Yongguang; Wang, Zhiqiang; Zhao, Mingzhi; Su, Na; Zhang, Tao; Chen, Lingsheng; Wei, Wei; Luo, Jing; Zhou, Yanxia; Xu, Yongru; Xu, Ping; Li, Wenjun; Tao, Yong

    2016-01-04

    The genus Nocardiopsis is one of the most dominant Actinobacteria that survives in hypersaline environments. However, the adaptation mechanisms for halophilism are still unclear. Here, we performed isobaric tags for relative and absolute quantification based quantitative proteomics to investigate the functions of the membrane proteome after salt stress. A total of 683 membrane proteins were identified and quantified, of which 126 membrane proteins displayed salt-induced changes in abundance. Intriguingly, bioinformatics analyses indicated that these differential proteins showed two expression patterns, which were further validated by phenotypic changes and functional differences. The majority of ABC transporters, secondary active transporters, cell motility proteins, and signal transduction kinases were up-regulated with increasing salt concentration, whereas cell differentiation, small molecular transporter (ions and amino acids), and secondary metabolism proteins were significantly up-regulated at optimum salinity, but down-regulated or unchanged at higher salinity. The small molecule transporters and cell differentiation-related proteins acted as sensing proteins that played a more important biological role at optimum salinity. However, the ABC transporters for compatible solutes, Na(+)-dependent transporters, and cell motility proteins acted as adaptive proteins that actively counteracted higher salinity stress. Overall, regulation of membrane proteins may provide a major protection strategy against hyperosmotic stress.

  9. A Comparative Pan-Genome Perspective of Niche-Adaptable Cell-Surface Protein Phenotypes in Lactobacillus rhamnosus

    Science.gov (United States)

    Kant, Ravi; Sigvart-Mattila, Pia; Paulin, Lars; Mecklin, Jukka-Pekka; Saarela, Maria; Palva, Airi; von Ossowski, Ingemar

    2014-01-01

    Lactobacillus rhamnosus is a ubiquitously adaptable Gram-positive bacterium and as a typical commensal can be recovered from various microbe-accessible bodily orifices and cavities. Then again, other isolates are food-borne, with some of these having been long associated with naturally fermented cheeses and yogurts. Additionally, because of perceived health benefits to humans and animals, numerous L. rhamnosus strains have been selected for use as so-called probiotics and are often taken in the form of dietary supplements and functional foods. At the genome level, it is anticipated that certain genetic variances will have provided the niche-related phenotypes that augment the flexible adaptiveness of this species, thus enabling its strains to grow and survive in their respective host environments. For this present study, we considered it functionally informative to examine and catalogue the genotype-phenotype variation existing at the cell surface between different L. rhamnosus strains, with the presumption that this might be relatable to habitat preferences and ecological adaptability. Here, we conducted a pan-genomic study involving 13 genomes from L. rhamnosus isolates with various origins. In using a benchmark strain (gut-adapted L. rhamnosus GG) for our pan-genome comparison, we had focused our efforts on a detailed examination and description of gene products for certain functionally relevant surface-exposed proteins, each of which in effect might also play a part in niche adaptability among the other strains. Perhaps most significantly of the surface protein loci we had analyzed, it would appear that the spaCBA operon (known to encode SpaCBA-called pili having a mucoadhesive phenotype) is a genomic rarity and an uncommon occurrence in L. rhamnosus. However, for any of the so-piliated L. rhamnosus strains, they will likely possess an increased niche-specific fitness, which functionally might presumably be manifested by a protracted transient colonization of

  10. Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

    Science.gov (United States)

    Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

    2009-03-11

    Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene

  11. Proteomic analysis of endothelial cold-adaptation

    Directory of Open Access Journals (Sweden)

    Zieger Michael AJ

    2011-12-01

    Full Text Available Abstract Background Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource. Results Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine

  12. A Functional Toll-Interacting Protein Variant Is Associated with Bacillus Calmette-Guérin-Specific Immune Responses and Tuberculosis.

    Science.gov (United States)

    Shah, Javeed A; Musvosvi, Munyaradzi; Shey, Muki; Horne, David J; Wells, Richard D; Peterson, Glenna J; Cox, Jeffery S; Daya, Michelle; Hoal, Eileen G; Lin, Lin; Gottardo, Raphael; Hanekom, Willem A; Scriba, Thomas J; Hatherill, Mark; Hawn, Thomas R

    2017-08-15

    The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection. We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2 + CD4 + T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG

  13. The diagnostic adaptive behavior scale: evaluating its diagnostic sensitivity and specificity.

    Science.gov (United States)

    Balboni, Giulia; Tassé, Marc J; Schalock, Robert L; Borthwick-Duffy, Sharon A; Spreat, Scott; Thissen, David; Widaman, Keith F; Zhang, Dalun; Navas, Patricia

    2014-11-01

    The Diagnostic Adaptive Behavior Scale (DABS) was constructed with items across three domains--conceptual, social, and practical adaptive skills--and normed on a representative sample of American individuals from 4 to 21 years of age. The DABS was developed to focus its assessment around the decision point for determining the presence or absence of significant limitations of adaptive behavior for the diagnosis of Intellectual Disability (ID). The purpose of this study, which was composed of 125 individuals with and 933 without an ID-related diagnosis, was to determine the ability of the DABS to correctly identify the individuals with and without ID (i.e., sensitivity and specificity). The results indicate that the DABS sensitivity coefficients ranged from 81% to 98%, specificity coefficients ranged from 89% to 91%, and that the Area Under the Receiver Operating Characteristic Curve were excellent or good. These results indicate that the DABS has very good levels of diagnostic efficiency. Copyright © 2014. Published by Elsevier Ltd.

  14. General and specific lipid-protein interactions in Na,K-ATPase.

    Science.gov (United States)

    Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

    2015-09-01

    The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Practical analysis of specificity-determining residues in protein families.

    Science.gov (United States)

    Chagoyen, Mónica; García-Martín, Juan A; Pazos, Florencio

    2016-03-01

    Determining the residues that are important for the molecular activity of a protein is a topic of broad interest in biomedicine and biotechnology. This knowledge can help understanding the protein's molecular mechanism as well as to fine-tune its natural function eventually with biotechnological or therapeutic implications. Some of the protein residues are essential for the function common to all members of a family of proteins, while others explain the particular specificities of certain subfamilies (like binding on different substrates or cofactors and distinct binding affinities). Owing to the difficulty in experimentally determining them, a number of computational methods were developed to detect these functional residues, generally known as 'specificity-determining positions' (or SDPs), from a collection of homologous protein sequences. These methods are mature enough for being routinely used by molecular biologists in directing experiments aimed at getting insight into the functional specificity of a family of proteins and eventually modifying it. In this review, we summarize some of the recent discoveries achieved through SDP computational identification in a number of relevant protein families, as well as the main approaches and software tools available to perform this type of analysis. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  16. The complex becomes more complex: protein-protein interactions of SnRK1 with DUF581 family proteins provide a framework for cell- and stimulus type-specific SnRK1 signaling in plants

    Directory of Open Access Journals (Sweden)

    Madlen eNietzsche

    2014-02-01

    Full Text Available In plants, SNF1-related kinase (SnRK1 responds to the availability of carbohydrates as well as to environmental stresses by down-regulating ATP consuming biosynthetic processes, while stimulating energy-generating catabolic reactions through gene expression and post-transcriptional regulation. The functional SnRK1 complex is a heterotrimer where the catalytic alpha subunit associates with a regulatory beta subunit and an activating gamma subunit. Several different metabolites as well as the hormone abscisic acid (ABA have been shown to modulate SnRK1 activity in a cell- and stimulus-type specific manner. It has been proposed that tissue- or stimulus-specific expression of adapter proteins mediating SnRK1 regulation can at least partly explain the differences observed in SnRK1 signaling. By using yeast two-hybrid and in planta bi-molecular fluorescence complementation assays we were able to demonstrate that proteins containing the domain of unknown function (DUF 581 could interact with both isoforms of the SnRK1 alpha subunit (AKIN10/11 of Arabidopsis. A structure/function analysis suggests that the DUF581 is a generic SnRK1 interaction module and co-expression with DUF581 proteins in plant cells leads to reallocation of the kinase to specific regions within the nucleus. Yeast two-hybrid analyses suggest that SnRK1 and DUF581 proteins can share common interaction partners inside the nucleus. The analysis of available microarray data implies that expression of the 19 members of the DUF581 encoding gene family in Arabidopsis is differentially regulated by hormones and environmental cues, indicating specialized functions of individual family members. We hypothesize that DUF581 proteins could act as mediators conferring tissue- and stimulus-type specific differences in SnRK1 regulation.

  17. Identification of species- and tissue-specific proteins using proteomic strategy

    Science.gov (United States)

    Chernukha, I. M.; Vostrikova, N. L.; Kovalev, L. I.; Shishkin, S. S.; Kovaleva, M. A.; Manukhin, Y. S.

    2017-09-01

    Proteomic technologies have proven to be very effective for detecting biochemical changes in meat products, such as changes in tissue- and species-specific proteins. In the tissues of cattle, pig, horse and camel M. longissimus dorsi both tissue- and species specific proteins were detected using two dimensional electrophoresis. Species-specific isoforms of several muscle proteins were also identified. The identified and described proteins of cattle, pig, horse and camel skeletal muscles (including mass spectra of the tryptic peptides) were added to the national free access database “Muscle organ proteomics”. This research has enabled the development of new highly sensitive technologies for meat product quality control against food fraud.

  18. Effects of practice schedule and task specificity on the adaptive process of motor learning.

    Science.gov (United States)

    Barros, João Augusto de Camargo; Tani, Go; Corrêa, Umberto Cesar

    2017-10-01

    This study investigated the effects of practice schedule and task specificity based on the perspective of adaptive process of motor learning. For this purpose, tasks with temporal and force control learning requirements were manipulated in experiments 1 and 2, respectively. Specifically, the task consisted of touching with the dominant hand the three sequential targets with specific movement time or force for each touch. Participants were children (N=120), both boys and girls, with an average age of 11.2years (SD=1.0). The design in both experiments involved four practice groups (constant, random, constant-random, and random-constant) and two phases (stabilisation and adaptation). The dependent variables included measures related to the task goal (accuracy and variability of error of the overall movement and force patterns) and movement pattern (macro- and microstructures). Results revealed a similar error of the overall patterns for all groups in both experiments and that they adapted themselves differently in terms of the macro- and microstructures of movement patterns. The study concludes that the effects of practice schedules on the adaptive process of motor learning were both general and specific to the task. That is, they were general to the task goal performance and specific regarding the movement pattern. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. An adaptive bin framework search method for a beta-sheet protein homopolymer model

    Directory of Open Access Journals (Sweden)

    Hoos Holger H

    2007-04-01

    Full Text Available Abstract Background The problem of protein structure prediction consists of predicting the functional or native structure of a protein given its linear sequence of amino acids. This problem has played a prominent role in the fields of biomolecular physics and algorithm design for over 50 years. Additionally, its importance increases continually as a result of an exponential growth over time in the number of known protein sequences in contrast to a linear increase in the number of determined structures. Our work focuses on the problem of searching an exponentially large space of possible conformations as efficiently as possible, with the goal of finding a global optimum with respect to a given energy function. This problem plays an important role in the analysis of systems with complex search landscapes, and particularly in the context of ab initio protein structure prediction. Results In this work, we introduce a novel approach for solving this conformation search problem based on the use of a bin framework for adaptively storing and retrieving promising locally optimal solutions. Our approach provides a rich and general framework within which a broad range of adaptive or reactive search strategies can be realized. Here, we introduce adaptive mechanisms for choosing which conformations should be stored, based on the set of conformations already stored in memory, and for biasing choices when retrieving conformations from memory in order to overcome search stagnation. Conclusion We show that our bin framework combined with a widely used optimization method, Monte Carlo search, achieves significantly better performance than state-of-the-art generalized ensemble methods for a well-known protein-like homopolymer model on the face-centered cubic lattice.

  20. Specificity and evolvability in eukaryotic protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Pedro Beltrao

    2007-02-01

    Full Text Available Progress in uncovering the protein interaction networks of several species has led to questions of what underlying principles might govern their organization. Few studies have tried to determine the impact of protein interaction network evolution on the observed physiological differences between species. Using comparative genomics and structural information, we show here that eukaryotic species have rewired their interactomes at a fast rate of approximately 10(-5 interactions changed per protein pair, per million years of divergence. For Homo sapiens this corresponds to 10(3 interactions changed per million years. Additionally we find that the specificity of binding strongly determines the interaction turnover and that different biological processes show significantly different link dynamics. In particular, human proteins involved in immune response, transport, and establishment of localization show signs of positive selection for change of interactions. Our analysis suggests that a small degree of molecular divergence can give rise to important changes at the network level. We propose that the power law distribution observed in protein interaction networks could be partly explained by the cell's requirement for different degrees of protein binding specificity.

  1. Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

    Science.gov (United States)

    Ohta, Shinya; Kimura, Michiko; Takagi, Shunsuke; Toramoto, Iyo; Ishihama, Yasushi

    2016-09-02

    During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

  2. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    Science.gov (United States)

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  3. Solar adaptive optics: specificities, lessons learned, and open alternatives

    Science.gov (United States)

    Montilla, I.; Marino, J.; Asensio Ramos, A.; Collados, M.; Montoya, L.; Tallon, M.

    2016-07-01

    First on sky adaptive optics experiments were performed on the Dunn Solar Telescope on 1979, with a shearing interferometer and limited success. Those early solar adaptive optics efforts forced to custom-develop many components, such as Deformable Mirrors and WaveFront Sensors, which were not available at that time. Later on, the development of the correlation Shack-Hartmann marked a breakthrough in solar adaptive optics. Since then, successful Single Conjugate Adaptive Optics instruments have been developed for many solar telescopes, i.e. the National Solar Observatory, the Vacuum Tower Telescope and the Swedish Solar Telescope. Success with the Multi Conjugate Adaptive Optics systems for GREGOR and the New Solar Telescope has proved to be more difficult to attain. Such systems have a complexity not only related to the number of degrees of freedom, but also related to the specificities of the Sun, used as reference, and the sensing method. The wavefront sensing is performed using correlations on images with a field of view of 10", averaging wavefront information from different sky directions, affecting the sensing and sampling of high altitude turbulence. Also due to the low elevation at which solar observations are performed we have to include generalized fitting error and anisoplanatism, as described by Ragazzoni and Rigaut, as non-negligible error sources in the Multi Conjugate Adaptive Optics error budget. For the development of the next generation Multi Conjugate Adaptive Optics systems for the Daniel K. Inouye Solar Telescope and the European Solar Telescope we still need to study and understand these issues, to predict realistically the quality of the achievable reconstruction. To improve their designs other open issues have to be assessed, i.e. possible alternative sensing methods to avoid the intrinsic anisoplanatism of the wide field correlation Shack-Hartmann, new parameters to estimate the performance of an adaptive optics solar system, alternatives to

  4. Computational design, construction, and characterization of a set of specificity determining residues in protein-protein interactions.

    Science.gov (United States)

    Nagao, Chioko; Izako, Nozomi; Soga, Shinji; Khan, Samia Haseeb; Kawabata, Shigeki; Shirai, Hiroki; Mizuguchi, Kenji

    2012-10-01

    Proteins interact with different partners to perform different functions and it is important to elucidate the determinants of partner specificity in protein complex formation. Although methods for detecting specificity determining positions have been developed previously, direct experimental evidence for these amino acid residues is scarce, and the lack of information has prevented further computational studies. In this article, we constructed a dataset that is likely to exhibit specificity in protein complex formation, based on available crystal structures and several intuitive ideas about interaction profiles and functional subclasses. We then defined a "structure-based specificity determining position (sbSDP)" as a set of equivalent residues in a protein family showing a large variation in their interaction energy with different partners. We investigated sequence and structural features of sbSDPs and demonstrated that their amino acid propensities significantly differed from those of other interacting residues and that the importance of many of these residues for determining specificity had been verified experimentally. Copyright © 2012 Wiley Periodicals, Inc.

  5. Platyhelminth Venom Allergen-Like (VAL) proteins: revealing structural diversity, class-specific features and biological associations across the phylum

    Science.gov (United States)

    CHALMERS, IAIN W.; HOFFMANN, KARL F.

    2012-01-01

    SUMMARY During platyhelminth infection, a cocktail of proteins is released by the parasite to aid invasion, initiate feeding, facilitate adaptation and mediate modulation of the host immune response. Included amongst these proteins is the Venom Allergen-Like (VAL) family, part of the larger sperm coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) superfamily. To explore the significance of this protein family during Platyhelminthes development and host interactions, we systematically summarize all published proteomic, genomic and immunological investigations of the VAL protein family to date. By conducting new genomic and transcriptomic interrogations to identify over 200 VAL proteins (228) from species in all 4 traditional taxonomic classes (Trematoda, Cestoda, Monogenea and Turbellaria), we further expand our knowledge related to platyhelminth VAL diversity across the phylum. Subsequent phylogenetic and tertiary structural analyses reveal several class-specific VAL features, which likely indicate a range of roles mediated by this protein family. Our comprehensive analysis of platyhelminth VALs represents a unifying synopsis for understanding diversity within this protein family and a firm context in which to initiate future functional characterization of these enigmatic members. PMID:22717097

  6. Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex

    Directory of Open Access Journals (Sweden)

    Lisa M. Tuttle

    2018-03-01

    Full Text Available Summary: Transcription activation domains (ADs are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. : Tuttle et al. report a “fuzzy free-for-all” interaction mechanism that explains how seemingly unrelated transcription activators converge on a limited number of coactivator targets. The mechanism provides a rationale for the observation that individually weak and low-specificity interactions can combine to produce biologically critical function without requiring highly ordered structure. Keywords: transcription activation, intrinsically disordered proteins, fuzzy binding

  7. Optimizing scoring function of protein-nucleic acid interactions with both affinity and specificity.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Yan

    Full Text Available Protein-nucleic acid (protein-DNA and protein-RNA recognition is fundamental to the regulation of gene expression. Determination of the structures of the protein-nucleic acid recognition and insight into their interactions at molecular level are vital to understanding the regulation function. Recently, quantitative computational approach has been becoming an alternative of experimental technique for predicting the structures and interactions of biomolecular recognition. However, the progress of protein-nucleic acid structure prediction, especially protein-RNA, is far behind that of the protein-ligand and protein-protein structure predictions due to the lack of reliable and accurate scoring function for quantifying the protein-nucleic acid interactions. In this work, we developed an accurate scoring function (named as SPA-PN, SPecificity and Affinity of the Protein-Nucleic acid interactions for protein-nucleic acid interactions by incorporating both the specificity and affinity into the optimization strategy. Specificity and affinity are two requirements of highly efficient and specific biomolecular recognition. Previous quantitative descriptions of the biomolecular interactions considered the affinity, but often ignored the specificity owing to the challenge of specificity quantification. We applied our concept of intrinsic specificity to connect the conventional specificity, which circumvents the challenge of specificity quantification. In addition to the affinity optimization, we incorporated the quantified intrinsic specificity into the optimization strategy of SPA-PN. The testing results and comparisons with other scoring functions validated that SPA-PN performs well on both the prediction of binding affinity and identification of native conformation. In terms of its performance, SPA-PN can be widely used to predict the protein-nucleic acid structures and quantify their interactions.

  8. Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

    KAUST Repository

    Kim, Dongjin; Ntui, Valentine Otang; Zhang, Nianshu; Xiong, Liming

    2015-01-01

    Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

  9. Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

    KAUST Repository

    Kim, Dongjin

    2015-10-09

    Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

  10. Structural adaptations of proteins to different biological membranes

    Science.gov (United States)

    Pogozheva, Irina D.; Tristram-Nagle, Stephanie; Mosberg, Henry I.; Lomize, Andrei L.

    2013-01-01

    To gain insight into adaptations of proteins to their membranes, intrinsic hydrophobic thicknesses, distributions of different chemical groups and profiles of hydrogen-bonding capacities (α and β) and the dipolarity/polarizability parameter (π*) were calculated for lipid-facing surfaces of 460 integral α-helical, β-barrel and peripheral proteins from eight types of biomembranes. For comparison, polarity profiles were also calculated for ten artificial lipid bilayers that have been previously studied by neutron and X-ray scattering. Estimated hydrophobic thicknesses are 30-31 Å for proteins from endoplasmic reticulum, thylakoid, and various bacterial plasma membranes, but differ for proteins from outer bacterial, inner mitochondrial and eukaryotic plasma membranes (23.9, 28.6 and 33.5 Å, respectively). Protein and lipid polarity parameters abruptly change in the lipid carbonyl zone that matches the calculated hydrophobic boundaries. Maxima of positively charged protein groups correspond to the location of lipid phosphates at 20-22 Å distances from the membrane center. Locations of Tyr atoms coincide with hydrophobic boundaries, while distributions maxima of Trp rings are shifted by 3-4 Å toward the membrane center. Distributions of Trp atoms indicate the presence of two 5-8 Å-wide midpolar regions with intermediate π* values within the hydrocarbon core, whose size and symmetry depend on the lipid composition of membrane leaflets. Midpolar regions are especially asymmetric in outer bacterial membranes and cell membranes of mesophilic but not hyperthermophilic archaebacteria, indicating the larger width of the central nonpolar region in the later case. In artificial lipid bilayers, midpolar regions are observed up to the level of acyl chain double bonds. PMID:23811361

  11. Branch-specific plasticity of a bifunctional dopamine circuit encodes protein hunger.

    Science.gov (United States)

    Liu, Qili; Tabuchi, Masashi; Liu, Sha; Kodama, Lay; Horiuchi, Wakako; Daniels, Jay; Chiu, Lucinda; Baldoni, Daniel; Wu, Mark N

    2017-05-05

    Free-living animals must not only regulate the amount of food they consume but also choose which types of food to ingest. The shifting of food preference driven by nutrient-specific hunger can be essential for survival, yet little is known about the underlying mechanisms. We identified a dopamine circuit that encodes protein-specific hunger in Drosophila The activity of these neurons increased after substantial protein deprivation. Activation of this circuit simultaneously promoted protein intake and restricted sugar consumption, via signaling to distinct downstream neurons. Protein starvation triggered branch-specific plastic changes in these dopaminergic neurons, thus enabling sustained protein consumption. These studies reveal a crucial circuit mechanism by which animals adjust their dietary strategy to maintain protein homeostasis. Copyright © 2017, American Association for the Advancement of Science.

  12. Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.

    Science.gov (United States)

    Tuttle, Lisa M; Pacheco, Derek; Warfield, Linda; Luo, Jie; Ranish, Jeff; Hahn, Steven; Klevit, Rachel E

    2018-03-20

    Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    KAUST Repository

    Cannistraci, Carlo Vittorio

    2015-01-26

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet\\'s performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis.

  14. Median Modified Wiener Filter for nonlinear adaptive spatial denoising of protein NMR multidimensional spectra

    KAUST Repository

    Cannistraci, Carlo Vittorio; Abbas, Ahmed; Gao, Xin

    2015-01-01

    Denoising multidimensional NMR-spectra is a fundamental step in NMR protein structure determination. The state-of-the-art method uses wavelet-denoising, which may suffer when applied to non-stationary signals affected by Gaussian-white-noise mixed with strong impulsive artifacts, like those in multi-dimensional NMR-spectra. Regrettably, Wavelet's performance depends on a combinatorial search of wavelet shapes and parameters; and multi-dimensional extension of wavelet-denoising is highly non-trivial, which hampers its application to multidimensional NMR-spectra. Here, we endorse a diverse philosophy of denoising NMR-spectra: less is more! We consider spatial filters that have only one parameter to tune: the window-size. We propose, for the first time, the 3D extension of the median-modified-Wiener-filter (MMWF), an adaptive variant of the median-filter, and also its novel variation named MMWF*. We test the proposed filters and the Wiener-filter, an adaptive variant of the mean-filter, on a benchmark set that contains 16 two-dimensional and three-dimensional NMR-spectra extracted from eight proteins. Our results demonstrate that the adaptive spatial filters significantly outperform their non-adaptive versions. The performance of the new MMWF* on 2D/3D-spectra is even better than wavelet-denoising. Noticeably, MMWF* produces stable high performance almost invariant for diverse window-size settings: this signifies a consistent advantage in the implementation of automatic pipelines for protein NMR-spectra analysis.

  15. Tendon Adaptation to Sport-specific Loading in Adolescent Athletes.

    Science.gov (United States)

    Cassel, M; Carlsohn, A; Fröhlich, K; John, M; Riegels, N; Mayer, F

    2016-02-01

    Tendon adaptation due to mechanical loading is controversially discussed. However, data concerning the development of tendon thickness in adolescent athletes is sparse. The purpose of this study was to examine possible differences in Achilles (AT) and patellar tendon (PT) thickness in adolescent athletes while considering age, gender and sport-specific loading. In 500 adolescent competitive athletes of 16 different sports and 40 recreational controls both ATs and PTs were sonographically measured. Subjects were divided into 2 age groups (sport type categories (ball, combat, and water sports, combined disciplines, cycling, controls). In addition, 3 risk groups (low, moderate, high) were created according to the athlete's risk of developing tendinopathy. AT and PT thickness did not significantly differ between age groups (AT/PT:sports/cyclists and lowest in controls (p≤0.002). PT thickness was greatest in water sports and lowest in controls (p=0.02). High risk athletes presented slightly higher AT thickness compared to the low risk group (p=0.03). Increased AT and PT thickness in certain sport types compared to controls supports the hypothesis of structural tendon adaptation due to sport-specific loading. © Georg Thieme Verlag KG Stuttgart · New York.

  16. Sequence-Based Analysis of Thermal Adaptation and Protein Energy Landscapes in an Invasive Blue Mussel (Mytilus galloprovincialis).

    Science.gov (United States)

    Saarman, Norah P; Kober, Kord M; Simison, W Brian; Pogson, Grant H

    2017-10-01

    Adaptive responses to thermal stress in poikilotherms plays an important role in determining competitive ability and species distributions. Amino acid substitutions that affect protein stability and modify the thermal optima of orthologous proteins may be particularly important in this context. Here, we examine a set of 2,770 protein-coding genes to determine if proteins in a highly invasive heat tolerant blue mussel (Mytilus galloprovincialis) contain signals of adaptive increases in protein stability relative to orthologs in a more cold tolerant M. trossulus. Such thermal adaptations might help to explain, mechanistically, the success with which the invasive marine mussel M. galloprovincialis has displaced native species in contact zones in the eastern (California) and western (Japan) Pacific. We tested for stabilizing amino acid substitutions in warm tolerant M. galloprovincialis relative to cold tolerant M. trossulus with a generalized linear model that compares in silico estimates of recent changes in protein stability among closely related congeners. Fixed substitutions in M. galloprovincialis were 3,180.0 calories per mol per substitution more stabilizing at genes with both elevated dN/dS ratios and transcriptional responses to heat stress, and 705.8 calories per mol per substitution more stabilizing across all 2,770 loci investigated. Amino acid substitutions concentrated in a small number of genes were more stabilizing in M. galloprovincialis compared with cold tolerant M. trossulus. We also tested for, but did not find, enrichment of a priori GO terms in genes with elevated dN/dS ratios in M. galloprovincialis. This might indicate that selection for thermodynamic stability is generic across all lineages, and suggests that the high change in estimated protein stability that we observed in M. galloprovincialis is driven by selection for extra stabilizing substitutions, rather than by higher incidence of selection in a greater number of genes in this lineage

  17. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

    Directory of Open Access Journals (Sweden)

    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  18. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

    Directory of Open Access Journals (Sweden)

    Eric K Johnson

    Full Text Available Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

  19. Aerobic Exercise Training Adaptations Are Increased by Postexercise Carbohydrate-Protein Supplementation

    Directory of Open Access Journals (Sweden)

    Lisa Ferguson-Stegall

    2011-01-01

    Full Text Available Carbohydrate-protein supplementation has been found to increase the rate of training adaptation when provided postresistance exercise. The present study compared the effects of a carbohydrate and protein supplement in the form of chocolate milk (CM, isocaloric carbohydrate (CHO, and placebo on training adaptations occurring over 4.5 weeks of aerobic exercise training. Thirty-two untrained subjects cycled 60 min/d, 5 d/wk for 4.5 wks at 75–80% of maximal oxygen consumption (VO2 max. Supplements were ingested immediately and 1 h after each exercise session. VO2 max and body composition were assessed before the start and end of training. VO2 max improvements were significantly greater in CM than CHO and placebo. Greater improvements in body composition, represented by a calculated lean and fat mass differential for whole body and trunk, were found in the CM group compared to CHO. We conclude supplementing with CM postexercise improves aerobic power and body composition more effectively than CHO alone.

  20. A reduced amino acid alphabet for understanding and designing protein adaptation to mutation.

    Science.gov (United States)

    Etchebest, C; Benros, C; Bornot, A; Camproux, A-C; de Brevern, A G

    2007-11-01

    Protein sequence world is considerably larger than structure world. In consequence, numerous non-related sequences may adopt similar 3D folds and different kinds of amino acids may thus be found in similar 3D structures. By grouping together the 20 amino acids into a smaller number of representative residues with similar features, sequence world simplification may be achieved. This clustering hence defines a reduced amino acid alphabet (reduced AAA). Numerous works have shown that protein 3D structures are composed of a limited number of building blocks, defining a structural alphabet. We previously identified such an alphabet composed of 16 representative structural motifs (5-residues length) called Protein Blocks (PBs). This alphabet permits to translate the structure (3D) in sequence of PBs (1D). Based on these two concepts, reduced AAA and PBs, we analyzed the distributions of the different kinds of amino acids and their equivalences in the structural context. Different reduced sets were considered. Recurrent amino acid associations were found in all the local structures while other were specific of some local structures (PBs) (e.g Cysteine, Histidine, Threonine and Serine for the alpha-helix Ncap). Some similar associations are found in other reduced AAAs, e.g Ile with Val, or hydrophobic aromatic residues Trp with Phe and Tyr. We put into evidence interesting alternative associations. This highlights the dependence on the information considered (sequence or structure). This approach, equivalent to a substitution matrix, could be useful for designing protein sequence with different features (for instance adaptation to environment) while preserving mainly the 3D fold.

  1. Redox stress proteins are involved in adaptation response of the hyperthermoacidophilic archaeon Sulfolobus solfataricus to nickel challenge

    Directory of Open Access Journals (Sweden)

    Scaloni Andrea

    2007-08-01

    Full Text Available Abstract Background Exposure to nickel (Ni and its chemical derivatives has been associated with severe health effects in human. On the contrary, poor knowledge has been acquired on target physiological processes or molecular mechanisms of this metal in model organisms, including Bacteria and Archaea. In this study, we describe an analysis focused at identifying proteins involved in the recovery of the archaeon Sulfolobus solfataricus strain MT4 from Ni-induced stress. Results To this purpose, Sulfolobus solfataricus was grown in the presence of the highest nickel sulphate concentration still allowing cells to survive; crude extracts from treated and untreated cells were compared at the proteome level by using a bi-dimensional chromatography approach. We identified several proteins specifically repressed or induced as result of Ni treatment. Observed up-regulated proteins were largely endowed with the ability to trigger recovery from oxidative and osmotic stress in other biological systems. It is noteworthy that most of the proteins induced following Ni treatment perform similar functions and a few have eukaryal homologue counterparts. Conclusion These findings suggest a series of preferential gene expression pathways activated in adaptation response to metal challenge.

  2. Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

    International Nuclear Information System (INIS)

    Pastoureau, P.; Merle, B.; Delmas, P.D.

    1988-01-01

    We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of pufified bone gla-protein was in agreement with a previous publication. The competitive RIA uses 125 I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound 125 I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P<0.001) decreased from 532 ± 169 μg/l at birth, to 240 ± 43 μg/l at 45 days, 152 ± 44 μg/l at 90 days, and 5.9 ± 0.7 μg/l at 7 years age. In addition, bone gla-protein levels at birth were higher in normal birth weight than in hypotrophic lambs with low birth weight (535 ± 169 vs 271 ± 156 μg/l, P<0.0001). Furthermore, lambs raised outside in free conditions tended to have higher serum bone gla-protein levels than lambs raised under shelter (1984 ± 53 vs 137 ± 34 μg/l), suggesting a role of breeding factors such as diet or relative immobilization on bone gla-protein levels. These results emphasize the interest of a RIA for the bone-specific protein bone gla-protein as a potential tool for experimental studies on skeletal growth and bone remodelling in a

  3. Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein

    International Nuclear Information System (INIS)

    Lee, G.; Ronai, Z.A.; Pincus, M.R.; Brandt-Rauf, P.W.; Weinstein, I.B.; Murphy, R.B.; Delohery, T.M.; Nishimura, S.; Yamaizumi, Z.

    1989-01-01

    An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with modified p21 protein, the cells were pulsed with [ 35 S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21 - protein complexes. By using this technique, the authors found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. They suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes

  4. Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

    Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

  5. Identification of group specific motifs in Beta-lactamase family of proteins

    Directory of Open Access Journals (Sweden)

    Saxena Akansha

    2009-12-01

    Full Text Available Abstract Background Beta-lactamases are one of the most serious threats to public health. In order to combat this threat we need to study the molecular and functional diversity of these enzymes and identify signatures specific to these enzymes. These signatures will enable us to develop inhibitors and diagnostic probes specific to lactamases. The existing classification of beta-lactamases was developed nearly 30 years ago when few lactamases were available. DLact database contain more than 2000 beta-lactamase, which can be used to study the molecular diversity and to identify signatures specific to this family. Methods A set of 2020 beta-lactamase proteins available in the DLact database http://59.160.102.202/DLact were classified using graph-based clustering of Best Bi-Directional Hits. Non-redundant (> 90 percent identical protein sequences from each group were aligned using T-Coffee and annotated using information available in literature. Motifs specific to each group were predicted using PRATT program. Results The graph-based classification of beta-lactamase proteins resulted in the formation of six groups (Four major groups containing 191, 726, 774 and 73 proteins while two minor groups containing 50 and 8 proteins. Based on the information available in literature, we found that each of the four major groups correspond to the four classes proposed by Ambler. The two minor groups were novel and do not contain molecular signatures of beta-lactamase proteins reported in literature. The group-specific motifs showed high sensitivity (> 70% and very high specificity (> 90%. The motifs from three groups (corresponding to class A, C and D had a high level of conservation at DNA as well as protein level whereas the motifs from the fourth group (corresponding to class B showed conservation at only protein level. Conclusion The graph-based classification of beta-lactamase proteins corresponds with the classification proposed by Ambler, thus there is

  6. Adaptability and stability of soybean genotypes in off-season cultivation.

    Science.gov (United States)

    Batista, R O; Hamawaki, R L; Sousa, L B; Nogueira, A P O; Hamawaki, O T

    2015-08-14

    The oil and protein contents of soybean grains are important quantitative traits for use in breeding. However, few breeding programs perform selection based on these traits in different environments. This study assessed the adaptability and stability of 14 elite early soybean breeding lines in off-season cultivation with respect to yield, and oil and protein contents. A range of statistical methods was applied and these analyses indicated that for off-season cultivation, the lines UFUS 5 and UFUS 10 could be recommended due to their superior performance in grain yield, oil content, and specific adaptability to unfavorable environments along with high stability in these characteristics. Also recommended were UFUS 06, which demonstrated superior performance in all three tested characteristics and showed adaptation to favorable environments, and UFUS 13, which showed high adaptability and stability and a superior performance for protein content.

  7. Recent Developments in the Site-Specific Immobilization of Proteins onto Solid Supports

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2007-02-21

    Immobilization of proteins onto surfaces is of great importance in numerous applications, including protein analysis, drug screening, and medical diagnostics, among others. The success of all these technologies relies on the immobilization technique employed to attach a protein to the corresponding surface. Non-specific physical adsorption or chemical cross-linking with appropriate surfaces results in the immobilization of the protein in random orientations. Site-specific covalent attachment, on the other hand, leads to molecules being arranged in a definite, orderly fashion and allows the use of spacers and linkers to help minimize steric hindrances between the protein and the surface. The present work reviews the latest chemical and biochemical developments for the site-specific covalent attachment of proteins onto solid supports.

  8. Domain requirements for the Dock adapter protein in growth- cone signaling.

    Science.gov (United States)

    Rao, Y; Zipursky, S L

    1998-03-03

    Tyrosine phosphorylation has been implicated in growth-cone guidance through genetic, biochemical, and pharmacological studies. Adapter proteins containing src homology 2 (SH2) domains and src homology 3 (SH3) domains provide a means of linking guidance signaling through phosphotyrosine to downstream effectors regulating growth-cone motility. The Drosophila adapter, Dreadlocks (Dock), the homolog of mammalian Nck containing three N-terminal SH3 domains and a single SH2 domain, is highly specialized for growth-cone guidance. In this paper, we demonstrate that Dock can couple signals in either an SH2-dependent or an SH2-independent fashion in photoreceptor (R cell) growth cones, and that Dock displays different domain requirements in different neurons.

  9. The limits of adaptation of functional protein synthesis to sever undernutrition

    International Nuclear Information System (INIS)

    Jahoor, F.; Bhattiprolu, S.; Reeds, P.; Forrester, T.; Boyne, M.

    1994-01-01

    Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child's prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab

  10. The limits of adaptation of functional protein synthesis to sever undernutrition

    Energy Technology Data Exchange (ETDEWEB)

    Jahoor, F; Bhattiprolu, S; Reeds, P [Baylor Coll. of Medicine, Houston, TX (United States). Children` s Nutrition Research Centre; Forrester, T; Boyne, M [West Indies Univ., Mona (Jamaica). Tropical Metabolism Research Unit

    1994-12-31

    Our goal is to determine how the stress of infections alters the adaptation to reduced food intake in children. We think that an important element is the need for hepatic synthesis of rapidly turning over acute-phase proteins, a critical factor in overall maintenance of host defenses. When the child`s prior intake has been adequate, even though growth may temporarily cease, the presence of adequate amino acid stores in tissues allows the hepatic response to stress to be maintained at the same time as an adequate rate of synthesis of nutrient transport proteins. However, when the immune system is activated in a children whose nutrition is already suboptimal the ability of the liver to synthesize nutrient transport proteins is compromised thereby further impeding nutrient utilization. We will use stable isotope tracer methodology to determine the effects of severe protein energy malnutrition, with and without infection, on the rates of synthesis of nutrient transport proteins and acute-phase proteins in undernourished children at three time points during treatment; in the early resuscitative period, after appetite has returned, and at the end of the catch-up growth phase when normal growth has resumed. (author). 12 refs, 1 fig., 1 tab.

  11. Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

    Science.gov (United States)

    Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

    1989-11-01

    An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

  12. Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

    Science.gov (United States)

    Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I

    2010-05-01

    The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.

  13. Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1.

    Science.gov (United States)

    Chen, Ming-Ju; Tang, Hsin-Yu; Chiang, Ming-Lun

    2017-09-01

    Lactobacillus kefiranofaciens M1 is a probiotic strain isolated from Taiwanese kefir grains. The present study evaluated the effects of heat, cold, acid and bile salt adaptations on the stress tolerance of L. kefiranofaciens M1. The regulation of protein expression of L. kefiranofaciens M1 under these adaptation conditions was also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat, cold, acid and bile salts induced homologous tolerance and cross-protection against heterologous challenge. The extent of induced tolerance varied depending on the type and condition of stress. Proteomic analysis revealed that 27 proteins exhibited differences in expression between non-adapted and stress-adapted L. kefiranofaciens M1 cells. Among these proteins, three proteins involved in carbohydrate metabolism (triosephosphate isomerase, enolase and NAD-dependent glycerol-3-phosphate dehydrogenase), two proteins involved in pH homeostasis (ATP synthase subunits AtpA and AtpB), two stress response proteins (chaperones DnaK and GroEL) and one translation-related protein (30S ribosomal protein S2) were up-regulated by three of the four adaptation treatments examined. The increased synthesis of these stress proteins might play a critical protective role in the cellular defense against heat, cold, acid and bile salt stresses. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Adaptive grid generation in a patient-specific cerebral aneurysm

    Science.gov (United States)

    Hodis, Simona; Kallmes, David F.; Dragomir-Daescu, Dan

    2013-11-01

    Adapting grid density to flow behavior provides the advantage of increasing solution accuracy while decreasing the number of grid elements in the simulation domain, therefore reducing the computational time. One method for grid adaptation requires successive refinement of grid density based on observed solution behavior until the numerical errors between successive grids are negligible. However, such an approach is time consuming and it is often neglected by the researchers. We present a technique to calculate the grid size distribution of an adaptive grid for computational fluid dynamics (CFD) simulations in a complex cerebral aneurysm geometry based on the kinematic curvature and torsion calculated from the velocity field. The relationship between the kinematic characteristics of the flow and the element size of the adaptive grid leads to a mathematical equation to calculate the grid size in different regions of the flow. The adaptive grid density is obtained such that it captures the more complex details of the flow with locally smaller grid size, while less complex flow characteristics are calculated on locally larger grid size. The current study shows that kinematic curvature and torsion calculated from the velocity field in a cerebral aneurysm can be used to find the locations of complex flow where the computational grid needs to be refined in order to obtain an accurate solution. We found that the complexity of the flow can be adequately described by velocity and vorticity and the angle between the two vectors. For example, inside the aneurysm bleb, at the bifurcation, and at the major arterial turns the element size in the lumen needs to be less than 10% of the artery radius, while at the boundary layer, the element size should be smaller than 1% of the artery radius, for accurate results within a 0.5% relative approximation error. This technique of quantifying flow complexity and adaptive remeshing has the potential to improve results accuracy and reduce

  15. Mechanism of Genome Interrogation: How CRISPR RNA-Guided Cas9 Proteins Locate Specific Targets on DNA.

    Science.gov (United States)

    Shvets, Alexey A; Kolomeisky, Anatoly B

    2017-10-03

    The ability to precisely edit and modify a genome opens endless opportunities to investigate fundamental properties of living systems as well as to advance various medical techniques and bioengineering applications. This possibility is now close to reality due to a recent discovery of the adaptive bacterial immune system, which is based on clustered regularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas) that utilize RNA to find and cut the double-stranded DNA molecules at specific locations. Here we develop a quantitative theoretical approach to analyze the mechanism of target search on DNA by CRISPR RNA-guided Cas9 proteins, which is followed by a selective cleavage of nucleic acids. It is based on a discrete-state stochastic model that takes into account the most relevant physical-chemical processes in the system. Using a method of first-passage processes, a full dynamic description of the target search is presented. It is found that the location of specific sites on DNA by CRISPR Cas9 proteins is governed by binding first to protospacer adjacent motif sequences on DNA, which is followed by reversible transitions into DNA interrogation states. In addition, the search dynamics is strongly influenced by the off-target cutting. Our theoretical calculations allow us to explain the experimental observations and to give experimentally testable predictions. Thus, the presented theoretical model clarifies some molecular aspects of the genome interrogation by CRISPR RNA-guided Cas9 proteins. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  17. Prioritizing disease candidate proteins in cardiomyopathy-specific protein-protein interaction networks based on "guilt by association" analysis.

    Directory of Open Access Journals (Sweden)

    Wan Li

    Full Text Available The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial. Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on "guilt by association" analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on "guilt by association" analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.

  18. Selective radiolabeling of cell surface proteins to a high specific activity

    International Nuclear Information System (INIS)

    Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

    1987-01-01

    A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125 I-(hydroxyphenyl)propionyl groups via 125 I-sulfosuccinimidyl (hydroxyphenyl)propionate ( 125 II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[ 125 I]iodide labeling technique revealed that 125 I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125 I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane

  19. Ubiquitination of specific mitochondrial matrix proteins

    International Nuclear Information System (INIS)

    Lehmann, Gilad; Ziv, Tamar; Braten, Ori; Admon, Arie; Udasin, Ronald G.; Ciechanover, Aaron

    2016-01-01

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  20. Ubiquitination of specific mitochondrial matrix proteins

    Energy Technology Data Exchange (ETDEWEB)

    Lehmann, Gilad [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ziv, Tamar [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Braten, Ori [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Admon, Arie [The Smoler Proteomics Center, Faculty of Biology – Technion-Israel Institute of Technology, Haifa, 32000 (Israel); Udasin, Ronald G. [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel); Ciechanover, Aaron, E-mail: aaroncie@tx.technion.ac.il [The Janet and David Polak Tumor and Vascular Biology Research Center and the Technion Integrated Cancer Center (TICC), The Rappaport Faculty of Medicine and Research Institute, Haifa, 31096 (Israel)

    2016-06-17

    Several protein quality control systems in bacteria and/or mitochondrial matrix from lower eukaryotes are absent in higher eukaryotes. These are transfer-messenger RNA (tmRNA), The N-end rule ATP-dependent protease ClpAP, and two more ATP-dependent proteases, HslUV and ClpXP (in yeast). The lost proteases resemble the 26S proteasome and the role of tmRNA and the N-end rule in eukaryotic cytosol is performed by the ubiquitin proteasome system (UPS). Therefore, we hypothesized that the UPS might have substituted these systems – at least partially – in the mitochondrial matrix of higher eukaryotes. Using three independent experimental approaches, we demonstrated the presence of ubiquitinated proteins in the matrix of isolated yeast mitochondria. First, we show that isolated mitochondria contain ubiquitin (Ub) conjugates, which remained intact after trypsin digestion. Second, we demonstrate that the mitochondrial soluble fraction contains Ub-conjugates, several of which were identified by mass spectrometry and are localized to the matrix. Third, using immunoaffinity enrichment by specific antibodies recognizing digested ubiquitinated peptides, we identified a group of Ub-modified matrix proteins. The modification was further substantiated by separation on SDS-PAGE and immunoblots. Last, we attempted to identify the ubiquitin ligase(s) involved, and identified Dma1p as a trypsin-resistant protein in our mitochondrial preparations. Taken together, these data suggest a yet undefined role for the UPS in regulation of the mitochondrial matrix proteins. -- Highlights: •Mitochondrial matrix contains ubiquitinated proteins. •Ubiquitination occurs most probably in the matrix. •Dma1p is a ubiquitin ligase present in mitochondrial preparations.

  1. A study on climatic adaptation of dipteran mitochondrial protein coding genes

    Directory of Open Access Journals (Sweden)

    Debajyoti Kabiraj

    2017-10-01

    Full Text Available Diptera, the true flies are frequently found in nature and their habitat is found all over the world including Antarctica and Polar Regions. The number of documented species for order diptera is quite high and thought to be 14% of the total animal present in the earth [1]. Most of the study in diptera has focused on the taxa of economic and medical importance, such as the fruit flies Ceratitis capitata and Bactrocera spp. (Tephritidae, which are serious agricultural pests; the blowflies (Calliphoridae and oestrid flies (Oestridae, which can cause myiasis; the anopheles mosquitoes (Culicidae, are the vectors of malaria; and leaf-miners (Agromyzidae, vegetable and horticultural pests [2]. Insect mitochondrion consists of 13 protein coding genes, 22 tRNAs and 2 rRNAs, are the remnant portion of alpha-proteobacteria is responsible for simultaneous function of energy production and thermoregulation of the cell through the bi-genomic system thus different adaptability in different climatic condition might have compensated by complementary changes is the both genomes [3,4]. In this study we have collected complete mitochondrial genome and occurrence data of one hundred thirteen such dipteran insects from different databases and literature survey. Our understanding of the genetic basis of climatic adaptation in diptera is limited to the basic information on the occurrence location of those species and mito genetic factors underlying changes in conspicuous phenotypes. To examine this hypothesis, we have taken an approach of Nucleotide substitution analysis for 13 protein coding genes of mitochondrial DNA individually and combined by different software for monophyletic group as well as paraphyletic group of dipteran species. Moreover, we have also calculated codon adaptation index for all dipteran mitochondrial protein coding genes. Following this work, we have classified our sample organisms according to their location data from GBIF (https

  2. Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

    Science.gov (United States)

    Schumacher, Dominik; Helma, Jonas; Mann, Florian A; Pichler, Garwin; Natale, Francesco; Krause, Eberhard; Cardoso, M Cristina; Hackenberger, Christian P R; Leonhardt, Heinrich

    2015-11-09

    A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

    Science.gov (United States)

    Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

    2008-01-01

    The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

  4. Site-Specific Infrared Probes of Proteins

    Science.gov (United States)

    Ma, Jianqiang; Pazos, Ileana M.; Zhang, Wenkai; Culik, Robert M.; Gai, Feng

    2015-01-01

    Infrared spectroscopy has played an instrumental role in studying a wide variety of biological questions. However, in many cases it is impossible or difficult to rely on the intrinsic vibrational modes of biological molecules of interest, such as proteins, to reveal structural and/or environmental information in a site-specific manner. To overcome this limitation, many recent efforts have been dedicated to the development and application of various extrinsic vibrational probes that can be incorporated into biological molecules and used to site-specifically interrogate their structural and/or environmental properties. In this Review, we highlight some recent advancements of this rapidly growing research area. PMID:25580624

  5. Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

    Science.gov (United States)

    Allison, Andrew B.; Kohler, Dennis J.; Ortega, Alicia; Hoover, Elizabeth A.; Grove, Daniel M.; Holmes, Edward C.; Parrish, Colin R.

    2014-01-01

    Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range. PMID:25375184

  6. Host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species.

    Directory of Open Access Journals (Sweden)

    Andrew B Allison

    2014-11-01

    Full Text Available Canine parvovirus (CPV emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV, a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that>95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR, the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.

  7. UV-C Adaptation of Shigella: Morphological, Outer Membrane Proteins, Secreted Proteins, and Lipopolysaccharides Effects.

    Science.gov (United States)

    Chourabi, Kalthoum; Campoy, Susana; Rodriguez, Jesus A; Kloula, Salma; Landoulsi, Ahmed; Chatti, Abdelwaheb

    2017-11-01

    Water UV disinfection remains extremely important, particularly in developing countries where drinking and reclaimed crop irrigation water may spread devastating infectious diseases. Enteric bacterial pathogens, among which Shigella, are possible contaminants of drinking and bathing water and foods. To study the effect of UV light on Shigella, four strains were exposed to different doses in a laboratory-made irradiation device, given that the ultraviolet radiation degree of inactivation is directly related to the UV dose applied to water. Our results showed that the UV-C rays are effective against all the tested Shigella strains. However, UV-C doses appeared as determinant factors for Shigella eradication. On the other hand, Shigella-survived strains changed their outer membrane protein profiles, secreted proteins, and lipopolysaccharides. Also, as shown by electron microscopy transmission, morphological alterations were manifested by an internal cytoplasm disorganized and membrane envelope breaks. Taken together, the focus of interest of our study is to know the adaptive mechanism of UV-C resistance of Shigella strains.

  8. Cancer-specific self-efficacy and psychosocial and functional adaptation to early stage breast cancer.

    Science.gov (United States)

    Manne, Sharon L; Ostroff, Jamie S; Norton, Tina R; Fox, Kevin; Grana, Generosa; Goldstein, Lori

    2006-04-01

    Although self-efficacy is considered a key psychological resource in adapting to chronic physical illness, this construct has received less attention among individuals coping with cancer. To examine changes in cancer self-efficacy over time among women with early stage breast cancer and associations between task-specific domains of self-efficacy and specific psychological, relationship, and functional outcomes. Ninety-five women diagnosed with early stage breast cancer completed surveys postsurgery and 1 year later. Cancer-related self-efficacy was relatively stable over 1 year, with only 2 domains of efficacy-(a) Activity Management and (b) Self-Satisfaction-evidencing significant increases over the 1-year time period. Cross-sectional findings were relatively consistent with predictions and suggested that specific domains of self-efficacy were more strongly related to relevant domains of adaptation. Longitudinal findings were not as consistent with the domain-specificity hypothesis but did suggest several predictive associations between self-efficacy and outcomes. Personal Management self-efficacy was associated with higher relationship satisfaction, higher Communication Self-Efficacy was associated with less functional impairment, and higher Affective Management self-efficacy was associated with higher self-esteem 1 year later. Specific domains of cancer-related self-efficacy are most closely related to relevant areas of adaptation when considered cross-sectionally, but further study is needed to clarify the nature of these relationships over time.

  9. Proteins labelling with 125I and experimental determination of their specific activity

    International Nuclear Information System (INIS)

    Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

    1975-11-01

    A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

  10. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    OpenAIRE

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  11. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants.

    Science.gov (United States)

    Baudisch, Bianca; Pfort, Ingrid; Sorge, Eberhard; Conrad, Udo

    2018-01-01

    Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  12. Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

    Science.gov (United States)

    Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and

  13. Nanobody-Directed Specific Degradation of Proteins by the 26S-Proteasome in Plants

    Directory of Open Access Journals (Sweden)

    Bianca Baudisch

    2018-02-01

    Full Text Available Here, we present data showing the directed degradation of target proteins recognized by a specific nanobody in transgenic plants. Green fluorescent protein was depleted by a chimeric nanobody fused to a distinct F-box domain, which enables protein degradation via the ubiquitin proteasome pathway. This technique could thus be used to knock out other proteins of interest in planta using specific, high-affinity binding proteins.

  14. Specificity, cross-talk and adaptation in Interferon signaling

    Science.gov (United States)

    Zilman, Anton

    Innate immune system is the first line of defense of higher organisms against pathogens. It coordinates the behavior of millions of cells of multiple types, achieved through numerous signaling molecules. This talk focuses on the signaling specificity of a major class of signaling molecules - Type I Interferons - which are also used therapeutically in the treatment of a number of diseases, such as Hepatitis C, multiple sclerosis and some cancers. Puzzlingly, different Interferons act through the same cell surface receptor but have different effects on the target cells. They also exhibit a strange pattern of temporal cross-talk resulting in a serious clinical problem - loss of response to Interferon therapy. We combined mathematical modeling with quantitative experiments to develop a quantitative model of specificity and adaptation in the Interferon signaling pathway. The model resolves several outstanding experimental puzzles and directly affects the clinical use of Type I Interferons in treatment of viral hepatitis and other diseases.

  15. SPEER-SERVER: a web server for prediction of protein specificity determining sites.

    Science.gov (United States)

    Chakraborty, Abhijit; Mandloi, Sapan; Lanczycki, Christopher J; Panchenko, Anna R; Chakrabarti, Saikat

    2012-07-01

    Sites that show specific conservation patterns within subsets of proteins in a protein family are likely to be involved in the development of functional specificity. These sites, generally termed specificity determining sites (SDS), might play a crucial role in binding to a specific substrate or proteins. Identification of SDS through experimental techniques is a slow, difficult and tedious job. Hence, it is very important to develop efficient computational methods that can more expediently identify SDS. Herein, we present Specificity prediction using amino acids' Properties, Entropy and Evolution Rate (SPEER)-SERVER, a web server that predicts SDS by analyzing quantitative measures of the conservation patterns of protein sites based on their physico-chemical properties and the heterogeneity of evolutionary changes between and within the protein subfamilies. This web server provides an improved representation of results, adds useful input and output options and integrates a wide range of analysis and data visualization tools when compared with the original standalone version of the SPEER algorithm. Extensive benchmarking finds that SPEER-SERVER exhibits sensitivity and precision performance that, on average, meets or exceeds that of other currently available methods. SPEER-SERVER is available at http://www.hpppi.iicb.res.in/ss/.

  16. Optimal Multitrial Prediction Combination and Subject-Specific Adaptation for Minimal Training Brain Switch Designs

    NARCIS (Netherlands)

    Spyrou, L.; Blokland, Y.M.; Farquhar, J.D.R.; Bruhn, J.

    2016-01-01

    Brain-Computer Interface (BCI) systems are traditionally designed by taking into account user-specific data to enable practical use. More recently, subject independent (SI) classification algorithms have been developed which bypass the subject specific adaptation and enable rapid use of the system.

  17. Optimal multitrial prediction combination and subject-specific adaptation for minimal training brain switch designs

    NARCIS (Netherlands)

    Spyrou, L.; Blokland, Y.M.; Farquhar, J.D.R.; Bruhn, J.

    2016-01-01

    Brain-Computer Interface systems are traditionally designed by taking into account user-specific data to enable practical use. More recently, subject independent (SI) classification algorithms have been developed which bypass the subject specific adaptation and enable rapid use of the system. A

  18. New developments for the site-specific attachment of protein to surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Camarero, J A

    2005-05-12

    Protein immobilization on surfaces is of great importance in numerous applications in biology and biophysics. The key for the success of all these applications relies on the immobilization technique employed to attach the protein to the corresponding surface. Protein immobilization can be based on covalent or noncovalent interaction of the molecule with the surface. Noncovalent interactions include hydrophobic interactions, hydrogen bonding, van der Waals forces, electrostatic forces, or physical adsorption. However, since these interactions are weak, the molecules can get denatured or dislodged, thus causing loss of signal. They also result in random attachment of the protein to the surface. Site-specific covalent attachment of proteins onto surfaces, on the other hand, leads to molecules being arranged in a definite, orderly fashion and uses spacers and linkers to help minimize steric hindrances between the protein surface. This work reviews in detail some of the methods most commonly used as well as the latest developments for the site-specific covalent attachment of protein to solid surfaces.

  19. Direct association between the Ret receptor tyrosine kinase and the Src homology 2-containing adapter protein Grb7.

    Science.gov (United States)

    Pandey, A; Liu, X; Dixon, J E; Di Fiore, P P; Dixit, V M

    1996-05-03

    Adapter proteins containing Src homology 2 (SH2) domains link transmembrane receptor protein-tyrosine kinases to downstream signal transducing molecules. A family of SH2 containing adapter proteins including Grb7 and Grb10 has been recently identified. We had previously shown that Grb10 associates with Ret via its SH2 domain in an activation-dependent manner (Pandey, A., Duan, H., Di Fiore, P.P., and Dixit, V.M. (1995) J. Biol, Chem. 270, 21461-21463). We now demonstrate that the related adapter molecule Grb7 also associates with Ret in vitro and in vivo, and that the binding of the SH2 domain of Grb7 to Ret is direct. This binding is dependent upon Ret autophosphorylation since Grb7 is incapable of binding a kinase-defective mutant of Ret. Thus two members of the Grb family, Grb7 and Grb10, likely relay signals emanating from Ret to other, as yet, unidentified targets within the cell.

  20. Protein Engineering for Nicotinamide Coenzyme Specificity in Oxidoreductases: Attempts and Challenges.

    Science.gov (United States)

    Chánique, Andrea M; Parra, Loreto P

    2018-01-01

    Oxidoreductases are ubiquitous enzymes that catalyze an extensive range of chemical reactions with great specificity, efficiency, and selectivity. Most oxidoreductases are nicotinamide cofactor-dependent enzymes with a strong preference for NADP or NAD. Because these coenzymes differ in stability, bioavailability and costs, the enzyme preference for a specific coenzyme is an important issue for practical applications. Different approaches for the manipulation of coenzyme specificity have been reported, with different degrees of success. Here we present various attempts for the switching of nicotinamide coenzyme preference in oxidoreductases by protein engineering. This review covers 103 enzyme engineering studies from 82 articles and evaluates the accomplishments in terms of coenzyme specificity and catalytic efficiency compared to wild type enzymes of different classes. We analyzed different protein engineering strategies and related them with the degree of success in inverting the cofactor specificity. In general, catalytic activity is compromised when coenzyme specificity is reversed, however when switching from NAD to NADP, better results are obtained. In most of the cases, rational strategies were used, predominantly with loop exchange generating the best results. In general, the tendency of removing acidic residues and incorporating basic residues is the strategy of choice when trying to change specificity from NAD to NADP, and vice versa . Computational strategies and algorithms are also covered as helpful tools to guide protein engineering strategies. This mini review aims to give a general introduction to the topic, giving an overview of tools and information to work in protein engineering for the reversal of coenzyme specificity.

  1. Mouse CCDC79 (TERB1) is a meiosis-specific telomere associated protein.

    Science.gov (United States)

    Daniel, Katrin; Tränkner, Daniel; Wojtasz, Lukasz; Shibuya, Hiroki; Watanabe, Yoshinori; Alsheimer, Manfred; Tóth, Attila

    2014-05-22

    Telomeres have crucial meiosis-specific roles in the orderly reduction of chromosome numbers and in ensuring the integrity of the genome during meiosis. One such role is the attachment of telomeres to trans-nuclear envelope protein complexes that connect telomeres to motor proteins in the cytoplasm. These trans-nuclear envelope connections between telomeres and cytoplasmic motor proteins permit the active movement of telomeres and chromosomes during the first meiotic prophase. Movements of chromosomes/telomeres facilitate the meiotic recombination process, and allow high fidelity pairing of homologous chromosomes. Pairing of homologous chromosomes is a prerequisite for their correct segregation during the first meiotic division. Although inner-nuclear envelope proteins, such as SUN1 and potentially SUN2, are known to bind and recruit meiotic telomeres, these proteins are not meiosis-specific, therefore cannot solely account for telomere-nuclear envelope attachment and/or for other meiosis-specific characteristics of telomeres in mammals. We identify CCDC79, alternatively named TERB1, as a meiosis-specific protein that localizes to telomeres from leptotene to diplotene stages of the first meiotic prophase. CCDC79 and SUN1 associate with telomeres almost concurrently at the onset of prophase, indicating a possible role for CCDC79 in telomere-nuclear envelope interactions and/or telomere movements. Consistent with this scenario, CCDC79 is missing from most telomeres that fail to connect to SUN1 protein in spermatocytes lacking the meiosis-specific cohesin SMC1B. SMC1B-deficient spermatocytes display both reduced efficiency in telomere-nuclear envelope attachment and reduced stability of telomeres specifically during meiotic prophase. Importantly, CCDC79 associates with telomeres in SUN1-deficient spermatocytes, which strongly indicates that localization of CCDC79 to telomeres does not require telomere-nuclear envelope attachment. CCDC79 is a meiosis-specific telomere

  2. PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum.

    Science.gov (United States)

    van Lith, Marcel; Hartigan, Nichola; Hatch, Jennifer; Benham, Adam M

    2005-01-14

    Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.

  3. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Directory of Open Access Journals (Sweden)

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  4. Changes associated with Ebola virus adaptation to novel species.

    OpenAIRE

    Pappalardo, Morena; Reddin, Ian; Cantoni, Diego; Rossman, Jeremy S.; Michaelis, Martin; Wass, Mark N.

    2017-01-01

    Motivation: Ebola viruses are not pathogenic but can be adapted to replicate and cause disease in rodents. Here, we used a structural bioinformatics approach to analyze the mutations associated with Ebola virus adaptation to rodents to elucidate the determinants of host-specific Ebola virus pathogenicity.\\ud Results: We identified 33 different mutations associated with Ebola virus adaptation to rodents in the proteins GP, NP, L, VP24, and VP35. Only VP24, GP and NP were consistently found mut...

  5. Not letting the left leg know what the right leg is doing: limb-specific locomotor adaptation to sensory-cue conflict.

    Science.gov (United States)

    Durgin, Frank H; Fox, Laura F; Hoon Kim, Dong

    2003-11-01

    We investigated the phenomenon of limb-specific locomotor adaptation in order to adjudicate between sensory-cue-conflict theory and motor-adaptation theory. The results were consistent with cue-conflict theory in demonstrating that two different leg-specific hopping aftereffects are modulated by the presence of conflicting estimates of self-motion from visual and nonvisual sources. Experiment 1 shows that leg-specific increases in forward drift during attempts to hop in place on one leg while blindfolded vary according to the relationship between visual information and motor activity during an adaptation to outdoor forward hopping. Experiment 2 shows that leg-specific changes in performance on a blindfolded hopping-to-target task are similarly modulated by the presence of cue conflict during adaptation to hopping on a treadmill. Experiment 3 shows that leg-specific aftereffects from hopping additionally produce inadvertent turning during running in place while blindfolded. The results of these experiments suggest that these leg-specific locomotor aftereffects are produced by sensory-cue conflict rather than simple motor adaptation.

  6. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein.

    Science.gov (United States)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu; Nielsen, Maria E O; Chiang, Yueh-Chin; Kierkegaard, Morten; Wang, Xin; Lee, Darren J; Andersen, Jens S; Yao, Gang

    2012-09-01

    Poly(A) binding protein (PAB1) is involved in a number of RNA metabolic functions in eukaryotic cells and correspondingly is suggested to associate with a number of proteins. We have used mass spectrometric analysis to identify 55 non-ribosomal proteins that specifically interact with PAB1 from Saccharomyces cerevisiae. Because many of these factors may associate only indirectly with PAB1 by being components of the PAB1-mRNP structure, we additionally conducted mass spectrometric analyses on seven metabolically defined PAB1 deletion derivatives to delimit the interactions between these proteins and PAB1. These latter analyses identified 13 proteins whose associations with PAB1 were reduced by deleting one or another of PAB1's defined domains. Included in this list of 13 proteins were the translation initiation factors eIF4G1 and eIF4G2, translation termination factor eRF3, and PBP2, all of whose previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense-mediated decay, was confirmed to interact with PAB1 through the RRM1 domain. We additionally established that while the RRM1 domain of PAB1 was required for UPF1-induced acceleration of deadenylation during nonsense-mediated decay, it was not required for the more critical step of acceleration of mRNA decapping. These results begin to identify the proteins most likely to interact with PAB1 and the domains of PAB1 through which these contacts are made.

  7. Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

    Directory of Open Access Journals (Sweden)

    Johan Nilvebrant

    Full Text Available The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

  8. The adaptive evolution of the mammalian mitochondrial genome

    Directory of Open Access Journals (Sweden)

    O'Brien Stephen J

    2008-03-01

    Full Text Available Abstract Background The mitochondria produce up to 95% of a eukaryotic cell's energy through oxidative phosphorylation. The proteins involved in this vital process are under high functional constraints. However, metabolic requirements vary across species, potentially modifying selective pressures. We evaluate the adaptive evolution of 12 protein-coding mitochondrial genes in 41 placental mammalian species by assessing amino acid sequence variation and exploring the functional implications of observed variation in secondary and tertiary protein structures. Results Wide variation in the properties of amino acids were observed at functionally important regions of cytochrome b in species with more-specialized metabolic requirements (such as adaptation to low energy diet or large body size, such as in elephant, dugong, sloth, and pangolin, and adaptation to unusual oxygen requirements, for example diving in cetaceans, flying in bats, and living at high altitudes in alpacas. Signatures of adaptive variation in the NADH dehydrogenase complex were restricted to the loop regions of the transmembrane units which likely function as protons pumps. Evidence of adaptive variation in the cytochrome c oxidase complex was observed mostly at the interface between the mitochondrial and nuclear-encoded subunits, perhaps evidence of co-evolution. The ATP8 subunit, which has an important role in the assembly of F0, exhibited the highest signal of adaptive variation. ATP6, which has an essential role in rotor performance, showed a high adaptive variation in predicted loop areas. Conclusion Our study provides insight into the adaptive evolution of the mtDNA genome in mammals and its implications for the molecular mechanism of oxidative phosphorylation. We present a framework for future experimental characterization of the impact of specific mutations in the function, physiology, and interactions of the mtDNA encoded proteins involved in oxidative phosphorylation.

  9. Cartilage oligomeric matrix protein specific antibodies are pathogenic

    DEFF Research Database (Denmark)

    Geng, Hui; Nandakumar, Kutty Selva; Pramhed, Anna

    2012-01-01

    -specific monoclonal antibodies (mAbs). METHODS: B cell immunodominant regions on the COMP molecule were measured with a novel enzyme-linked immunosorbent assay using mammalian expressed full-length mouse COMP as well as a panel of recombinant mouse COMP fragments. 18 mAbs specific to COMP were generated......ABSTRACT: INTRODUCTION: Cartilage oligomeric matrix protein (COMP) is a major non-collagenous component of cartilage. Earlier, we developed a new mouse model for rheumatoid arthritis using COMP. This study was undertaken to investigate the epitope specificity and immunopathogenicity of COMP...

  10. Adaptability and specificity of inhibition processes in distractor-induced blindness.

    Science.gov (United States)

    Winther, Gesche N; Niedeggen, Michael

    2017-12-01

    In a rapid serial visual presentation task, inhibition processes cumulatively impair processing of a target possessing distractor properties. This phenomenon-known as distractor-induced blindness-has thus far only been elicited using dynamic visual features, such as motion and orientation changes. In three ERP experiments, we used a visual object feature-color-to test for the adaptability and specificity of the effect. In Experiment I, participants responded to a color change (target) in the periphery whose onset was signaled by a central cue. Presentation of irrelevant color changes prior to the cue (distractors) led to reduced target detection, accompanied by a frontal ERP negativity that increased with increasing number of distractors, similar to the effects previously found for dynamic targets. This suggests that distractor-induced blindness is adaptable to color features. In Experiment II, the target consisted of coherent motion contrasting the color distractors. Correlates of distractor-induced blindness were found neither in the behavioral nor in the ERP data, indicating a feature specificity of the process. Experiment III confirmed the strict distinction between congruent and incongruent distractors: A single color distractor was embedded in a stream of motion distractors with the target consisting of a coherent motion. While behavioral performance was affected by the distractors, the color distractor did not elicit a frontal negativity. The experiments show that distractor-induced blindness is also triggered by visual stimuli predominantly processed in the ventral stream. The strict specificity of the central inhibition process also applies to these stimulus features. © 2017 Society for Psychophysiological Research.

  11. Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

    Science.gov (United States)

    2014-01-01

    While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, α-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

  12. A lepidopteran-specific gene family encoding valine-rich midgut proteins.

    Directory of Open Access Journals (Sweden)

    Jothini Odman-Naresh

    Full Text Available Many lepidopteran larvae are serious agricultural pests due to their feeding activity. Digestion of the plant diet occurs mainly in the midgut and is facilitated by the peritrophic matrix (PM, an extracellular sac-like structure, which lines the midgut epithelium and creates different digestive compartments. The PM is attracting increasing attention to control lepidopteran pests by interfering with this vital function. To identify novel PM components and thus potential targets for insecticides, we performed an immunoscreening with anti-PM antibodies using an expression library representing the larval midgut transcriptome of the tobacco hornworm, Manduca sexta. We identified three cDNAs encoding valine-rich midgut proteins of M. sexta (MsVmps, which appear to be loosely associated with the PM. They are members of a lepidopteran-specific family of nine VMP genes, which are exclusively expressed in larval stages in M. sexta. Most of the MsVMP transcripts are detected in the posterior midgut, with the highest levels observed for MsVMP1. To obtain further insight into Vmp function, we expressed MsVMP1 in insect cells and purified the recombinant protein. Lectin staining and glycosidase treatment indicated that MsVmp1 is highly O-glycosylated. In line with results from qPCR, immunoblots revealed that MsVmp1 amounts are highest in feeding larvae, while MsVmp1 is undetectable in starving and molting larvae. Finally using immunocytochemistry, we demonstrated that MsVmp1 localizes to the cytosol of columnar cells, which secrete MsVmp1 into the ectoperitrophic space in feeding larvae. In starving and molting larvae, MsVmp1 is found in the gut lumen, suggesting that the PM has increased its permeability. The present study demonstrates that lepidopteran species including many agricultural pests have evolved a set of unique proteins that are not found in any other taxon and thus may reflect an important adaptation in the highly specialized lepidopteran

  13. Specific protein-protein interactions of calsequestrin with junctional sarcoplasmic reticulum of skeletal muscle

    International Nuclear Information System (INIS)

    Damiani, E.; Margreth, A.

    1990-01-01

    Minor protein components of triads and of sarcoplasmic reticulum (SR) terminal cisternae (TC), i.e. 47 and 37 kDa peptides and 31-30 kDa and 26-25 kDa peptide doublets, were identified from their ability to bind 125 I calsequestrin (CS) in the presence of EGTA. The CS-binding peptides are specifically associated with the junctional membrane of TC, since they could not be detected in junctional transverse tubules and in longitudinal SR fragments. The 31-30 kDa peptide doublet, exclusively, did not bind CS in the presence of Ca 2+ . Thus, different types of protein-protein interactions appear to be involved in selective binding of CS to junctional TC

  14. Evidence of molecular adaptation to extreme environments and applicability to space environments

    Directory of Open Access Journals (Sweden)

    Filipović M.

    2008-01-01

    Full Text Available This is initial investigation of gene signatures responsible for adapting microscopic life to the extreme Earth environments. We present preliminary results on identification of the clusters of orthologous groups (COGs common to several hyperthermophiles and exclusion of those common to a mesophile (non-hyperthermophile: Escherichia coli (E. coli K12, will yield a group of proteins possibly involved in adaptation to life under extreme temperatures. Comparative genome analyses represent a powerful tool in discovery of novel genes responsible for adaptation to specific extreme environments. Methanogens stand out as the only group of organisms that have species capable of growth at 0ºC (Metarhizium frigidum (M. frigidum and Methanococcoides burtonii (M. burtonii and 110ºC (Methanopyrus kandleri (M. kandleri. Although not all the components of heat adaptation can be attributed to novel genes, the chaperones known as heat shock proteins stabilize the enzymes under elevated temperature. However, highly conserved chaperons found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique chaperone TF55. Our aim was to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The following hyperthermophile genomes incorporated in this software were used for these studies: Methanocaldococcus jannaschii (M. jannaschii, M. kandleri, Archaeoglobus fulgidus (A. fulgidus and three species of Pyrococcus. Common genes were annotated and grouped according to their roles in cellular processes where such information was available and proteins not previously implicated in the heat-adaptation of hyperthermophiles were identified. Additional experimental data are needed in order to learn more about these proteins. To address non-gene based components of thermal adaptation, all sequenced extremophiles were

  15. The effects of whey protein with or without carbohydrates on resistance training adaptations.

    Science.gov (United States)

    Hulmi, Juha J; Laakso, Mia; Mero, Antti A; Häkkinen, Keijo; Ahtiainen, Juha P; Peltonen, Heikki

    2015-01-01

    Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of whey proteins (n = 22), isocaloric carbohydrates (maltodextrin, n = 21), or protein + carbohydrates (n = 25). Within these groups, the subjects were further randomized into two whole-body 12-week RT regimens aiming either for muscle hypertrophy and maximal strength or muscle strength, hypertrophy and power. The post-exercise drink was always ingested immediately after the exercise bout, 2-3 times per week depending on the training period. Body composition (by DXA), quadriceps femoris muscle cross-sectional area (by panoramic ultrasound), maximal strength (by dynamic and isometric leg press) and serum lipids as basic markers of cardiovascular health, were analysed before and after the intervention. Twelve-week RT led to increased fat-free mass, muscle size and strength independent of post-exercise nutrient intake (P carbohydrate group independent of the type of RT (P carbohydrate group (P carbohydrates or combination of proteins and carbohydrates did not have a major effect on muscle size or strength when ingested two to three times a week. However, whey proteins may increase abdominal fat loss and relative fat-free mass adaptations in response to resistance training when compared to fast-acting carbohydrates.

  16. Anti-HmuY antibodies specifically recognize Porphyromonas gingivalis HmuY protein but not homologous proteins in other periodontopathogens.

    Directory of Open Access Journals (Sweden)

    Michał Śmiga

    Full Text Available Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins and T. forsythia (Tfo protein and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium.

  17. Arabinogalactan Proteins Are Involved in Salt-Adaptation and Vesicle Trafficking in Tobacco by-2 Cell Cultures.

    Science.gov (United States)

    Olmos, Enrique; García De La Garma, Jesús; Gomez-Jimenez, Maria C; Fernandez-Garcia, Nieves

    2017-01-01

    Arabinogalactan proteins (AGPs) are a highly diverse family of glycoproteins that are commonly found in most plant species. However, little is known about the physiological and molecular mechanisms of their function. AGPs are involved in different biological processes such as cell differentiation, cell expansion, tissue development and somatic embryogenesis. AGPs are also involved in abiotic stress response such as salinity modulating cell wall expansion. In this study, we describe how salt-adaptation in tobacco BY-2 cell cultures induces important changes in arabinogalactan proteins distribution and contents. Using the immuno-dot blot technique with different anti-AGP antibodies (JIM13, JIM15, and others), we observed that AGPs were highly accumulated in the culture medium of salt-adapted tobacco cells, probably due to the action of phospholipases. We located these AGP epitopes using immunogold labeling in the cytoplasm associated to the endoplasmic reticulum, the golgi apparatus, and vesicles, plasma membrane and tonoplast. Our results show that salt-adaptation induced a significant reduction of the cytoplasm, plasma membrane and tonoplast content of these epitopes. Yariv reagent was added to the control and salt-adapted tobacco cell cultures, leading to cell death induction in control cells but not in salt-adapted cells. Ultrastructural and immunogold labeling revealed that cell death induced by Yariv reagent in control cells was due to the interaction of Yariv reagent with the AGPs linked to the plasma membranes. Finally, we propose a new function of AGPs as a possible sodium carrier through the mechanism of vesicle trafficking from the apoplast to the vacuoles in salt-adapted tobacco BY-2 cells. This mechanism may contribute to sodium homeostasis during salt-adaptation to high saline concentrations.

  18. Protein and DNA sequence determinants of thermophilic adaptation.

    Directory of Open Access Journals (Sweden)

    Konstantin B Zeldovich

    2007-01-01

    Full Text Available There have been considerable attempts in the past to relate phenotypic trait--habitat temperature of organisms--to their genotypes, most importantly compositions of their genomes and proteomes. However, despite accumulation of anecdotal evidence, an exact and conclusive relationship between the former and the latter has been elusive. We present an exhaustive study of the relationship between amino acid composition of proteomes, nucleotide composition of DNA, and optimal growth temperature (OGT of prokaryotes. Based on 204 complete proteomes of archaea and bacteria spanning the temperature range from -10 degrees C to 110 degrees C, we performed an exhaustive enumeration of all possible sets of amino acids and found a set of amino acids whose total fraction in a proteome is correlated, to a remarkable extent, with the OGT. The universal set is Ile, Val, Tyr, Trp, Arg, Glu, Leu (IVYWREL, and the correlation coefficient is as high as 0.93. We also found that the G + C content in 204 complete genomes does not exhibit a significant correlation with OGT (R = -0.10. On the other hand, the fraction of A + G in coding DNA is correlated with temperature, to a considerable extent, due to codon patterns of IVYWREL amino acids. Further, we found strong and independent correlation between OGT and the frequency with which pairs of A and G nucleotides appear as nearest neighbors in genome sequences. This adaptation is achieved via codon bias. These findings present a direct link between principles of proteins structure and stability and evolutionary mechanisms of thermophylic adaptation. On the nucleotide level, the analysis provides an example of how nature utilizes codon bias for evolutionary adaptation to extreme conditions. Together these results provide a complete picture of how compositions of proteomes and genomes in prokaryotes adjust to the extreme conditions of the environment.

  19. Surviving colorectal cancer: long-term, persistent ostomy-specific concerns and adaptations.

    Science.gov (United States)

    Sun, Virginia; Grant, Marcia; McMullen, Carmit K; Altschuler, Andrea; Mohler, M Jane; Hornbrook, Mark C; Herrinton, Lisa J; Baldwin, Carol M; Krouse, Robert S

    2013-01-01

    The purpose of this article was to describe persistent ostomy-specific concerns and adaptations in long-term (>5 years) colorectal cancer survivors with ostomies. Thirty-three colorectal cancer survivors who participated in 8 gender- and health-related quality of life stratified focus groups and 130 colorectal cancer survivors who provided written comments to 2 open-ended questions on ostomy location and pouch problems participated in the study. Data were collected on health maintenance organization members in Oregon, southwestern Washington, and northern California. Qualitative data were analyzed for the 8 focus groups and written comments from 2 open-ended survey questions. Discussions from the focu s groups were recorded, transcribed, and analyzed using content analysis. Written content from the open-ended questions was derived from a mailed questionnaire on health-related quality of life in survivors with ostomies and analyzed using content analysis. Discussions related to persistent ostomy-related issues more than 5 years after formation were common. Persistent ostomy-related issues were focused on clothing restrictions and adaptations, dietary concerns, issues related to ostomy equipment and self-care, and the constant need to find solutions to adjust and readjust to living with an ostomy. Ostomy-specific concerns persist 5 years and more for long-term colorectal cancer survivors after initial ostomy formation. Adaptations tend to be individualized and based on trial and error. Findings underscore the need to develop long-term support mechanisms that survivors can access to promote better coping and adjustment to living with an ostomy.

  20. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  1. Identification of proteins specific for human herpesvirus 6-infected human T cells

    International Nuclear Information System (INIS)

    Balachandran, N.; Amelse, R.E.; Zhou, W.W.; Chang, C.K.

    1989-01-01

    Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [ 35 S]methionine- and [ 3 H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [ 35 S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 135,000-M r polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105k and gp82k, gp116k, gp64k, and gp54k, and gp102k

  2. Identification of proteins specific for human herpesvirus 6-infected human T cells

    International Nuclear Information System (INIS)

    Balachandran, N.; Amelse, R.E.; Zhou, W.W.; Chang, C.K.

    1989-01-01

    Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [ 35 S]methionine- and [ 3 H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [ 35 S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpes viruses reacted with few HHV-6-infected cell proteins, and only a 135,000-M/sub r/ polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gp105K and gp92k, gp116k, gp64k, and gp54k, and gp102k

  3. Rapid detection and purification of sequence specific DNA binding proteins using magnetic separation

    Directory of Open Access Journals (Sweden)

    TIJANA SAVIC

    2006-02-01

    Full Text Available In this paper, a method for the rapid identification and purification of sequence specific DNA binding proteins based on magnetic separation is presented. This method was applied to confirm the binding of the human recombinant USF1 protein to its putative binding site (E-box within the human SOX3 protomer. It has been shown that biotinylated DNA attached to streptavidin magnetic particles specifically binds the USF1 protein in the presence of competitor DNA. It has also been demonstrated that the protein could be successfully eluted from the beads, in high yield and with restored DNA binding activity. The advantage of these procedures is that they could be applied for the identification and purification of any high-affinity sequence-specific DNA binding protein with only minor modifications.

  4. Specific detection of proteins using Nanomechanical resonators

    DEFF Research Database (Denmark)

    Fischer, Lee MacKenzie; Wright, V.A.; Guthy, C.

    2008-01-01

    of probes onto their surfaces in order to enable the specificity of the detection. Such nanoresonator-based specific detection of proteins is here reported using streptavidin as target system, and immobilized biotin as probe. Nanomechanical resonators resistant to stiction were first realized from silicon...... carbonitride using a novel fabrication method. Vapor-phase deposition of mercaptopropyl trimethoxysilane was performed, and an added mass of 2.22 +/- 0.07 fg/mu m(2) was measured. This linker molecule was used to attach biotin onto the devices, enabling the specific detection of streptavidin. A mass of 3.6 fg....../mu m(2) was attributed to the added streptavidin, corresponding to one molecule per 27 nm(2). The specificity of this recognition was confirmed by exposing the devices to a solution of streptavidin that was already saturated with biotin. An additional negative control was also performed by also...

  5. Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome

    Directory of Open Access Journals (Sweden)

    Suh Moo-Jin

    2012-04-01

    Full Text Available Abstract Background The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process. Results To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development. Four different stages were examined: dormant conidia, isotropically expanding conidia, hyphae in which germ tube emergence has just begun, and pre-septation hyphae. To enrich for glycan-linked cell wall proteins we used an alkaline cell extraction method. Shotgun proteomic resulted in the identification of 375 unique gene products with high confidence, with no evidence for enrichment of cell wall-immobilized and secreted proteins. The most interesting discovery was the identification of 52 proteins enriched in dormant conidia including 28 proteins that have never been detected in the A. fumigatus conidial proteome such as signaling protein Pil1, chaperones BipA and calnexin, and transcription factor HapB. Additionally we found many small, Aspergillus specific proteins of unknown function including 17 hypothetical proteins. Thus, the most abundant protein, Grg1 (AFUA_5G14210, was also one of the smallest proteins detected in this study (M.W. 7,367. Among previously characterized proteins were melanin pigment and pseurotin A biosynthesis enzymes, histones H3 and H4.1, and other proteins involved in conidiation and response to oxidative or hypoxic stress. In contrast, expanding conidia, hyphae with early germ tubes, and pre-septation hyphae samples were enriched for proteins responsible for

  6. SH2-Balpha is an insulin-receptor adapter protein and substrate that interacts with the activation loop of the insulin-receptor kinase.

    OpenAIRE

    Kotani, K; Wilden, P; Pillay, T S

    1998-01-01

    We identified SH2-Balpha as an insulin-receptor-binding protein based on interaction screening in yeast hybrid systems and co-precipitation in cells. SH2-Balpha contains pleckstrin-homology ('PH') and Src homology 2 (SH2) domains and is closely related to APS (adapter protein with a PH domain and an SH2 domain) and lnk, adapter proteins first identified in lymphocytes. SH2-Balpha is ubiquitously expressed and is present in rat epididymal adipose tissue, liver and skeletal muscle, physiologica...

  7. CRISPR-Cas: Adapting to change.

    Science.gov (United States)

    Jackson, Simon A; McKenzie, Rebecca E; Fagerlund, Robert D; Kieper, Sebastian N; Fineran, Peter C; Brouns, Stan J J

    2017-04-07

    Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems. Copyright © 2017, American Association for the Advancement of Science.

  8. Specific Internalisation of Gold Nanoparticles into Engineered Porous Protein Cages via Affinity Binding.

    Science.gov (United States)

    Paramelle, David; Peng, Tao; Free, Paul; Fernig, David G; Lim, Sierin; Tomczak, Nikodem

    2016-01-01

    Porous protein cages are supramolecular protein self-assemblies presenting pores that allow the access of surrounding molecules and ions into their core in order to store and transport them in biological environments. Protein cages' pores are attractive channels for the internalisation of inorganic nanoparticles and an alternative for the preparation of hybrid bioinspired nanoparticles. However, strategies based on nanoparticle transport through the pores are largely unexplored, due to the difficulty of tailoring nanoparticles that have diameters commensurate with the pores size and simultaneously displaying specific affinity to the cages' core and low non-specific binding to the cages' outer surface. We evaluated the specific internalisation of single small gold nanoparticles, 3.9 nm in diameter, into porous protein cages via affinity binding. The E2 protein cage derived from the Geobacillus stearothermophilus presents 12 pores, 6 nm in diameter, and an empty core of 13 nm in diameter. We engineered the E2 protein by site-directed mutagenesis with oligohistidine sequences exposing them into the cage's core. Dynamic light scattering and electron microscopy analysis show that the structures of E2 protein cages mutated with bis- or penta-histidine sequences are well conserved. The surface of the gold nanoparticles was passivated with a self-assembled monolayer made of a mixture of short peptidols and thiolated alkane ethylene glycol ligands. Such monolayers are found to provide thin coatings preventing non-specific binding to proteins. Further functionalisation of the peptide coated gold nanoparticles with Ni2+ nitrilotriacetic moieties enabled the specific binding to oligohistidine tagged cages. The internalisation via affinity binding was evaluated by electron microscopy analysis. From the various mutations tested, only the penta-histidine mutated E2 protein cage showed repeatable and stable internalisation. The present work overcomes the limitations of currently

  9. Stimulation of Protein Expression Through the Harmonic Resonance of Frequency-Specific Music.

    Science.gov (United States)

    Orhan, Ibrahim Y; Gulbahar, Burak A

    2016-12-01

    The use of specific frequencies for specific individual amino acids may increase the potential energy of protein molecules in the medium [1]. The resonance would also increase the movement of particles in the cytosol, increasing the collisions necessary for the conduction of protein expression. The clash of two waves that share frequencies will exhibit an increase in energy through an increase in amplitude [2]. The increase in energy would in turn increase the number of collisions forming the tRNA-amino acid, increasing the amino acid acquiry for ribosomes to improve intracellular efficiency in gene expression. To test the hypothesis, Red Fluorescent Protein (RFP) in transformated BL-21 strains of E. coli and p53 protein of MCF-7 were examined after exposure to sounds of specific frequencies. Through the exposure of the experimental systems to a sequence of sounds that match the frequencies of specific amino acids, the levels of RFP exhibition respective to the control groups in the bacterial medium increased two-fold in terms of RFU. The experiments that targeted the p53 protein with the 'music' showed a decrease in the cell prevalence in the MCF-7 type breast cancer cells by 28%, by decreasing the speed of tumour formation. Exposure to 'music' that was designed through assigning a musical note for every single one of the twenty unique amino acids, produced both an analytical and a visible shift in protein synthesis, making it as potential tool for reducing procedural time uptake.

  10. Growth-Phase-Specific Modulation of Cell Morphology and Gene Expression by an Archaeal Histone Protein.

    Science.gov (United States)

    Dulmage, Keely A; Todor, Horia; Schmid, Amy K

    2015-09-08

    In all three domains of life, organisms use nonspecific DNA-binding proteins to compact and organize the genome as well as to regulate transcription on a global scale. Histone is the primary eukaryotic nucleoprotein, and its evolutionary roots can be traced to the archaea. However, not all archaea use this protein as the primary DNA-packaging component, raising questions regarding the role of histones in archaeal chromatin function. Here, quantitative phenotyping, transcriptomic, and proteomic assays were performed on deletion and overexpression mutants of the sole histone protein of the hypersaline-adapted haloarchaeal model organism Halobacterium salinarum. This protein is highly conserved among all sequenced haloarchaeal species and maintains hallmark residues required for eukaryotic histone functions. Surprisingly, despite this conservation at the sequence level, unlike in other archaea or eukaryotes, H. salinarum histone is required to regulate cell shape but is not necessary for survival. Genome-wide expression changes in histone deletion strains were global, significant but subtle in terms of fold change, bidirectional, and growth phase dependent. Mass spectrometric proteomic identification of proteins from chromatin enrichments yielded levels of histone and putative nucleoid-associated proteins similar to those of transcription factors, consistent with an open and transcriptionally active genome. Taken together, these data suggest that histone in H. salinarum plays a minor role in DNA compaction but important roles in growth-phase-dependent gene expression and regulation of cell shape. Histone function in haloarchaea more closely resembles a regulator of gene expression than a chromatin-organizing protein like canonical eukaryotic histone. Histones comprise the major protein component of eukaryotic chromatin and are required for both genome packaging and global regulation of expression. The current paradigm maintains that archaea whose genes encode

  11. Dietary protein intake and distribution patterns of well-trained Dutch athletes

    NARCIS (Netherlands)

    Gillen, Jenna B.; Trommelen, Jorn; Wardenaar, Floris C.; Brinkmans, Naomi Y.J.; Versteegen, Joline J.; Jonvik, Kristin L.; Kapp, Christoph; Vries, de Jeanne; Borne, van den Joost J.G.C.; Gibala, Martin J.; Loon, van Luc J.C.

    2017-01-01

    Dietary protein intake should be optimized in all athletes to ensure proper recovery and enhance the skeletal muscle adaptive response to exercise training. In addition to total protein intake, the use of specific proteincontaining food sources and the distribution of protein throughout the day

  12. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific

  13. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  14. Organ-specific gene expression: the bHLH protein Sage provides tissue specificity to Drosophila FoxA.

    Science.gov (United States)

    Fox, Rebecca M; Vaishnavi, Aria; Maruyama, Rika; Andrew, Deborah J

    2013-05-01

    FoxA transcription factors play major roles in organ-specific gene expression, regulating, for example, glucagon expression in the pancreas, GLUT2 expression in the liver, and tyrosine hydroxylase expression in dopaminergic neurons. Organ-specific gene regulation by FoxA proteins is achieved through cooperative regulation with a broad array of transcription factors with more limited expression domains. Fork head (Fkh), the sole Drosophila FoxA family member, is required for the development of multiple distinct organs, yet little is known regarding how Fkh regulates tissue-specific gene expression. Here, we characterize Sage, a bHLH transcription factor expressed exclusively in the Drosophila salivary gland (SG). We show that Sage is required for late SG survival and normal tube morphology. We find that many Sage targets, identified by microarray analysis, encode SG-specific secreted cargo, transmembrane proteins, and the enzymes that modify these proteins. We show that both Sage and Fkh are required for the expression of Sage target genes, and that co-expression of Sage and Fkh is sufficient to drive target gene expression in multiple cell types. Sage and Fkh drive expression of the bZip transcription factor Senseless (Sens), which boosts expression of Sage-Fkh targets, and Sage, Fkh and Sens colocalize on SG chromosomes. Importantly, expression of Sage-Fkh target genes appears to simply add to the tissue-specific gene expression programs already established in other cell types, and Sage and Fkh cannot alter the fate of most embryonic cell types even when expressed early and continuously.

  15. A human-specific de novo protein-coding gene associated with human brain functions.

    Directory of Open Access Journals (Sweden)

    Chuan-Yun Li

    2010-03-01

    Full Text Available To understand whether any human-specific new genes may be associated with human brain functions, we computationally screened the genetic vulnerable factors identified through Genome-Wide Association Studies and linkage analyses of nicotine addiction and found one human-specific de novo protein-coding gene, FLJ33706 (alternative gene symbol C20orf203. Cross-species analysis revealed interesting evolutionary paths of how this gene had originated from noncoding DNA sequences: insertion of repeat elements especially Alu contributed to the formation of the first coding exon and six standard splice junctions on the branch leading to humans and chimpanzees, and two subsequent substitutions in the human lineage escaped two stop codons and created an open reading frame of 194 amino acids. We experimentally verified FLJ33706's mRNA and protein expression in the brain. Real-Time PCR in multiple tissues demonstrated that FLJ33706 was most abundantly expressed in brain. Human polymorphism data suggested that FLJ33706 encodes a protein under purifying selection. A specifically designed antibody detected its protein expression across human cortex, cerebellum and midbrain. Immunohistochemistry study in normal human brain cortex revealed the localization of FLJ33706 protein in neurons. Elevated expressions of FLJ33706 were detected in Alzheimer's brain samples, suggesting the role of this novel gene in human-specific pathogenesis of Alzheimer's disease. FLJ33706 provided the strongest evidence so far that human-specific de novo genes can have protein-coding potential and differential protein expression, and be involved in human brain functions.

  16. Similar rates of protein adaptation in Drosophila miranda and D. melanogaster, two species with different current effective population sizes

    Directory of Open Access Journals (Sweden)

    Bachtrog Doris

    2008-12-01

    Full Text Available Abstract Background Adaptive protein evolution is common in several Drosophila species investigated. Some studies point to very weak selection operating on amino-acid mutations, with average selection intensities on the order of Nes ~ 5 in D. melanogaster and D. simulans. Species with lower effective population sizes should undergo less adaptation since they generate fewer mutations and selection is ineffective on a greater proportion of beneficial mutations. Results Here I study patterns of polymorphism and divergence at 91 X-linked loci in D. miranda, a species with a roughly 5-fold smaller effective population size than D. melanogaster. Surprisingly, I find a similar fraction of amino-acid mutations being driven to fixation by positive selection in D. miranda and D. melanogaster. Genes with higher rates of amino-acid evolution show lower levels of neutral diversity, a pattern predicted by recurrent adaptive protein evolution. I fit a hitchhiking model to patterns of polymorphism in D. miranda and D. melanogaster and estimate an order of magnitude higher selection coefficients for beneficial mutations in D. miranda. Conclusion This analysis suggests that effective population size may not be a major determinant in rates of protein adaptation. Instead, adaptation may not be mutation-limited, or the distribution of fitness effects for beneficial mutations might differ vastly between different species or populations. Alternative explanation such as biases in estimating the fraction of beneficial mutations or slightly deleterious mutation models are also discussed.

  17. Prespacer processing and specific integration in a Type I-A CRISPR system

    Science.gov (United States)

    Rollie, Clare; Graham, Shirley; Rouillon, Christophe

    2018-01-01

    Abstract The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types. PMID:29228332

  18. To Be Specific or Not: The Critical Relationship Between Hox And TALE Proteins.

    Science.gov (United States)

    Merabet, Samir; Mann, Richard S

    2016-06-01

    Hox proteins are key regulatory transcription factors that act in different tissues of the embryo to provide specific spatial and temporal coordinates to each cell. These patterning functions often depend on the presence of the TALE-homeodomain class cofactors, which form cooperative DNA-binding complexes with all Hox proteins. How this family of cofactors contributes to the highly diverse and specific functions of Hox proteins in vivo remains an important unsolved question. We review here the most recent advances in understanding the molecular mechanisms underlying Hox-TALE function. In particular, we discuss the role of DNA shape, DNA-binding affinity, and protein-protein interaction flexibility in dictating Hox-TALE specificity. We propose several models to explain how these mechanisms are integrated with each other in the context of the many distinct functions that Hox and TALE factors carry out in vivo. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Specific radioimmunoassay for ovine bone gla-protein (osteocalcin)

    Energy Technology Data Exchange (ETDEWEB)

    Pastoureau, P; Merle, B; Delmas, P D

    1988-01-01

    We developed a sensitive and specific radioimmunoassay for ovine bone gla-protein (osteocalcin) using a polyclonal rabbit antibody raised against ovine bone gla-protein. Bone from lambs was extracted in 0.5 mol/l EDTA and desalted on Sephadex G-25. Bone gla-protein was purified by gel filtration chromatography over Sephadex G-100 and ion-exchange chromatography on DEAE-Sephadex A-25. The protein, subjected to monodimensional electrophoresis migrated as a single spot in SDS PAGE with the same apparent molecular weight of 12 kD as bovine bone gla-protein. The amino acid composition of pufified bone gla-protein was in agreement with a previous publication. The competitive RIA uses /sup 125/I-labelled bone gla-protein as a tracer and a complex of a second antibody and polyethylene glycol to separate free and antibody-bound /sup 125/I-labelled bone gla-protein. The intra- and inter-assay variations are less than 6 and 10%, respectively. There is no reactivity of our antisera with dog sera. The cross-reactivity is only partial with calf and human sera and complete with ovine sera. We measured bone gla-protein levels in serum of 96 normal male sheep of different ages. Serum bone gla-protein rapidly and significantly (P<0.001) decreased from 532 +- 169 ..mu..g/l at birth, to 240 +- 43 ..mu..g/l at 45 days, 152 +- 44 ..mu..g/l at 90 days, and 5.9 +- 0.7 ..mu..g/l at 7 years age. In addition, bone gla-protein levels at birth were higher in normal birth weight than in hypotrophic lambs with low birth weight (535 +- 169 vs 271 +- 156 ..mu..g/l, P<0.0001). Furthermore, lambs raised outside in free conditions tended to have higher serum bone gla-protein levels than lambs raised under shelter (1984 +- 53 vs 137 +- 34 ..mu..g/l), suggesting a role of breeding factors such as diet or relative immobilization on bone gla-protein levels. (Abstract Truncated)

  20. Characterization of Macrophage Endogenous S-Nitrosoproteome Using a Cysteine-Specific Phosphonate Adaptable Tag in Combination with TiO2 Chromatography.

    Science.gov (United States)

    Ibáñez-Vea, María; Huang, Honggang; Martínez de Morentin, Xabier; Pérez, Estela; Gato, Maria; Zuazo, Miren; Arasanz, Hugo; Fernández-Irigoyen, Joaquin; Santamaría, Enrique; Fernandez-Hinojal, Gonzalo; Larsen, Martin R; Escors, David; Kochan, Grazyna

    2018-03-02

    Protein S-nitrosylation is a cysteine post-translational modification mediated by nitric oxide. An increasing number of studies highlight S-nitrosylation as an important regulator of signaling involved in numerous cellular processes. Despite the significant progress in the development of redox proteomic methods, identification and quantification of endogeneous S-nitrosylation using high-throughput mass-spectrometry-based methods is a technical challenge because this modification is highly labile. To overcome this drawback, most methods induce S-nitrosylation chemically in proteins using nitrosylating compounds before analysis, with the risk of introducing nonphysiological S-nitrosylation. Here we present a novel method to efficiently identify endogenous S-nitrosopeptides in the macrophage total proteome. Our approach is based on the labeling of S-nitrosopeptides reduced by ascorbate with a cysteine specific phosphonate adaptable tag (CysPAT), followed by titanium dioxide (TiO 2 ) chromatography enrichment prior to nLC-MS/MS analysis. To test our procedure, we performed a large-scale analysis of this low-abundant modification in a murine macrophage cell line. We identified 569 endogeneous S-nitrosylated proteins compared with 795 following exogenous chemically induced S-nitrosylation. Importantly, we discovered 579 novel S-nitrosylation sites. The large number of identified endogenous S-nitrosylated peptides allowed the definition of two S-nitrosylation consensus sites, highlighting protein translation and redox processes as key S-nitrosylation targets in macrophages.

  1. Study on Fusion Protein and Its gene in Baculovirus Specificity

    International Nuclear Information System (INIS)

    Nemr, W.A.H.

    2012-01-01

    Baculoviruses are subdivided into two groups depending on the type of budded virus envelop fusion protein; group I utilized gp64 which include the most of nucleopolyhedroviruses (NPVs), group II utilized F protein which include the remnants of NPVs and all Granuloviruses (GVs). Recent studies reported the viral F protein coding gene as a host cellular sourced gene and may evolutionary acquired from the host genome referring to phylogeny analysis of fusion proteins. Thus, it was deduced that F protein coding gene is species- specific nucleotide sequence related to the type of the specific host and if virus could infect an unexpected host, the resulted virus may encode a vary F gene. In this regard, the present study utilized the mentioned properties of F gene in an attempt to produce a model of specific and more economic wider range granulovirus bio- pesticide able to infect both Spodoptera littoralis and Phthorimaea operculella larvae. Multiple sequence alignment and phylogeny analysis were performed on six members of group II baculovirus, novel universal PCR primers were manually designed from the conserved regions in the alignment graph, targeted to amplify species- specific sequence entire F gene open reading frame (ORF) which is useful in molecular identification of baculovirus in unknown samples. So, the PCR product of SpliGV used to prepare a specific probe for the F gene of this type of virus. Results reflected that it is possible to infect S. littoralis larvae by PhopGV if injected into larval haemocoel, the resulted virus of this infection showed by using DNA hybridization technique to be encode to F gene homologous with the F gene of Spli GV, which is revealed that the resulted virus acquired this F gene sequence from the host genome after infection. Consequently, these results may infer that if genetic aberrations occur in the host genome, this may affect in baculoviral infectivity. So, this study aimed to investigate the effect of gamma radiation at

  2. Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a

    International Nuclear Information System (INIS)

    Miranda, Tina Branscombe; Webb, Kristofor J.; Edberg, Dale D.; Reeves, Raymond; Clarke, Steven

    2005-01-01

    The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation

  3. Recent advances in covalent, site-specific protein immobilization [version 1; referees

    DEFF Research Database (Denmark)

    Meldal, Morten Peter; Schoffelen, Sanne

    2016-01-01

    The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control...

  4. Prediction of membrane transport proteins and their substrate specificities using primary sequence information.

    Directory of Open Access Journals (Sweden)

    Nitish K Mishra

    Full Text Available Membrane transport proteins (transporters move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task.Support vector machine (SVM-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM, were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC of 0.49 and a receiver operating characteristic area under the curve (AUC of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset.Our analyses suggest that evolutionary information (i.e., the PSSM and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide accurate predictions

  5. Molecular evolution and thermal adaptation

    Science.gov (United States)

    Chen, Peiqiu

    2011-12-01

    In this thesis, we address problems in molecular evolution, thermal adaptation, and the kinetics of adaptation of bacteria and viruses to elevated environmental temperatures. We use a nearly neutral fitness model where the replication speed of an organism is proportional to the copy number of folded proteins. Our model reproduces the distribution of stabilities of natural proteins in excellent agreement with experiment. We find that species with high mutation rates tend to have less stable proteins compared to species with low mutation rate. We found that a broad distribution of protein stabilities observed in the model and in experiment is the key determinant of thermal response for viruses and bacteria. Our results explain most of the earlier experimental observations: striking asymmetry of thermal response curves, the absence of evolutionary trade-off which was expected but not found in experiments, correlation between denaturation temperature for several protein families and the Optimal Growth Temperature (OGT) of their carrier organisms, and proximity of bacterial or viral OGTs to their evolutionary temperatures. Our theory quantitatively and with high accuracy described thermal response curves for 35 bacterial species. The model also addresses the key to adaptation is in weak-link genes (WLG), which encode least thermodynamically stable essential proteins in the proteome. We observe, as in experiment, a two-stage adaptation process. The first stage is a Luria-Delbruck type of selection, whereby rare WLG alleles, whose proteins are more stable than WLG proteins of the majority of the population (either due to standing genetic variation or due to an early acquired mutation), rapidly rise to fixation. The second stage constitutes subsequent slow accumulation of mutations in an adapted population. As adaptation progresses, selection regime changes from positive to neutral: Selection coefficient of beneficial mutations scales as a negative power of number of

  6. Intracellular Protein Delivery System Using a Target-Specific Repebody and Translocation Domain of Bacterial Exotoxin.

    Science.gov (United States)

    Kim, Hee-Yeon; Kang, Jung Ae; Ryou, Jeong-Hyun; Lee, Gyeong Hee; Choi, Dae Seong; Lee, Dong Eun; Kim, Hak-Sung

    2017-11-17

    With the high efficacy of protein-based therapeutics and plenty of intracellular drug targets, cytosolic protein delivery in a cell-specific manner has attracted considerable attention in the field of precision medicine. Herein, we present an intracellular protein delivery system based on a target-specific repebody and the translocation domain of Pseudomonas aeruginosa exotoxin A. The delivery platform was constructed by genetically fusing an EGFR-specific repebody as a targeting moiety to the translocation domain, while a protein cargo was fused to the C-terminal end of the delivery platform. The delivery platform was revealed to efficiently translocate a protein cargo to the cytosol in a target-specific manner. We demonstrate the utility and potential of the delivery platform by showing a remarkable tumor regression with negligible toxicity in a xenograft mice model when gelonin was used as the cytotoxic protein cargo. The present platform can find wide applications to the cell-selective cytosolic delivery of diverse proteins in many areas.

  7. Kinetic Stability of Proteins in Beans and Peas: Implications for Protein Digestibility, Seed Germination, and Plant Adaptation.

    Science.gov (United States)

    Xia, Ke; Pittelli, Sandy; Church, Jennifer; Colón, Wilfredo

    2016-10-12

    Kinetically stable proteins (KSPs) are resistant to the denaturing detergent sodium dodecyl sulfate (SDS). Such resilience makes KSPs resistant to proteolytic degradation and may have arisen in nature as a mechanism for organismal adaptation and survival against harsh conditions. Legumes are well-known for possessing degradation-resistant proteins that often diminish their nutritional value. Here we applied diagonal two-dimensional (D2D) SDS-polyacrylamide gel electrophoresis (PAGE), a method that allows for the proteomics-level identification of KSPs, to a group of 12 legumes (mostly beans and peas) of agricultural and nutritional importance. Our proteomics results show beans that are more difficult to digest, such as soybean, lima beans, and various common beans, have high contents of KSPs. In contrast, mung bean, red lentil, and various peas that are highly digestible contain low amounts of KSPs. Identified proteins with high kinetic stability are associated with warm-season beans, which germinate at higher temperatures. In contrast, peas and red lentil, which are cool-season legumes, contain low levels of KSPs. Thus, our results show protein kinetic stability is an important factor in the digestibility of legume proteins and may relate to nutrition efficiency, timing of seed germination, and legume resistance to biotic stressors. Furthermore, we show D2D SDS-PAGE is a powerful method that could be applied for determining the abundance and identity of KSPs in engineered and wild legumes and for advancing basic research and associated applications.

  8. Site-specific labeling of proteins with NMR-active unnatural amino acids

    International Nuclear Information System (INIS)

    Jones, David H.; Cellitti, Susan E.; Hao Xueshi; Zhang Qiong; Jahnz, Michael; Summerer, Daniel; Schultz, Peter G.; Uno, Tetsuo; Geierstanger, Bernhard H.

    2010-01-01

    A large number of amino acids other than the canonical amino acids can now be easily incorporated in vivo into proteins at genetically encoded positions. The technology requires an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is added to the media while a TAG amber or frame shift codon specifies the incorporation site in the protein to be studied. These unnatural amino acids can be isotopically labeled and provide unique opportunities for site-specific labeling of proteins for NMR studies. In this perspective, we discuss these opportunities including new photocaged unnatural amino acids, outline usage of metal chelating and spin-labeled unnatural amino acids and expand the approach to in-cell NMR experiments.

  9. Sequencing of 50 human exomes reveals adaptation to high altitude

    DEFF Research Database (Denmark)

    Yi, Xin; Liang, Yu; Huerta-Sanchez, Emilia

    2010-01-01

    Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which repres...... in genetic adaptation to high altitude.......Residents of the Tibetan Plateau show heritable adaptations to extreme altitude. We sequenced 50 exomes of ethnic Tibetans, encompassing coding sequences of 92% of human genes, with an average coverage of 18x per individual. Genes showing population-specific allele frequency changes, which...... represent strong candidates for altitude adaptation, were identified. The strongest signal of natural selection came from endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), a transcription factor involved in response to hypoxia. One single-nucleotide polymorphism (SNP) at EPAS1 shows a 78% frequency...

  10. Evolutionary reprograming of protein-protein interaction specificity.

    Science.gov (United States)

    Akiva, Eyal; Babbitt, Patricia C

    2015-10-22

    Using mutation libraries and deep sequencing, Aakre et al. study the evolution of protein-protein interactions using a toxin-antitoxin model. The results indicate probable trajectories via "intermediate" proteins that are promiscuous, thus avoiding transitions via non-interactions. These results extend observations about other biological interactions and enzyme evolution, suggesting broadly general principles. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Evaluation of epididymal function through specific protein on spermatozoa.

    Science.gov (United States)

    Del Río, A G; De Sánchez, L Z; Sirena, A

    1984-01-01

    Investigations were focused on the characterization of specific epididymal proteins on the human spermatozoa as a representative parameter for epididymal function. An easy and attainable method, suitable for investigators and clinical use, is proposed in this article.

  12. A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

    Science.gov (United States)

    Kumar, Priyadarsini; Walsh, Donal A

    2002-03-15

    We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

  13. Fusion peptides from oncogenic chimeric proteins as putative specific biomarkers of cancer.

    Science.gov (United States)

    Conlon, Kevin P; Basrur, Venkatesha; Rolland, Delphine; Wolfe, Thomas; Nesvizhskii, Alexey I; MacCoss, Michael J; Lim, Megan S; Elenitoba-Johnson, Kojo S J

    2013-10-01

    Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.

  14. Neurotrophin and FGF Signaling Adapter Proteins, FRS2 and FRS3, Regulate Dentate Granule Cell Maturation and Excitatory Synaptogenesis.

    Science.gov (United States)

    Nandi, Sayan; Alviña, Karina; Lituma, Pablo J; Castillo, Pablo E; Hébert, Jean M

    2018-01-15

    Dentate granule cells (DGCs) play important roles in cognitive processes. Knowledge about how growth factors such as FGFs and neurotrophins contribute to the maturation and synaptogenesis of DGCs is limited. Here, using brain-specific and germline mouse mutants we show that a module of neurotrophin and FGF signaling, the FGF Receptor Substrate (FRS) family of intracellular adapters, FRS2 and FRS3, are together required for postnatal brain development. In the hippocampus, FRS promotes dentate gyrus morphogenesis and DGC maturation during developmental neurogenesis, similar to previously published functions for both neurotrophins and FGFs. Consistent with a role in DGC maturation, two-photon imaging revealed that Frs2,3-double mutants have reduced numbers of dendritic branches and spines in DGCs. Functional analysis further showed that double-mutant mice exhibit fewer excitatory synaptic inputs onto DGCs. These observations reveal roles for FRS adapters in DGC maturation and synaptogenesis and suggest that FRS proteins may act as an important node for FGF and neurotrophin signaling in postnatal hippocampal development. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  15. Shared and Unique Proteins in Human, Mouse and Rat Saliva Proteomes: Footprints of Functional Adaptation

    Directory of Open Access Journals (Sweden)

    Robert C. Karn

    2013-12-01

    Full Text Available The overall goal of our study was to compare the proteins found in the saliva proteomes of three mammals: human, mouse and rat. Our first objective was to compare two human proteomes with very different analysis depths. The 89 shared proteins in this comparison apparently represent a core of highly-expressed human salivary proteins. Of the proteins unique to each proteome, one-half to 2/3 lack signal peptides and probably are contaminants instead of less highly-represented salivary proteins. We recently published the first rodent saliva proteomes with saliva collected from the genome mouse (C57BL/6 and the genome rat (BN/SsNHsd/Mcwi. Our second objective was to compare the proteins in the human proteome with those we identified in the genome mouse and rat to determine those common to all three mammals, as well as the specialized rodent subset. We also identified proteins unique to each of the three mammals, because differences in the secreted protein constitutions can provide clues to differences in the evolutionary adaptation of the secretions in the three different mammals.

  16. Campylobacter jejuni transducer like proteins: Chemotaxis and beyond.

    Science.gov (United States)

    Chandrashekhar, Kshipra; Kassem, Issmat I; Rajashekara, Gireesh

    2017-07-04

    Chemotaxis, a process that mediates directional motility toward or away from chemical stimuli (chemoeffectors/ligands that can be attractants or repellents) in the environment, plays an important role in the adaptation of Campylobacter jejuni to disparate niches. The chemotaxis system consists of core signal transduction proteins and methyl-accepting-domain-containing Transducer like proteins (Tlps). Ligands binding to Tlps relay a signal to chemotaxis proteins in the cytoplasm which initiate a signal transduction cascade, culminating into a directional flagellar movement. Tlps facilitate substrate-specific chemotaxis in C. jejuni, which plays an important role in the pathogen's adaptation, pathobiology and colonization of the chicken gastrointestinal tract. However, the role of Tlps in C. jejuni's host tissue specific colonization, physiology and virulence remains not completely understood. Based on recent studies, it can be predicted that Tlps might be important targets for developing strategies to control C. jejuni via vaccines and antimicrobials.

  17. DUF581 is plant specific FCS-like zinc finger involved in protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Muhammed Jamsheer K

    Full Text Available Zinc fingers are a ubiquitous class of protein domain with considerable variation in structure and function. Zf-FCS is a highly diverged group of C2-C2 zinc finger which is present in animals, prokaryotes and viruses, but not in plants. In this study we identified that a plant specific domain of unknown function, DUF581 is a zf-FCS type zinc finger. Based on HMM-HMM comparison and signature motif similarity we named this domain as FCS-Like Zinc finger (FLZ domain. A genome wide survey identified that FLZ domain containing genes are bryophytic in origin and this gene family is expanded in spermatophytes. Expression analysis of selected FLZ gene family members of A. thaliana identified an overlapping expression pattern suggesting a possible redundancy in their function. Unlike the zf-FCS domain, the FLZ domain found to be highly conserved in sequence and structure. Using a combination of bioinformatic and protein-protein interaction tools, we identified that FLZ domain is involved in protein-protein interaction.

  18. Molecular tweezers modulate 14-3-3 protein-protein interactions

    Science.gov (United States)

    Bier, David; Rose, Rolf; Bravo-Rodriguez, Kenny; Bartel, Maria; Ramirez-Anguita, Juan Manuel; Dutt, Som; Wilch, Constanze; Klärner, Frank-Gerrit; Sanchez-Garcia, Elsa; Schrader, Thomas; Ottmann, Christian

    2013-03-01

    Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins—a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)—in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

  19. Odor-Specific Habituation Arises from Interaction of Afferent Synaptic Adaptation and Intrinsic Synaptic Potentiation in Olfactory Cortex

    Science.gov (United States)

    Linster, Christiane; Menon, Alka V.; Singh, Christopher Y.; Wilson, Donald A.

    2009-01-01

    Segmentation of target odorants from background odorants is a fundamental computational requirement for the olfactory system and is thought to be behaviorally mediated by olfactory habituation memory. Data from our laboratory have shown that odor-specific adaptation in piriform neurons, mediated at least partially by synaptic adaptation between…

  20. BACH transcription factors in innate and adaptive immunity.

    Science.gov (United States)

    Igarashi, Kazuhiko; Kurosaki, Tomohiro; Roychoudhuri, Rahul

    2017-07-01

    BTB and CNC homology (BACH) proteins are transcriptional repressors of the basic region leucine zipper (bZIP) transcription factor family. Recent studies indicate widespread roles of BACH proteins in controlling the development and function of the innate and adaptive immune systems, including the differentiation of effector and memory cells of the B and T cell lineages, CD4 + regulatory T cells and macrophages. Here, we emphasize similarities at a molecular level in the cell-type-specific activities of BACH factors, proposing that competitive interactions of BACH proteins with transcriptional activators of the bZIP family form a common mechanistic theme underlying their diverse actions. The findings contribute to a general understanding of how transcriptional repressors shape lineage commitment and cell-type-specific functions through repression of alternative lineage programmes.

  1. The effects of whey protein with or without carbohydrates on resistance training adaptations

    OpenAIRE

    Hulmi, Juha; Laakso, Mia; Mero, Antti; Häkkinen, Keijo; Ahtiainen, Juha; Peltonen, Heikki

    2015-01-01

    Background: Nutrition intake in the context of a resistance training (RT) bout may affect body composition and muscle strength. However, the individual and combined effects of whey protein and carbohydrates on long-term resistance training adaptations are poorly understood. Methods: A four-week preparatory RT period was conducted in previously untrained males to standardize the training background of the subjects. Thereafter, the subjects were randomized into three groups: 30 g of...

  2. Binding proteins enhance specific uptake rate by increasing the substrate-transporter encounter rate.

    Science.gov (United States)

    Bosdriesz, Evert; Magnúsdóttir, Stefanía; Bruggeman, Frank J; Teusink, Bas; Molenaar, Douwe

    2015-06-01

    Microorganisms rely on binding-protein assisted, active transport systems to scavenge for scarce nutrients. Several advantages of using binding proteins in such uptake systems have been proposed. However, a systematic, rigorous and quantitative analysis of the function of binding proteins is lacking. By combining knowledge of selection pressure and physiochemical constraints, we derive kinetic, thermodynamic, and stoichiometric properties of binding-protein dependent transport systems that enable a maximal import activity per amount of transporter. Under the hypothesis that this maximal specific activity of the transport complex is the selection objective, binding protein concentrations should exceed the concentration of both the scarce nutrient and the transporter. This increases the encounter rate of transporter with loaded binding protein at low substrate concentrations, thereby enhancing the affinity and specific uptake rate. These predictions are experimentally testable, and a number of observations confirm them. © 2015 FEBS.

  3. Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

    Science.gov (United States)

    Kazakov, Alexei S; Sokolov, Andrei S; Vologzhannikova, Alisa A; Permyakova, Maria E; Khorn, Polina A; Ismailov, Ramis G; Denessiouk, Konstantin A; Denesyuk, Alexander I; Rastrygina, Victoria A; Baksheeva, Viktoriia E; Zernii, Evgeni Yu; Zinchenko, Dmitry V; Glazatov, Vladimir V; Uversky, Vladimir N; Mirzabekov, Tajib A; Permyakov, Eugene A; Permyakov, Sergei E

    2017-01-01

    Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 μM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

  4. Structural adaptations of octaheme nitrite reductases from haloalkaliphilic Thioalkalivibrio bacteria to alkaline pH and high salinity.

    Directory of Open Access Journals (Sweden)

    Anna Popinako

    Full Text Available Bacteria Tv. nitratireducens and Tv. paradoxus from soda lakes grow optimally in sodium carbonate/NaCl brines at pH range from 9.5 to 10 and salinity from 0.5 to 1.5 M Na+. Octaheme nitrite reductases (ONRs from haloalkaliphilic bacteria of genus Thioalkalivibrio are stable and active in a wide range of pH (up to 11 and salinity (up to 1 M NaCl. To establish adaptation mechanisms of ONRs from haloalkaliphilic bacteria a comparative analysis of amino acid sequences and structures of ONRs from haloalkaliphilic bacteria and their homologues from non-halophilic neutrophilic bacteria was performed. The following adaptation strategies were observed: (1 strategies specific for halophilic and alkaliphilic proteins (an increase in the number of aspartate and glutamate residues and a decrease in the number of lysine residues on the protein surface, (2 strategies specific for halophilic proteins (an increase in the arginine content and a decrease in the number of hydrophobic residues on the solvent-accessible protein surface, (3 strategies specific for alkaliphilic proteins (an increase in the area of intersubunit hydrophobic contacts. Unique adaptation mechanism inherent in the ONRs from bacteria of genus Thioalkalivibrio was revealed (an increase in the core in the number of tryptophan and phenylalanine residues, and an increase in the number of small side chain residues, such as alanine and valine, in the core.

  5. Tick-Host Range Adaptation: Changes in Protein Profiles in Unfed Adult Ixodes scapularis and Amblyomma americanum Saliva Stimulated to Feed on Different Hosts

    Directory of Open Access Journals (Sweden)

    Lucas Tirloni

    2017-12-01

    Full Text Available Understanding the molecular basis of how ticks adapt to feed on different animal hosts is central to understanding tick and tick-borne disease (TBD epidemiology. There is evidence that ticks differentially express specific sets of genes when stimulated to start feeding. This study was initiated to investigate if ticks such as Ixodes scapularis and Amblyomma americanum that are adapted to feed on multiple hosts utilized the same sets of proteins to prepare for feeding. We exposed I. scapularis and A. americanum to feeding stimuli of different hosts (rabbit, human, and dog by keeping unfed adult ticks enclosed in a perforated microfuge in close contact with host skin, but not allowing ticks to attach on host. Our data suggest that ticks of the same species differentially express tick saliva proteins (TSPs when stimulated to start feeding on different hosts. SDS-PAGE and silver staining analysis revealed unique electrophoretic profiles in saliva of I. scapularis and A. americanum that were stimulated to feed on different hosts: rabbit, human, and dog. LC-MS/MS sequencing and pairwise analysis demonstrated that I. scapularis and A. americanum ticks expressed unique protein profiles in their saliva when stimulated to start feeding on different hosts: rabbit, dog, or human. Specifically, our data revealed TSPs that were unique to each treatment and those that were shared between treatments. Overall, we identified a total of 276 and 340 non-redundant I. scapularis and A. americanum TSPs, which we have classified into 28 functional classes including: secreted conserved proteins (unknown functions, proteinase inhibitors, lipocalins, extracellular matrix/cell adhesion, heme/iron metabolism, signal transduction and immunity-related proteins being the most predominant in saliva of unfed ticks. With exception of research on vaccines against Rhipicephalus microplus, which its natural host, cattle, research on vaccine against other ticks relies feeding ticks

  6. Adaptation of Lactobacillus casei Zhang to Gentamycin Involves an Alkaline Shock Protein

    Directory of Open Access Journals (Sweden)

    Wenyi Zhang

    2017-11-01

    Full Text Available Lactobacillus (L. casei Zhang is a koumiss-originated probiotic strain, which was used as a model in a long-term antibiotics-driven evolution experiment to reveal bacterial evolutionary dynamics; and we isolated gentamycin-resistant L. casei Zhang descendents. To decipher the gentamycin resistance mechanism, here we cultivated the parental L. casei Zhang and its descendent cells in an antibiotics-containing environment to compare their global protein expression profiles using the iTRAQ-based proteomic approach. A total of 72 proteins were significantly up-regulated (>2.0-fold, P < 0.05, whilst 32 proteins were significantly down-regulated <−2.0-fold, P < 0.05 in the descendent line. The gentamycin-resistant descendent line showed elevated expression in some carbohydrates, amino acids, and purine metabolic pathways. Several stress-related proteins were also differentially expressed. Among them, one alkaline shock protein, asp23, was up-regulated most in the gentamycin-resistant strain (21.9-fold increase compared with the parental strain. The asp23 gene disruption mutant was significantly more sensitive to gentamycin compared with the wild type, suggesting an important role of this gene in developing the gentamycin-resistant phenotype in L. casei. Our report has described the adaptation of a probiotic strain that has acquired antibiotics resistance through long-term antibiotics exposure at the proteome level, and we revealed a novel mechanism of gentamycin resistance.

  7. Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

    International Nuclear Information System (INIS)

    Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

    1986-01-01

    Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

  8. Development of antifertility vaccine using sperm specific proteins

    Directory of Open Access Journals (Sweden)

    A H Bandivdekar

    2014-01-01

    Full Text Available Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4, protein hyaluronidase (PH-20, and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80kDa HSA is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80kDa HSA (Peptide NT and its peptides (Peptides 1, 2, 3 and 4 obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80kDA HAS suggest that the synthetic Peptide-1 of 80kDa HSA is the promising candidate for development of male contraceptive vaccine.

  9. Intramuscular Immunisation with Chlamydial Proteins Induces Chlamydia trachomatis Specific Ocular Antibodies.

    Directory of Open Access Journals (Sweden)

    Alexander Badamchi-Zadeh

    Full Text Available Ocular infection with Chlamydia trachomatis can cause trachoma, which is the leading cause of blindness due to infection worldwide. Despite the large-scale implementation of trachoma control programmes in the majority of countries where trachoma is endemic, there remains a need for a vaccine. Since C. trachomatis infects the conjunctival epithelium and stimulates an immune response in the associated lymphoid tissue, vaccine regimens that enhance local antibody responses could be advantageous. In experimental infections of non-human primates (NHPs, antibody specificity to C. trachomatis antigens was found to change over the course of ocular infection. The appearance of major outer membrane protein (MOMP specific antibodies correlated with a reduction in ocular chlamydial burden, while subsequent generation of antibodies specific for PmpD and Pgp3 correlated with C. trachomatis eradication.We used a range of heterologous prime-boost vaccinations with DNA, Adenovirus, modified vaccinia Ankara (MVA and protein vaccines based on the major outer membrane protein (MOMP as an antigen, and investigated the effect of vaccine route, antigen and regimen on the induction of anti-chlamydial antibodies detectable in the ocular lavage fluid of mice.Three intramuscular vaccinations with recombinant protein adjuvanted with MF59 induced significantly greater levels of anti-MOMP ocular antibodies than the other regimens tested. Intranasal delivery of vaccines induced less IgG antibody in the eye than intramuscular delivery. The inclusion of the antigens PmpD and Pgp3, singly or in combination, induced ocular antigen-specific IgG antibodies, although the anti-PmpD antibody response was consistently lower and attenuated by combination with other antigens.If translatable to NHPs and/or humans, this investigation of the murine C. trachomatis specific ocular antibody response following vaccination provides a potential mouse model for the rapid and high throughput

  10. Adaptive Evolution of Eel Fluorescent Proteins from Fatty Acid Binding Proteins Produces Bright Fluorescence in the Marine Environment.

    Directory of Open Access Journals (Sweden)

    David F Gruber

    Full Text Available We report the identification and characterization of two new members of a family of bilirubin-inducible fluorescent proteins (FPs from marine chlopsid eels and demonstrate a key region of the sequence that serves as an evolutionary switch from non-fluorescent to fluorescent fatty acid-binding proteins (FABPs. Using transcriptomic analysis of two species of brightly fluorescent Kaupichthys eels (Kaupichthys hyoproroides and Kaupichthys n. sp., two new FPs were identified, cloned and characterized (Chlopsid FP I and Chlopsid FP II. We then performed phylogenetic analysis on 210 FABPs, spanning 16 vertebrate orders, and including 163 vertebrate taxa. We show that the fluorescent FPs diverged as a protein family and are the sister group to brain FABPs. Our results indicate that the evolution of this family involved at least three gene duplication events. We show that fluorescent FABPs possess a unique, conserved tripeptide Gly-Pro-Pro sequence motif, which is not found in non-fluorescent fatty acid binding proteins. This motif arose from a duplication event of the FABP brain isoforms and was under strong purifying selection, leading to the classification of this new FP family. Residues adjacent to the motif are under strong positive selection, suggesting a further refinement of the eel protein's fluorescent properties. We present a phylogenetic reconstruction of this emerging FP family and describe additional fluorescent FABP members from groups of distantly related eels. The elucidation of this class of fish FPs with diverse properties provides new templates for the development of protein-based fluorescent tools. The evolutionary adaptation from fatty acid-binding proteins to fluorescent fatty acid-binding proteins raises intrigue as to the functional role of bright green fluorescence in this cryptic genus of reclusive eels that inhabit a blue, nearly monochromatic, marine environment.

  11. Development of a novel DDS for site-specific PEGylated proteins

    Directory of Open Access Journals (Sweden)

    Yoshioka Yasuo

    2011-05-01

    Full Text Available Abstract Because of the shifted focus in life science research from genome analyses to genetic and protein function analyses, we now know functions of numerous proteins. These analyses, including those of newly identified proteins, are expected to contribute to the identification of proteins of therapeutic value in various diseases. Consequently, pharmacoproteomic-based drug discovery and development of protein therapies attracted a great deal of attention in recent years. Clinical applications of most of these proteins are, however, limited because of their unexpectedly low therapeutic effects, resulting from the proteolytic degradation in vivo followed by rapid removal from the circulatory system. Therefore, frequent administration of excessively high dose of a protein is required to observe its therapeutic effect in vivo. This often results in impaired homeostasis in vivo and leads to severe adverse effects. To overcome these problems, we have devised a method for chemical modification of proteins with polyethylene glycol (PEGylation and other water-soluble polymers. In addition, we have established a method for creating functional mutant proteins (muteins with desired properties, and developed a site-specific polymer-conjugation method to further improve their therapeutic potency. In this review, we are introducing our original protein-drug innovation system mentioned above.

  12. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

    Directory of Open Access Journals (Sweden)

    Stefan M Ivanov

    Full Text Available An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  13. Protein-protein interactions in paralogues: Electrostatics modulates specificity on a conserved steric scaffold.

    Science.gov (United States)

    Ivanov, Stefan M; Cawley, Andrew; Huber, Roland G; Bond, Peter J; Warwicker, Jim

    2017-01-01

    An improved knowledge of protein-protein interactions is essential for better understanding of metabolic and signaling networks, and cellular function. Progress tends to be based on structure determination and predictions using known structures, along with computational methods based on evolutionary information or detailed atomistic descriptions. We hypothesized that for the case of interactions across a common interface, between proteins from a pair of paralogue families or within a family of paralogues, a relatively simple interface description could distinguish between binding and non-binding pairs. Using binding data for several systems, and large-scale comparative modeling based on known template complex structures, it is found that charge-charge interactions (for groups bearing net charge) are generally a better discriminant than buried non-polar surface. This is particularly the case for paralogue families that are less divergent, with more reliable comparative modeling. We suggest that electrostatic interactions are major determinants of specificity in such systems, an observation that could be used to predict binding partners.

  14. Mass spectrometric identification of proteins that interact through specific domains of the poly(A) binding protein

    DEFF Research Database (Denmark)

    Richardson, Roy; Denis, Clyde L; Zhang, Chongxu

    2012-01-01

    previously known direct interactions with specific PAB1 domains were either confirmed, delimited, or extended. The remaining nine proteins that interacted through a specific PAB1 domain were CBF5, SLF1, UPF1, CBC1, SSD1, NOP77, yGR250c, NAB6, and GBP2. In further study, UPF1, involved in nonsense...

  15. Sex-specific local life-history adaptation in surface- and cave-dwelling Atlantic mollies (Poecilia mexicana).

    Science.gov (United States)

    Riesch, Rüdiger; Reznick, David N; Plath, Martin; Schlupp, Ingo

    2016-03-10

    Cavefishes have long been used as model organisms showcasing adaptive diversification, but does adaptation to caves also facilitate the evolution of reproductive isolation from surface ancestors? We raised offspring of wild-caught surface- and cave-dwelling ecotypes of the neotropical fish Poecilia mexicana to sexual maturity in a 12-month common garden experiment. Fish were raised under one of two food regimes (high vs. low), and this was crossed with differences in lighting conditions (permanent darkness vs. 12:12 h light:dark cycle) in a 2 × 2 factorial design, allowing us to elucidate potential patterns of local adaptation in life histories. Our results reveal a pattern of sex-specific local life-history adaptation: Surface molly females had the highest fitness in the treatment best resembling their habitat of origin (high food and a light:dark cycle), and suffered from almost complete reproductive failure in darkness, while cave molly females were not similarly affected in any treatment. Males of both ecotypes, on the other hand, showed only weak evidence for local adaptation. Nonetheless, local life-history adaptation in females likely contributes to ecological diversification in this system and other cave animals, further supporting the role of local adaptation due to strong divergent selection as a major force in ecological speciation.

  16. Identification of the Specific Interactors of the Human Lariat RNA Debranching Enzyme 1 Protein

    Directory of Open Access Journals (Sweden)

    So Masaki

    2015-02-01

    Full Text Available In eukaryotes, pre-mRNA splicing is an essential step for gene expression. We have been analyzing post-splicing intron turnover steps in higher eukaryotes. Here, we report protein interaction between human Debranching enzyme 1 (hDbr1 and several factors found in the Intron Large (IL complex, which is an intermediate complex of the intron degradation pathway. The hDbr1 protein specifically interacts with xeroderma pigmentosum, complementeation group A (XPA-binding protein 2 (Xab2. We also attempted to identify specific interactors of hDbr1. Co-immunoprecipitation experiments followed by mass spectrometry analysis identified a novel protein as one of the specific interactors of hDbr1. This protein is well conserved among many species and shows the highest similarity to yeast Drn1, so it is designated as human Dbr1 associated ribonuclease 1 (hDrn1. hDrn1 directly interacts with hDbr1 through protein–protein interaction. Furthermore, hDrn1 shuttles between the nucleus and the cytoplasm, as hDbr1 protein does. These findings suggest that hDrn1 has roles in both the nucleus and the cytoplasm, which are highly likely to involve hDbr1.

  17. Biofilm-specific extracellular matrix proteins of non-typeable Haemophilus influenzae

    Science.gov (United States)

    Wu, Siva; Baum, Marc M.; Kerwin, James; Guerrero-Given, Debbie; Webster, Simon; Schaudinn, Christoph; VanderVelde, David; Webster, Paul

    2014-01-01

    Non-typeable Haemophilus influenzae (NTHi), a human respiratory tract pathogen can form colony biofilms in vitro. Bacterial cells and the amorphous extracellular matrix (ECM) constituting the biofilm can be separated using sonication. The ECM from 24 hr and 96 hr NTHi biofilms contained polysaccharides and proteinaceous components as detected by NMR and FTIR spectroscopy. More conventional chemical assays on the biofilm ECM confirmed the presence of these components and also DNA. Proteomics revealed eighteen proteins present in biofilm ECM that were not detected in planktonic bacteria. One ECM protein was unique to 24 hr biofilms, two were found only in 96 hr biofilms, and fifteen were present in the ECM of both 24 hr and 96 hr NTHi biofilms. All proteins identified were either associated with bacterial membranes or were cytoplasmic proteins. Immunocytochemistry showed two of the identified proteins, a DNA-directed RNA polymerase and the outer membrane protein OMP P2, associated with bacteria and biofilm ECM. Identification of biofilm-specific proteins present in immature biofilms is an important step in understanding the in vitro process of NTHi biofilm formation. The presence of a cytoplasmic protein and a membrane protein in the biofilm ECM of immature NTHi biofilms suggests that bacterial cell lysis may be a feature of early biofilm formation. PMID:24942343

  18. Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains.

    Science.gov (United States)

    Privat, Nicolas; Levavasseur, Etienne; Yildirim, Serfildan; Hannaoui, Samia; Brandel, Jean-Philippe; Laplanche, Jean-Louis; Béringue, Vincent; Seilhean, Danielle; Haïk, Stéphane

    2017-10-06

    Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrP Sc ). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrP Sc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. The specificity of interactions between proteins and sulfated polysaccharides

    Directory of Open Access Journals (Sweden)

    Barbara Mulloy

    2005-12-01

    Full Text Available Sulfated polysaccharides are capable of binding with proteins at several levels of specificity. As highly acidic macromolecules, they can bind non-specifically to any basic patch on a protein surface at low ionic strength, and such interactions are not likely to be physiologically significant. On the other hand, several systems have been identified in which very specific substructures of sulfated polysaccharides confer high affinity for particular proteins; the best-known example of this is the pentasaccharide in heparin with high affinity for antithrombin, but other examples may be taken from the study of marine invertebrates: the importance of the fine structure of dermatan sulfate (DS to its interaction with heparin cofactor II (HCII, and the involvement of sea urchin egg-jelly fucans in species specific fertilization. A third, intermediate, kind of specific interaction is described for the cell-surface glycosaminoglycan heparan sulfate (HS, in which patterns of sulfate substitution can show differential affinities for cytokines, growth factors, and morphogens at cell surfaces and in the intracellular matrix. This complex interplay of proteins and glycans is capable of influencing the diffusion of such proteins through tissue, as well as modulating cellular responses to them.Os polissacarídeos sulfatados são capazes de se ligar às proteínas com diferentes níveis de especificidade. São macromoléculas altamente ácidas que podem se ligar de forma inespecífica a qualquer domínio básico da superfície de uma proteína em soluções com baixa força iônica, contudo tais interações não parecem ser fisiologicamente significativas. Por outro lado, foram identificados vários sistemas nos quais componentes estruturais muito específicos dos polissacarídeos sulfatados conferem alta afinidade para algumas proteínas. O exemplo mais conhecido é o pentassacarídeo da heparina com alta afinidade pela antitrombina. Outros exemplos podem ser

  20. Nutrition and training adaptations in aquatic sports.

    Science.gov (United States)

    Mujika, Iñigo; Stellingwerff, Trent; Tipton, Kevin

    2014-08-01

    The adaptive response to training is determined by the combination of the intensity, volume, and frequency of the training. Various periodized approaches to training are used by aquatic sports athletes to achieve performance peaks. Nutritional support to optimize training adaptations should take periodization into consideration; that is, nutrition should also be periodized to optimally support training and facilitate adaptations. Moreover, other aspects of training (e.g., overload training, tapering and detraining) should be considered when making nutrition recommendations for aquatic athletes. There is evidence, albeit not in aquatic sports, that restricting carbohydrate availability may enhance some training adaptations. More research needs to be performed, particularly in aquatic sports, to determine the optimal strategy for periodizing carbohydrate intake to optimize adaptations. Protein nutrition is an important consideration for optimal training adaptations. Factors other than the total amount of daily protein intake should be considered. For instance, the type of protein, timing and pattern of protein intake and the amount of protein ingested at any one time influence the metabolic response to protein ingestion. Body mass and composition are important for aquatic sport athletes in relation to power-to-mass and for aesthetic reasons. Protein may be particularly important for athletes desiring to maintain muscle while losing body mass. Nutritional supplements, such as b-alanine and sodium bicarbonate, may have particular usefulness for aquatic athletes' training adaptation.

  1. Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

    Science.gov (United States)

    Getzenberg, R H; Coffey, D S

    1990-09-01

    The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

  2. Adaptive response to high LET radiations

    International Nuclear Information System (INIS)

    Dam, Annamaria; Bogdandi, E. Noemi; Polonyi, Istvan; Sardy, M. Marta; Balashazy, Imre; Palfalvy, Jozsef

    2001-01-01

    The biological consequences of exposure to ionizing radiation include gene mutation, chromosome aberrations, cellular transformation and cell death. These effects are attributed to the DNA damaging effects of the irradiation resulting in irreversible changes during DNA replication or during the processing of the DNA damage by enzymatic repair processes. These repair processes could initiate some adaptive mechanisms in the cell, which could lead to radioadaptive response (RAR). Adaptive responses have typically been detected by exposing cells to a low radiation dose (1-50 mGy) and then challenging the cells with a higher dose of radiation (2-4 Gy) and comparing the outcome to that seen with the challenge dose only. For adaptive response to be seen the challenge dose must be delivered within 24 hour of the inducing dose. Radio-adaptation is extensively studied for low LET radiation. Nevertheless, few data are available for high LET radiation at very low doses and dose rate. Our study was aimed to investigate the radioadaptive response to low-dose neutron irradiation by detection of the genotoxic damage i.e.: hprt-mutant colonies induced. Altered protein synthesis was also studied to identify stress proteins may responsible for radio-adaptation. New alpha particle irradiator system was also built up to study the biological effects of low dose alpha irradiation. The experiments were carried out on monolayers of human melanoma and CHO (Chines Hamster Ovary) cells irradiated by neutrons produced in the biological irradiation channel of the Research Reactor of Budapest Neutron Center. Cells were exposed to 0.5-50 mGy neutron doses with dose rates of 1.59-10 mGy/min. The challenge doses of 2-4 Gy gamma rays were administrated within 1-48 hours after priming treatment. The induced mutants at hprt locus were selected by adding 6-thioguanine and allow to grow for 10 days for expression of the phenotype. The protein synthesis was studied by PAGE, the molecular mass of specific

  3. Shape-specific nanostructured protein mimics from de novo designed chimeric peptides.

    Science.gov (United States)

    Jiang, Linhai; Yang, Su; Lund, Reidar; Dong, He

    2018-01-30

    Natural proteins self-assemble into highly-ordered nanoscaled architectures to perform specific functions. The intricate functions of proteins have provided great impetus for researchers to develop strategies for designing and engineering synthetic nanostructures as protein mimics. Compared to the success in engineering fibrous protein mimetics, the design of discrete globular protein-like nanostructures has been challenging mainly due to the lack of precise control over geometric packing and intermolecular interactions among synthetic building blocks. In this contribution, we report an effective strategy to construct shape-specific nanostructures based on the self-assembly of chimeric peptides consisting of a coiled coil dimer and a collagen triple helix folding motif. Under salt-free conditions, we showed spontaneous self-assembly of the chimeric peptides into monodisperse, trigonal bipyramidal-like nanoparticles with precise control over the stoichiometry of two folding motifs and the geometrical arrangements relative to one another. Three coiled coil dimers are interdigitated on the equatorial plane while the two collagen triple helices are located in the axial position, perpendicular to the coiled coil plane. A detailed molecular model was proposed and further validated by small angle X-ray scattering experiments and molecular dynamics (MD) simulation. The results from this study indicated that the molecular folding of each motif within the chimeric peptides and their geometric packing played important roles in the formation of discrete protein-like nanoparticles. The peptide design and self-assembly mechanism may open up new routes for the construction of highly organized, discrete self-assembling protein-like nanostructures with greater levels of control over assembly accuracy.

  4. Novel Cold-Adapted Esterase MHlip from an Antarctic Soil Metagenome

    Directory of Open Access Journals (Sweden)

    Moreno Galleni

    2013-01-01

    Full Text Available An Antarctic soil metagenomic library was screened for lipolytic enzymes and allowed for the isolation of a new cytosolic esterase from the a/b hydrolase family 6, named MHlip. This enzyme is related to hypothetical genes coding esterases, aryl-esterases and peroxydases, among others. MHlip was produced, purified and its activity was determined. The substrate profile of MHlip reveals a high specificity for short p-nitrophenyl-esters. The apparent optimal activity of MHlip was measured for p-nitrophenyl-acetate, at 33 °C, in the pH range of 6–9. The MHlip thermal unfolding was investigated by spectrophotometric methods, highlighting a transition (Tm at 50 °C. The biochemical characterization of this enzyme showed its adaptation to cold temperatures, even when it did not present evident signatures associated with cold-adapted proteins. Thus, MHlip adaptation to cold probably results from many discrete structural modifications, allowing the protein to remain active at low temperatures. Functional metagenomics is a powerful approach to isolate new enzymes with tailored biophysical properties (e.g., cold adaptation. In addition, beside the ever growing amount of sequenced DNA, the functional characterization of new catalysts derived from environment is still required, especially for poorly characterized protein families like α/b hydrolases.

  5. Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

    Directory of Open Access Journals (Sweden)

    Snoussi Sarra

    2012-02-01

    Full Text Available Abstract Background Salmonella enterica serovar Hadar (S. Hadar is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal, in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF, in the oxidative stress status (bacterioferritin, in virulence (OmpX, Yfgl or in motility (FlgE and UspF. Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms.

  6. Origins of Allostery and Evolvability in Proteins: A Case Study.

    Science.gov (United States)

    Raman, Arjun S; White, K Ian; Ranganathan, Rama

    2016-07-14

    Proteins display the capacity for adaptation to new functions, a property critical for evolvability. But what structural principles underlie the capacity for adaptation? Here, we show that adaptation to a physiologically distinct class of ligand specificity in a PSD95, DLG1, ZO-1 (PDZ) domain preferentially occurs through class-bridging intermediate mutations located distant from the ligand-binding site. These mutations provide a functional link between ligand classes and demonstrate the principle of "conditional neutrality" in mediating evolutionary adaptation. Structures show that class-bridging mutations work allosterically to open up conformational plasticity at the active site, permitting novel functions while retaining existing function. More generally, the class-bridging phenotype arises from mutations in an evolutionarily conserved network of coevolving amino acids in the PDZ family (the sector) that connects the active site to distant surface sites. These findings introduce the concept that allostery in proteins could have its origins not in protein function but in the capacity to adapt. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Accumulation of small heat shock proteins, including mitochondrial HSP22, induced by oxidative stress and adaptive response in tomato cells

    International Nuclear Information System (INIS)

    Banzet, N.; Richaud, C.; Deveaux, Y.; Kazmaier, M.; Gagnon, J.; Triantaphylides, C.

    1998-01-01

    Changes in gene expression, by application of H2O2, O2.- generating agents (methyl viologen, digitonin) and gamma irradiation to tomato suspension cultures, were investigated and compared to the well-described heat shock response. Two-dimensional gel protein mapping analyses gave the first indication that at least small heat shock proteins (smHSP) accumulated in response to application of H2O2 and gamma irradiation, but not to O2.- generating agents. While some proteins seemed to be induced specifically by each treatment, only part of the heat shock response was observed. On the basis of Northern hybridization experiments performed with four heterologous cDNA, corresponding to classes I-IV of pea smHSP, it could be concluded that significant amounts of class I and II smHSP mRNA are induced by H2O2 and by irradiation. Taken together, these results demonstrate that in plants some HSP genes are inducible by oxidative stresses, as in micro-organisms and other eukaryotic cells. HSP22, the main stress protein that accumulates following H2O2 action or gamma irradiation, was also purified. Sequence homology of amino terminal and internal sequences, and immunoreactivity with Chenopodium rubrum mitochondrial smHSP antibody, indicated that the protein belongs to the recently discovered class of plant mitochondrial smHSP. Heat shock or a mild H2O2 pretreatment was also shown to lead to plant cell protection against oxidative injury. Therefore, the synthesis of these stress proteins can be considered as an adaptive mechanism in which mitochondrial protection could be essential

  8. Emerging functions of ribosomal proteins in gene-specific transcription and translation

    International Nuclear Information System (INIS)

    Lindstroem, Mikael S.

    2009-01-01

    Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-κB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

  9. Specification and Generation of Adapters for System Integration

    NARCIS (Netherlands)

    Mooij, A.J.; Voorhoeve, M.

    2013-01-01

    Large systems-of-systems are developed by integrating several smaller systems that have been developed independently. System integration often requires adaptation mechanisms for bridging any technical incompatibilities between the systems. In order to develop adapters in a faster way, we study ways

  10. Evidence of Molecular Adaptation to Extreme Environments and Applicability to Space Environments

    Directory of Open Access Journals (Sweden)

    Filipović, M. D.

    2008-06-01

    Full Text Available This is initial investigation of gene signatures responsible for adapting microscopic life to the extreme Earth environments. We present preliminary results on identification of the clusters of orthologous groups (COGs common to several hyperthermophiles and exclusion of those common to a mesophile (non-hyperthermophile: {it Escherichia coli (E. coli K12}, will yield a group of proteins possibly involved in adaptation to life under extreme temperatures. Comparative genome analyses represent a powerful tool in discovery of novel genes responsible for adaptation to specific extreme environments. Methanogens stand out as the only group of organisms that have species capable of growth at 0D C ({it Metarhizium frigidum (M.~frigidum} and {it Methanococcoides burtonii (M.~burtonii} and 110D C ({it Methanopyrus kandleri (M.~kandleri}. Although not all the components of heat adaptation can be attributed to novel genes, the {it chaperones} known as heat shock proteins stabilize the enzymes under elevated temperature. However, highly conserved {it chaperons} found in bacteria and eukaryots are not present in hyperthermophilic Archea, rather, they have a unique {it chaperone TF55}. Our aim was to use software which we specifically developed for extremophile genome comparative analyses in order to search for additional novel genes involved in hyperthermophile adaptation. The followinghyperthermophile genomes incorporated in this software were used forthese studies: {it Methanocaldococcus jannaschii (M.~jannaschii, M.~kandleri, Archaeoglobus fulgidus (A.~fulgidus} and threespecies of {it Pyrococcus}. Common genes were annotated and groupedaccording to their roles in cellular processes where such informationwas available and proteins not previously implicated in theheat-adaptation of hyperthermophiles were identified. Additionalexperimental data are needed in order to learn more about theseproteins. To address non-gene based components of thermaladaptation

  11. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor

    Science.gov (United States)

    Zhang, Xirui; Daaboul, George G.; Spuhler, Philipp S.; Dröge, Peter; Ünlü, M. Selim

    2016-03-01

    DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood. Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein-DNA complexes. Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate. In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein-DNA interactions.DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are

  12. Clinical importance of non-specific lipid transfer proteins as food allergens

    NARCIS (Netherlands)

    van Ree, R.

    2002-01-01

    Non-specific lipid transfer proteins (nsLTPs) have recently been identified as plant food allergens. They are good examples of true food allergens, in the sense that they are capable of sensitizing, i.e. inducing specific IgE, as well as of eliciting severe symptoms. This is in contrast with most

  13. Personalized disease-specific protein corona influences the therapeutic impact of graphene oxide

    Science.gov (United States)

    Hajipour, Mohammad Javad; Raheb, Jamshid; Akhavan, Omid; Arjmand, Sareh; Mashinchian, Omid; Rahman, Masoud; Abdolahad, Mohammad; Serpooshan, Vahid; Laurent, Sophie; Mahmoudi, Morteza

    2015-05-01

    The hard corona, the protein shell that is strongly attached to the surface of nano-objects in biological fluids, is recognized as the first layer that interacts with biological objects (e.g., cells and tissues). The decoration of the hard corona (i.e., the type, amount, and conformation of the attached proteins) can define the biological fate of the nanomaterial. Recent developments have revealed that corona decoration strongly depends on the type of disease in human patients from which the plasma is obtained as a protein source for corona formation (referred to as the `personalized protein corona'). In this study, we demonstrate that graphene oxide (GO) sheets can trigger different biological responses in the presence of coronas obtained from various types of diseases. GO sheets were incubated with plasma from human subjects with different diseases/conditions, including hypofibrinogenemia, blood cancer, thalassemia major, thalassemia minor, rheumatism, fauvism, hypercholesterolemia, diabetes, and pregnancy. Identical sheets coated with varying protein corona decorations exhibited significantly different cellular toxicity, apoptosis, and uptake, reactive oxygen species production, lipid peroxidation and nitrogen oxide levels. The results of this report will help researchers design efficient and safe, patient-specific nano biomaterials in a disease type-specific manner for clinical and biological applications.The hard corona, the protein shell that is strongly attached to the surface of nano-objects in biological fluids, is recognized as the first layer that interacts with biological objects (e.g., cells and tissues). The decoration of the hard corona (i.e., the type, amount, and conformation of the attached proteins) can define the biological fate of the nanomaterial. Recent developments have revealed that corona decoration strongly depends on the type of disease in human patients from which the plasma is obtained as a protein source for corona formation (referred

  14. Specific DNA-binding proteins and DNA sequences involved in steroid hormone regulation of gene expression

    International Nuclear Information System (INIS)

    Spelsberg, T.; Hora, J.; Horton, M.; Goldberger, A.; Littlefield, B.; Seelke, R.; Toyoda, H.

    1987-01-01

    Steroid hormones circulate in the blood and are taken by target cells via complexes with intracellular binding proteins termed receptors, that are hormone and tissue specific. Each receptor binds it specific steroid with very high affinity, having an equilibrium dissociation constant (K/sub d/) in the range of 10 -9 to 10 -10 M. Once bound by their specific steroid hormones, the steroid receptors undergo a conformational change which allows them to bind with high affinity to sites on chromatin, termed nuclear acceptor sites. There are estimated 5,000 to 10,000 of these sites expressed with an equal number not expressed (''masked'') in intact chromatin. The result of the binding to nuclear acceptor sites is an alteration of gene transcription or, in some cases, gene expression as measured by the changing levels of specific RNAs and proteins in that target tissue. Each steroid regulates specific effects on the RNA and protein profiles. The chronology of the above mechanism of action after injection of radiolabelled steroid as is follows: Steroid-receptor complex formation (1 minute), nuclear acceptor sites (2 minutes), effects on RNA synthesis (10 to 30 minutes), and finally the changing protein profiles via changes in protein synthesis and protein turnover (1 to 6 hours). Thus steroid receptors represent one of the first identified intracellular gene regulation proteins. The receptor molecules themselves are regulated by the presence or absence of the steroid molecule

  15. Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme.

    Science.gov (United States)

    Ekman, Martin; Sandh, Gustaf; Nenninger, Anja; Oliveira, Paulo; Stensjö, Karin

    2014-03-01

    Ferritin-like proteins constitute a remarkably heterogeneous protein family, including ferritins, bacterioferritins and Dps proteins. The genome of the filamentous heterocyst-forming cyanobacterium Nostoc punctiforme encodes five ferritin-like proteins. In the present paper, we report a multidimensional characterization of these proteins. Our phylogenetic and bioinformatics analyses suggest both structural and physiological differences among the ferritin-like proteins. The expression of these five genes responded differently to hydrogen peroxide treatment, with a significantly higher rise in transcript level for Npun_F3730 as compared with the other four genes. A specific role for Npun_F3730 in the cells tolerance against hydrogen peroxide was also supported by the inactivation of Npun_F3730, Npun_R5701 and Npun_R6212; among these, only the ΔNpun_F3730 strain showed an increased sensitivity to hydrogen peroxide compared with wild type. Analysis of promoter-GFP reporter fusions of the ferritin-like genes indicated that Npun_F3730 and Npun_R5701 were expressed in all cell types of a diazotrophic culture, while Npun_F6212 was expressed specifically in heterocysts. Our study provides the first comprehensive analysis combining functional differentiation and cellular specificity within this important group of proteins in a multicellular cyanobacterium. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Molecular adaptation during adaptive radiation in the Hawaiian endemic genus Schiedea.

    Directory of Open Access Journals (Sweden)

    Maxim V Kapralov

    2006-12-01

    Full Text Available "Explosive" adaptive radiations on islands remain one of the most puzzling evolutionary phenomena. The rate of phenotypic and ecological adaptations is extremely fast during such events, suggesting that many genes may be under fairly strong selection. However, no evidence for adaptation at the level of protein coding genes was found, so it has been suggested that selection may work mainly on regulatory elements. Here we report the first evidence that positive selection does operate at the level of protein coding genes during rapid adaptive radiations. We studied molecular adaptation in Hawaiian endemic plant genus Schiedea (Caryophyllaceae, which includes closely related species with a striking range of morphological and ecological forms, varying from rainforest vines to woody shrubs growing in desert-like conditions on cliffs. Given the remarkable difference in photosynthetic performance between Schiedea species from different habitats, we focused on the "photosynthetic" Rubisco enzyme, the efficiency of which is known to be a limiting step in plant photosynthesis.We demonstrate that the chloroplast rbcL gene, encoding the large subunit of Rubisco enzyme, evolved under strong positive selection in Schiedea. Adaptive amino acid changes occurred in functionally important regions of Rubisco that interact with Rubisco activase, a chaperone which promotes and maintains the catalytic activity of Rubisco. Interestingly, positive selection acting on the rbcL might have caused favorable cytotypes to spread across several Schiedea species.We report the first evidence for adaptive changes at the DNA and protein sequence level that may have been associated with the evolution of photosynthetic performance and colonization of new habitats during a recent adaptive radiation in an island plant genus. This illustrates how small changes at the molecular level may change ecological species performance and helps us to understand the molecular bases of extremely

  17. Mitochondrial protein acetylation mediates nutrient sensing of mitochondrial protein synthesis and mitonuclear protein balance.

    Science.gov (United States)

    Di Domenico, Antonella; Hofer, Annette; Tundo, Federica; Wenz, Tina

    2014-11-01

    Changes in nutrient supply require global metabolic reprogramming to optimize the utilization of the nutrients. Mitochondria as a central component of the cellular metabolism play a key role in this adaptive process. Since mitochondria harbor their own genome, which encodes essential enzymes, mitochondrial protein synthesis is a determinant of metabolic adaptation. While regulation of cytoplasmic protein synthesis in response to metabolic challenges has been studied in great detail, mechanisms which adapt mitochondrial translation in response to metabolic challenges remain elusive. Our results suggest that the mitochondrial acetylation status controlled by Sirt3 and its proposed opponent GCN5L1 is an important regulator of the metabolic adaptation of mitochondrial translation. Moreover, both proteins modulate regulators of cytoplasmic protein synthesis as well as the mitonuclear protein balance making Sirt3 and GCN5L1 key players in synchronizing mitochondrial and cytoplasmic translation. Our results thereby highlight regulation of mitochondrial translation as a novel component in the cellular nutrient sensing scheme and identify mitochondrial acetylation as a new regulatory principle for the metabolic competence of mitochondrial protein synthesis. © 2014 International Union of Biochemistry and Molecular Biology.

  18. Fat-specific protein 27 regulates storage of triacylglycerol

    DEFF Research Database (Denmark)

    Keller, P.; Petrie, J.T.; Rose, P. De

    2008-01-01

    FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins...... in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L...... decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement Udgivelsesdato: 2008/5/23...

  19. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Peng Liu

    2015-01-01

    Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

  20. Chromosome locations of genes encoding human signal transduction adapter proteins, Nck (NCK), Shc (SHC1), and Grb2 (GRB2)

    DEFF Research Database (Denmark)

    Huebner, K; Kastury, K; Druck, T

    1994-01-01

    "adapter" proteins, which are involved in transducing signals from receptor tyrosine kinases to downstream signal recipients such as ras, because adaptor protein genes could also, logically, serve as targets of mutation, rearrangement, or other aberration in disease. Therefore, DNAs from panels of rodent-human......Abnormalities due to chromosomal aberration or point mutation in gene products of growth factor receptors or in ras gene products, which lie on the same signaling pathway, can cause disease in animals and humans. Thus, it can be important to determine chromosomal map positions of genes encoding...... hybrids carrying defined complements of human chromosomes were assayed for the presence of the cognate genes for NCK, SHC, and GRB2, three SH2 or SH2/SH3 (Src homology 2 and 3) domain-containing adapter proteins. Additionally, NCK and SHC genes were more narrowly localized by chromosomal in situ...

  1. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    International Nuclear Information System (INIS)

    Guo Junqing; Chen Liu; Ai Hongwu; Jing Jiannian; Zhou Jiyong; Zhang Chuyu; You Shangyou

    2004-01-01

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-γ inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V H region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo

  2. Gene expression of a green fluorescent protein homolog as a host-specific biomarker of heat stress within a reef-building coral.

    Science.gov (United States)

    Smith-Keune, C; Dove, S

    2008-01-01

    Recent incidences of mass coral bleaching indicate that major reef building corals are increasingly suffering thermal stress associated with climate-related temperature increases. The development of pulse amplitude modulated (PAM) fluorometry has enabled rapid detection of the onset of thermal stress within coral algal symbionts, but sensitive biomarkers of thermal stress specific to the host coral have been slower to emerge. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to produce fingerprints of gene expression for the reef-building coral Acropora millepora exposed to 33 degrees C. Changes in the expression of 23 out of 399 putative genes occurred within 144 h. Down-regulation of one host-specific gene (AmA1a) occurred within just 6 h. Full-length sequencing revealed the product of this gene to be an all-protein chromatophore (green fluorescent protein [GFP]-homolog). RT-PCR revealed consistent down-regulation of this GFP-homolog for three replicate colonies within 6 h at both 32 degrees C and 33 degrees C but not at lower temperatures. Down-regulation of this host gene preceded significant decreases in the photosynthetic activity of photosystem II (dark-adapted F (v)/F (m)) of algal symbionts as measured by PAM fluorometry. Gene expression of host-specific genes such as GFP-homologs may therefore prove to be highly sensitive indicators for the onset of thermal stress within host coral cells.

  3. Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform.

    Science.gov (United States)

    Hui, Jingjing; Bao, Lei; Li, Siqiao; Zhang, Yi; Feng, Yimei; Ding, Lin; Ju, Huangxian

    2017-07-03

    Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Protein Detection with Aptamer Biosensors

    Directory of Open Access Journals (Sweden)

    Regina Stoltenburg

    2008-07-01

    Full Text Available Aptamers have been developed for different applications. Their use as new biological recognition elements in biosensors promises progress for fast and easy detection of proteins. This new generation of biosensor (aptasensors will be more stable and well adapted to the conditions of real samples because of the specific properties of aptamers.

  5. The effect of chloramphenicol on synthesis of ΦX 174-specific proteins and detection of the cistron A protein

    NARCIS (Netherlands)

    Mei, D. Van Der; Zandberg, J.; Jansz, H.S.

    1972-01-01

    Synthesis of ΦX 174-specific proteins in Escherichia coli H 502 was examined on sodium dodecyl sulphate-acrylamide gels by coelectrophoresis of proteins from [3H]leucine-labelled infected cells and [14C]leucine-labelled reference cells, which had been infected with ultraviolet-light irradiated

  6. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  7. Acetylation-Dependent Chromatin Reorganization by BRDT, a Testis-Specific Bromodomain-Containing Protein

    Science.gov (United States)

    Pivot-Pajot, Christophe; Caron, Cécile; Govin, Jérôme; Vion, Alexandre; Rousseaux, Sophie; Khochbin, Saadi

    2003-01-01

    The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor. PMID:12861021

  8. Neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

    International Nuclear Information System (INIS)

    Stevenson, K.B.; Nauseef, W.M.; Clark, R.A.

    1987-01-01

    The glucoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3 H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody. Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove adsorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils

  9. Protein-free transfection of CHO host cells with an IgG-fusion protein: selection and characterization of stable high producers and comparison to conventionally transfected clones.

    Science.gov (United States)

    Lattenmayer, Christine; Loeschel, Martina; Schriebl, Kornelia; Steinfellner, Willibald; Sterovsky, Thomas; Trummer, Evelyn; Vorauer-Uhl, Karola; Müller, Dethardt; Katinger, Hermann; Kunert, Renate

    2007-04-15

    In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development. (c) 2006 Wiley Periodicals, Inc.

  10. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    OpenAIRE

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

  11. Generalized theory on the mechanism of site-specific DNA-protein interactions

    Science.gov (United States)

    Niranjani, G.; Murugan, R.

    2016-05-01

    We develop a generalized theoretical framework on the binding of transcription factor proteins (TFs) with specific sites on DNA that takes into account the interplay of various factors regarding overall electrostatic potential at the DNA-protein interface, occurrence of kinetic traps along the DNA sequence, presence of other roadblock protein molecules along DNA and crowded environment, conformational fluctuations in the DNA binding domains (DBDs) of TFs, and the conformational state of the DNA. Starting from a Smolochowski type theoretical framework on site-specific binding of TFs we logically build our model by adding the effects of these factors one by one. Our generalized two-step model suggests that the electrostatic attractive forces present inbetween the positively charged DBDs of TFs and the negatively charged phosphate backbone of DNA, along with the counteracting shielding effects of solvent ions, is the core factor that creates a fluidic type environment at the DNA-protein interface. This in turn facilitates various one-dimensional diffusion (1Dd) processes such as sliding, hopping and intersegmental transfers. These facilitating processes as well as flipping dynamics of conformational states of DBDs of TFs between stationary and mobile states can enhance the 1Dd coefficient on a par with three-dimensional diffusion (3Dd). The random coil conformation of DNA also plays critical roles in enhancing the site-specific association rate. The extent of enhancement over the 3Dd controlled rate seems to be directly proportional to the maximum possible 1Dd length. We show that the overall site-specific binding rate scales with the length of DNA in an asymptotic way. For relaxed DNA, the specific binding rate will be independent of the length of DNA as length increases towards infinity. For condensed DNA as in in vivo conditions, the specific binding rate depends on the length of DNA in a turnover way with a maximum. This maximum rate seems to scale with the

  12. Species specificity in major urinary proteins by parallel evolution.

    Directory of Open Access Journals (Sweden)

    Darren W Logan

    Full Text Available Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1 diversity, to enable the signaling of multiple behaviors, 2 dynamic regulation, to indicate age and dominance, and 3 species-specificity. Recently, the major urinary proteins (Mups have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues.Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species-specific

  13. A novel fusion protein of IP10-scFv retains antibody specificity and chemokine function

    Energy Technology Data Exchange (ETDEWEB)

    Junqing, Guo; Liu, Chen; Hongwu, Ai; Jiannian, Jing; Jiyong, Zhou; Chuyu, Zhang; Shangyou, You

    2004-07-23

    We combined the specificity of tumor-specific antibody with the chemokine function of interferon-{gamma} inducible protein 10 (IP-10) to recruit immune effector cells in the vicinity of tumor cells. A novel fusion protein of IP10-scFv was constructed by fusing mouse IP-10 to V{sub H} region of single-chain Fv fragment (scFv) against acidic isoferritin (AIF), and expressed in NS0 murine myeloma cells. The IP10-scFv fusion protein was shown to maintain the specificity of the antiAIF scFv with similar affinity constant, and bind to the human hepatocarcinoma SMMC 7721 cells secreting AIF as well as the activated mouse T lymphocytes expressing CXCR3 receptor. Furthermore, the IP10-scFv protein either in solution or bound on the surface of SMMC 7721 cells induced significant chemotaxis of mouse T cells in vitro. The results indicate that the IP10-scFv fusion protein possesses both bioactivities of the tumor-specific antibody and IP-10 chemokine, suggesting its possibility to induce an enhanced immune response against the residual tumor cells in vivo.

  14. Genome-wide analysis of protein disorder in Arabidopsis thaliana: implications for plant environmental adaptation.

    Science.gov (United States)

    Pietrosemoli, Natalia; García-Martín, Juan A; Solano, Roberto; Pazos, Florencio

    2013-01-01

    Intrinsically disordered proteins/regions (IDPs/IDRs) are currently recognized as a widespread phenomenon having key cellular functions. Still, many aspects of the function of these proteins need to be unveiled. IDPs conformational flexibility allows them to recognize and interact with multiple partners, and confers them larger interaction surfaces that may increase interaction speed. For this reason, molecular interactions mediated by IDPs/IDRs are particularly abundant in certain types of protein interactions, such as those of signaling and cell cycle control. We present the first large-scale study of IDPs in Arabidopsis thaliana, the most widely used model organism in plant biology, in order to get insight into the biological roles of these proteins in plants. The work includes a comparative analysis with the human proteome to highlight the differential use of disorder in both species. Results show that while human proteins are in general more disordered, certain functional classes, mainly related to environmental response, are significantly more enriched in disorder in Arabidopsis. We propose that because plants cannot escape from environmental conditions as animals do, they use disorder as a simple and fast mechanism, independent of transcriptional control, for introducing versatility in the interaction networks underlying these biological processes so that they can quickly adapt and respond to challenging environmental conditions.

  15. Complete-proteome mapping of human influenza A adaptive mutations: implications for human transmissibility of zoonotic strains.

    Science.gov (United States)

    Miotto, Olivo; Heiny, A T; Albrecht, Randy; García-Sastre, Adolfo; Tan, Tin Wee; August, J Thomas; Brusic, Vladimir

    2010-02-03

    There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear

  16. Protein Stabilization and Enzyme Activation in Ionic Liquids: Specific Ion Effects

    Science.gov (United States)

    Zhao, Hua

    2015-01-01

    There are still debates on whether the hydration of ions perturbs the water structure, and what is the degree of such disturbance; therefore, the origin of Hofmeister effect on protein stabilization continues being questioned. For this reason, it is suggested to use the ‘specific ion effect’ instead of other misleading terms such as Hofmeister effect, Hofmeister series, lyotropic effect, and lyotropic series. In this review, we firstly discuss the controversial aspect of inorganic ion effects on water structures, and several possible contributors to the specific ion effect of protein stability. Due to recent overwhelming attraction of ionic liquids (ILs) as benign solvents in many enzymatic reactions, we further evaluate the structural properties and molecular-level interactions in neat ILs and their aqueous solutions. Next, we systematically compare the specific ion effects of ILs on enzyme stability and activity, and conclude that (a) the specificity of many enzymatic systems in diluted aqueous IL solutions is roughly in line with the traditional Hofmeister series albeit some exceptions; (b) however, the specificity follows a different track in concentrated or neat ILs because other factors (such as hydrogen-bond basicity, nucelophilicity, and hydrophobicity, etc) are playing leading roles. In addition, we demonstrate some examples of biocatalytic reactions in IL systems that are guided by the empirical specificity rule. PMID:26949281

  17. LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

    Science.gov (United States)

    Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

    2017-12-06

    An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

  18. Disruption of the acyl-coa binding protein gene delays hepatic adaptation to metabolic changes at weaning

    DEFF Research Database (Denmark)

    Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek

    2011-01-01

    The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an intracellular protein that binds C14-C22 acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however....... The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads...

  19. Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins.

    Science.gov (United States)

    Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

    2004-07-01

    Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

  20. Conformational adaptation of Asian macaque TRIMCyp directs lineage specific antiviral activity.

    Directory of Open Access Journals (Sweden)

    Laura M J Ylinen

    2010-08-01

    Full Text Available TRIMCyps are anti-retroviral proteins that have arisen independently in New World and Old World primates. All TRIMCyps comprise a CypA domain fused to the tripartite domains of TRIM5alpha but they have distinct lentiviral specificities, conferring HIV-1 restriction in New World owl monkeys and HIV-2 restriction in Old World rhesus macaques. Here we provide evidence that Asian macaque TRIMCyps have acquired changes that switch restriction specificity between different lentiviral lineages, resulting in species-specific alleles that target different viruses. Structural, thermodynamic and viral restriction analysis suggests that a single mutation in the Cyp domain, R69H, occurred early in macaque TRIMCyp evolution, expanding restriction specificity to the lentiviral lineages found in African green monkeys, sooty mangabeys and chimpanzees. Subsequent mutations have enhanced restriction to particular viruses but at the cost of broad specificity. We reveal how specificity is altered by a scaffold mutation, E143K, that modifies surface electrostatics and propagates conformational changes into the active site. Our results suggest that lentiviruses may have been important pathogens in Asian macaques despite the fact that there are no reported lentiviral infections in current macaque populations.

  1. Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

    Directory of Open Access Journals (Sweden)

    Martin H Schaefer

    2014-06-01

    Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

  2. Hominoid-specific de novo protein-coding genes originating from long non-coding RNAs.

    Directory of Open Access Journals (Sweden)

    Chen Xie

    2012-09-01

    Full Text Available Tinkering with pre-existing genes has long been known as a major way to create new genes. Recently, however, motherless protein-coding genes have been found to have emerged de novo from ancestral non-coding DNAs. How these genes originated is not well addressed to date. Here we identified 24 hominoid-specific de novo protein-coding genes with precise origination timing in vertebrate phylogeny. Strand-specific RNA-Seq analyses were performed in five rhesus macaque tissues (liver, prefrontal cortex, skeletal muscle, adipose, and testis, which were then integrated with public transcriptome data from human, chimpanzee, and rhesus macaque. On the basis of comparing the RNA expression profiles in the three species, we found that most of the hominoid-specific de novo protein-coding genes encoded polyadenylated non-coding RNAs in rhesus macaque or chimpanzee with a similar transcript structure and correlated tissue expression profile. According to the rule of parsimony, the majority of these hominoid-specific de novo protein-coding genes appear to have acquired a regulated transcript structure and expression profile before acquiring coding potential. Interestingly, although the expression profile was largely correlated, the coding genes in human often showed higher transcriptional abundance than their non-coding counterparts in rhesus macaque. The major findings we report in this manuscript are robust and insensitive to the parameters used in the identification and analysis of de novo genes. Our results suggest that at least a portion of long non-coding RNAs, especially those with active and regulated transcription, may serve as a birth pool for protein-coding genes, which are then further optimized at the transcriptional level.

  3. Interaction of hepatocyte nuclear factors in transcriptional regulation of tissue specific hormonal expression of human multidrug resistance-associated protein 2 (abcc2)

    International Nuclear Information System (INIS)

    Qadri, Ishtiaq; Hu, L.-J.; Iwahashi, Mieko; Al-Zuabi, Subhi; Quattrochi, Linda C.; Simon, Francis R.

    2009-01-01

    Multidrug resistance-associated protein 2 (MRP2) (ABCC2) is an ATP-binding cassette membrane protein located primarily on apical surface of hepatocytes that mediates transport of conjugated xenobiotics and endogenous compounds into bile. MRP2 is highly expressed in hepatocytes, and at lower levels in small intestines, stomach and kidney. Previous reports have characterized mammalian MRP2 promoters, but none have established the molecular mechanism(s) involved in liver enriched expression. This study aims to investigate the mechanism of hepatic MRP2 regulation. A 2130 bp of MRP2 promoter was cloned from PAC-1 clone P108G1-7, to identify putative liver specific/hormone responsive functional DNA binding sites. Using deletion analysis, site specific mutagenesis and co-transfection studies, liver specific expression was determined. MRP2 promoter-LUC constructs were highly expressed in liver cell lines compared to non-liver cells. The region extending from - 3 to+ 458 bp of MRP2 promoter starting from AUG contained the potential binding sites for CAAATT box enhancer binding protein (C/EBP), hepatocytes nuclear factor 1, 3 and 4 (HNF1, HNF3, and HNF4. Only HNF1 and HNF4 co-transfection with MRP2 luciferase increased expression. Site specific mutational analysis of HNF1 binding site indicated an important role for HNF1α. HNF4α induction of MRP2 was independent of HNF1 binding site. C/EBP, HNF3, and HNF6 inhibited HNF1α while HNF4α induced MRP2 luciferase expression and glucocorticoids stimulated MRP2 expression. This study emphasizes the complex regulation of MRP2 with HNF1α and HNF4α playing a central role. The coordinated regulation of xenobiotic transporters and oxidative conjugation may determine the adaptive responses to cellular detoxification processes

  4. Combinatorial synthesis and screening of cancer cell-specific nanomedicines targeted via phage fusion proteins

    Directory of Open Access Journals (Sweden)

    James W. Gillespie

    2015-06-01

    Full Text Available Active tumor targeting of nanomedicines has recently shown significant improvements in the therapeutic activity of currently existing drug delivery systems, such as liposomal doxorubicin (Doxil/Caelyx/Lipodox. Previously, we have shown that isolated pVIII major coat proteins of the fd tet filamentous phage vector, containing cancer cell-specific peptide fusions at their N terminus, can be used as active targeting ligands in a liposomal doxorubicin delivery system in vitro and in vivo. Here, we show a novel major coat protein isolation procedure in 2-propanol that allows spontaneous incorporation of the hydrophobic protein core into preformed liposomal doxorubicin with minimal damage or drug loss while still retaining the targeting ligand exposed for cell-specific targeting. Using a panel of 12 structurally unique ligands with specificity towards breast, lung, and/or pancreatic cancer, we showed the feasibility of pVIII major coat proteins to significantly increase the throughput of targeting ligand screening in a common nanomedicine core. Phage protein-modified Lipodox samples showed an average doxorubicin recovery of 82.8% across all samples with 100% of protein incorporation in the correct orientation (N-terminus exposed. Following cytotoxicity screening in a doxorubicin-sensitive breast cancer line (MCF-7, three major groups of ligands were identified. Ligands showing the most improved cytotoxicity included: DMPGTVLP, ANGRPSMT, VNGRAEAP, and ANDVYLD showing a 25-fold improvement (p < 0.05 in toxicity. Similarly DGQYLGSQ, ETYNQPYL, and GSSEQLYL ligands with specificity towards a doxorubicin-insensitive pancreatic cancer line (PANC-1 showed significant increases in toxicity (2-fold; p < 0.05. Thus, we demonstrated proof-of-concept that pVIII major coat proteins can be screened in significantly higher throughput to identify novel ligands displaying improved therapeutic activity in a desired cancer phenotype.

  5. Cold-Adapted Enzymes

    Science.gov (United States)

    Georlette, D.; Bentahir, M.; Claverie, P.; Collins, T.; D'amico, S.; Delille, D.; Feller, G.; Gratia, E.; Hoyoux, A.; Lonhienne, T.; Meuwis, M.-a.; Zecchinon, L.; Gerday, Ch.

    In the last few years, increased attention has been focused on enzymes produced by cold-adapted micro-organisms. It has emerged that psychrophilic enzymes represent an extremely powerful tool in both protein folding investigations and for biotechnological purposes. Such enzymes are characterised by an increased thermosensitivity and, most of them, by a higher catalytic efficiency at low and moderate temperatures, when compared to their mesophilic counterparts. The high thermosensitivity probably originates from an increased flexibility of either a selected area of the molecular edifice or the overall protein structure, providing enhanced abilities to undergo conformational changes during catalysis at low temperatures. Structure modelling and recent crystallographic data have allowed to elucidate the structural parameters that could be involved in this higher resilience. It was demonstrated that each psychrophilic enzyme adopts its own adaptive strategy. It appears, moreover, that there is a continuum in the strategy of protein adaptation to temperature, as the previously mentioned structural parameters are implicated in the stability of thermophilic proteins. Additional 3D crystal structures, site-directed and random mutagenesis experiments should now be undertaken to further investigate the stability-flexibility-activity relationship.

  6. The histone-like protein HU has a role in gene expression during the acid adaptation response in Helicobacter pylori.

    Science.gov (United States)

    Álvarez, Alhejandra; Toledo, Héctor

    2017-08-01

    Gastritis, ulcers, and gastric malignancy have been linked to human gastric epithelial colonization by Helicobacter pylori. Characterization of the mechanisms by which H. pylori adapts to the human stomach environment is of crucial importance to understand H. pylori pathogenesis. In an effort to extend our knowledge of these mechanisms, we used proteomic analysis and qRT-PCR to characterize the role of the histone-like protein HU in the response of H. pylori to low pH. Proteomic analysis revealed that genes involved in chemotaxis, oxidative stress, or metabolism are under control of the HU protein. Also, expression of the virulence factors Ggt and NapA is affected by the null mutation of hup gene both at neutral and acid pH, as evidenced by qRT-PCR analysis. Those results showed that H. pylori gene expression is altered by shift to low pH, thus confirming that acid exposure leads to profound changes in genomic expression, and suggest that the HU protein is a regulator that may help the bacterium adapt to the acid stress. In accordance with previous reports, we found that the HU protein participates in gene expression regulation when the microorganism is exposed to acid stress. Such transcriptional regulation underlies protein accumulation in the H. pylori cell. © 2017 John Wiley & Sons Ltd.

  7. The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.

    Science.gov (United States)

    Fujinami, Shun; Ito, Masahiro

    2018-01-01

    It is well known that the Na + cycle and the cell wall are essential for alkaline adaptation of Na + -dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na + concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na + concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.

  8. The age- and sex-specific decline of the 20s proteasome and the Nrf2/CncC signal transduction pathway in adaption and resistance to oxidative stress in Drosophila melanogaster.

    Science.gov (United States)

    Pomatto, Laura C D; Wong, Sarah; Carney, Caroline; Shen, Brenda; Tower, John; Davies, Kelvin J A

    2017-04-01

    Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C ( Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H 2 O 2 ) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H 2 O 2 , and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H 2 O 2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.

  9. Gene-specific correlation of RNA and protein levels in human cells and tissues

    DEFF Research Database (Denmark)

    Edfors, Fredrik; Danielsson, Frida; Hallström, Björn M.

    2016-01-01

    An important issue for molecular biology is to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. Here, we have developed a targeted proteomics approach for a set of human non-secreted proteins based on parallel reaction monitoring...... to measure, at steady-state conditions, absolute protein copy numbers across human tissues and cell lines and compared these levels with the corresponding mRNA levels using transcriptomics. The study shows that the transcript and protein levels do not correlate well unless a gene-specific RNA-to-protein (RTP...

  10. Protective effect of a non specific inflammation on bone marrow protein synthesis in irradiated mice

    International Nuclear Information System (INIS)

    Herodin, F.; Roques, P.; Court, L.

    1988-01-01

    Gamma radiations exert a decrease in mouse bone marrow total protein synthesis. A non-specific inflammatory process induced with polyacrylamide microbeads stimulates spleen and marrow protein synthesis and protects the medullar protein synthesis in irradiated mice [fr

  11. Extensive X-linked adaptive evolution in central chimpanzees

    DEFF Research Database (Denmark)

    Hvilsom, Christina; Qian, Yu; Bataillon, Thomas

    2012-01-01

    on the dominance of beneficial (adaptive) and deleterious mutations. Here we capture and sequence the complete exomes of 12 chimpanzees and present the largest set of protein-coding polymorphism to date. We report extensive adaptive evolution specifically targeting the X chromosome of chimpanzees with as much...... to humans, we find that purifying selection is stronger on the X chromosome than on the autosomes in chimpanzees. We therefore conclude that most adaptive mutations are recessive. We also document dramatically reduced synonymous diversity in the chimpanzee X chromosome relative to autosomes and stronger...... purifying selection than for the human X chromosome. If similar processes were operating in the human-chimpanzee ancestor as in central chimpanzees today, our results therefore provide an explanation for the much-discussed reduction in the human-chimpanzee divergence at the X chromosome....

  12. A constitutive damage specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells

    International Nuclear Information System (INIS)

    Hirschfeld, S.; Levine, A.S.; Ozato, K.; Protic, M.

    1990-01-01

    Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physiochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 6-4'-(pyrimidine-2'-one)-pyrimidine dimer: specific binding could not be detected with probes which contain -TT- cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts

  13. The Role of Non-specific and Specific Immune Systems in Poultry against Newcastle Disease

    Directory of Open Access Journals (Sweden)

    Dyah Ayu Hewajuli

    2015-09-01

    Full Text Available Newcastle disease (ND is caused by avian paramyxovirus-1 which belong to Avulavirus genus and Paramyxoviridae family. The birds have abnormalities in humoral (bursa fabricius and cellular (thymus and spleen lymphoid organs. Lesions decrease the immune system. Immune system consists of non-specific and specific immune systems. The main components of non-specific immunity are physical and chemical barrier (feather and skin or mucosa, phagocytic cells (macrophages and natural killer, protein complement and the mediator of inflammation and cytokines. Interferons (IFNs belong to a group of cytokines that play a major role in the nonspecific or innate (natural immunity. The virulent ND virus encodes protein of V gene can be suppressed IFN type I. This leads to non-specific immune system fail to respond to the virulent strains resulting in severe pathogenicity. The defense mechanism of the host is replaced by specific immunity (adaptive immunity when natural immunity fails to overcome the infection. The specific immune system consists of humoral mediated immunity (HMI and cell-mediated immunity (CMI. The cells of immune system that react specifically with the antigen are B lymphocytes producing the antibodies, T lymphocytes that regulate the synthesis of antibodies and T cells as effector or the direct cytotoxic cells. Both non-specific and specific immunities are complementary against the invasion of ND virus in the birds. The objective of this article is to discuss the role of non specific and specific immune system in ND.

  14. Selecting highly structure-specific antibodies using structured synthetic mimics of the cystine knot protein sclerostin

    NARCIS (Netherlands)

    Back, J.W.; Frisch, C.; Van Pee, K.; Boschert, V.; van Vught, R.; Puijk, W.; Mueller, T. D.; Knappik, A.; Timmerman, P.

    2012-01-01

    Antibodies directed against specific regions of a protein have traditionally been raised against full proteins, protein domains or simple unstructured peptides, containing contiguous stretches of primary sequence. We have used a new approach of selecting antibodies against restrained peptides

  15. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

    Directory of Open Access Journals (Sweden)

    Dawid Krokowski

    2017-12-01

    Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

  16. Site-specific covalent attachment of DNA to proteins using a photoactivatable Tus-Ter complex.

    Science.gov (United States)

    Dahdah, Dahdah B; Morin, Isabelle; Moreau, Morgane J J; Dixon, Nicholas E; Schaeffer, Patrick M

    2009-06-07

    Investigations into the photocrosslinking kinetics of the protein Tus with various bromodeoxyuridine-substituted Ter DNA variants highlight the potential use of this complex as a photoactivatable connector between proteins of interest and specific DNA sequences.

  17. Cross-Cultural Adaptation and Initial Validation of the Stroke-Specific Quality of Life Scale into the Yoruba Language

    Science.gov (United States)

    Akinpelu, Aderonke O.; Odetunde, Marufat O.; Odole, Adesola C.

    2012-01-01

    Stroke-Specific Quality of Life 2.0 (SS-QoL 2.0) scale is used widely and has been cross-culturally adapted to many languages. This study aimed at the cross-cultural adaptation of SS-QoL 2.0 to Yoruba, the indigenous language of south-western Nigeria, and to carry out an initial investigation on its validity. English SS-QoL 2.0 was first adapted…

  18. Cadmium induces the expression of specific stress proteins in sea urchin embryos

    International Nuclear Information System (INIS)

    Roccheri, Maria Carmela; Agnello, Maria; Bonaventura, Rosa; Matranga, Valeria

    2004-01-01

    Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72-70, 56, 28, and 25 kDa) was induced, with an increase in the rate of synthesis of 72-70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure

  19. Molecular Simulations of Sequence-Specific Association of Transmembrane Proteins in Lipid Bilayers

    Science.gov (United States)

    Doxastakis, Manolis; Prakash, Anupam; Janosi, Lorant

    2011-03-01

    Association of membrane proteins is central in material and information flow across the cellular membranes. Amino-acid sequence and the membrane environment are two critical factors controlling association, however, quantitative knowledge on such contributions is limited. In this work, we study the dimerization of helices in lipid bilayers using extensive parallel Monte Carlo simulations with recently developed algorithms. The dimerization of Glycophorin A is examined employing a coarse-grain model that retains a level of amino-acid specificity, in three different phospholipid bilayers. Association is driven by a balance of protein-protein and lipid-induced interactions with the latter playing a major role at short separations. Following a different approach, the effect of amino-acid sequence is studied using the four transmembrane domains of the epidermal growth factor receptor family in identical lipid environments. Detailed characterization of dimer formation and estimates of the free energy of association reveal that these helices present significant affinity to self-associate with certain dimers forming non-specific interfaces.

  20. Lipidomic Adaptations in White and Brown Adipose Tissue in Response to Exercise Demonstrate Molecular Species-Specific Remodeling

    Directory of Open Access Journals (Sweden)

    Francis J. May

    2017-02-01

    Full Text Available Exercise improves whole-body metabolic health through adaptations to various tissues, including adipose tissue, but the effects of exercise training on the lipidome of white adipose tissue (WAT and brown adipose tissue (BAT are unknown. Here, we utilize MS/MSALL shotgun lipidomics to determine the molecular signatures of exercise-induced adaptations to subcutaneous WAT (scWAT and BAT. Three weeks of exercise training decrease specific molecular species of phosphatidic acid (PA, phosphatidylcholines (PC, phosphatidylethanolamines (PE, and phosphatidylserines (PS in scWAT and increase specific molecular species of PC and PE in BAT. Exercise also decreases most triacylglycerols (TAGs in scWAT and BAT. In summary, exercise-induced changes to the scWAT and BAT lipidome are highly specific to certain molecular lipid species, indicating that changes in tissue lipid content reflect selective remodeling in scWAT and BAT of both phospholipids and glycerol lipids in response to exercise training, thus providing a comprehensive resource for future studies of lipid metabolism pathways.

  1. Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function".

    Science.gov (United States)

    Nestler, Eric J

    2016-08-15

    In 1991 we demonstrated that chronic morphine exposure increased levels of adenylyl cyclase and protein kinase A (PKA) in several regions of the rat central nervous system as inferred from measures of enzyme activity in crude extracts (Terwilliger et al., 1991). These findings led us to hypothesize that a concerted upregulation of the cAMP pathway is a general mechanism of opiate tolerance and dependence. Moreover, in the same study we showed similar induction of adenylyl cyclase and PKA activity in nucleus accumbens (NAc) in response to chronic administration of cocaine, but not of several non-abused psychoactive drugs. Morphine and cocaine also induced equivalent changes in inhibitory G protein subunits in this brain region. We thus extended our hypothesis to suggest that, particularly within brain reward regions such as NAc, cAMP pathway upregulation represents a common mechanism of reward tolerance and dependence shared by several classes of drugs of abuse. Research since that time, by many laboratories, has provided substantial support for these hypotheses. Specifically, opiates in several CNS regions including NAc, and cocaine more selectively in NAc, induce expression of certain adenylyl cyclase isoforms and PKA subunits via the transcription factor, CREB, and these transcriptional adaptations serve a homeostatic function to oppose drug action. In certain brain regions, such as locus coeruleus, these adaptations mediate aspects of physical opiate dependence and withdrawal, whereas in NAc they mediate reward tolerance and dependence that drives increased drug self-administration. This work has had important implications for understanding the molecular basis of addiction. "A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function". Previous studies have shown that chronic morphine increases levels of the G-protein subunits Giα and Goα, adenylate cyclase, cyclic AMP

  2. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Science.gov (United States)

    2011-01-01

    Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy) or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy). Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules. PMID:22192175

  3. Structural adaptation of extreme halophilic proteins through decrease of conserved hydrophobic contact surface

    Directory of Open Access Journals (Sweden)

    Siglioccolo Alessandro

    2011-12-01

    Full Text Available Abstract Background Halophiles are extremophilic microorganisms growing optimally at high salt concentrations. There are two strategies used by halophiles to maintain proper osmotic pressure in their cytoplasm: accumulation of molar concentrations of potassium and chloride with extensive adaptation of the intracellular macromolecules ("salt-in" strategy or biosynthesis and/or accumulation of organic osmotic solutes ("osmolyte" strategy. Our work was aimed at contributing to the understanding of the shared molecular mechanisms of protein haloadaptation through a detailed and systematic comparison of a sample of several three-dimensional structures of halophilic and non-halophilic proteins. Structural differences observed between the "salt-in" and the mesophilic homologous proteins were contrasted to those observed between the "osmolyte" and mesophilic pairs. Results The results suggest that haloadaptation strategy in the presence of molar salt concentration, but not of osmolytes, necessitates a weakening of the hydrophobic interactions, in particular at the level of conserved hydrophobic contacts. Weakening of these interactions counterbalances their strengthening by the presence of salts in solution and may help the structure preventing aggregation and/or loss of function in hypersaline environments. Conclusions Considering the significant increase of biotechnology applications of halophiles, the understanding of halophilicity can provide the theoretical basis for the engineering of proteins of great interest because stable at concentrations of salts that cause the denaturation or aggregation of the majority of macromolecules.

  4. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    Science.gov (United States)

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  5. Adaptive Evolution of CENP-A in Percid Fishes

    Directory of Open Access Journals (Sweden)

    Harriet N. A. Abbey

    2015-07-01

    Full Text Available Centromeric protein A (CENP-A is the epigenetic determinant of centromeres. This protein has been shown to be adaptively evolving in a number of animal and plant species. In a previous communication we were able to demonstrate that signs of adaptive evolution were detected in the comparison of CENP-A sequences from three percid fish species. In this study we isolated the CENP-A gene from eight additional species from the Percidae family. With these sequences and those previously obtained, we carried out a more robust statistical analysis of codon specific positive selection in CENP-A coding sequences of eleven percid species. We were able to demonstrate that at least two amino acid positions within the N-terminal tail are under strong positive selection and that one of these positions is potentially a substrate for phosphorylation. While nonsynonymous substitutions were detected in the histone fold domain, these were not statistically supported as resulting from positive selection.

  6. Machine Learning Identification of Protein Properties Useful for Specific Applications

    KAUST Repository

    Khamis, Abdullah

    2016-03-31

    Proteins play critical roles in cellular processes of living organisms. It is therefore important to identify and characterize their key properties associated with their functions. Correlating protein’s structural, sequence and physicochemical properties of its amino acids (aa) with protein functions could identify some of the critical factors governing the specific functionality. We point out that not all functions of even well studied proteins are known. This, complemented by the huge increase in the number of newly discovered and predicted proteins, makes challenging the experimental characterization of the whole spectrum of possible protein functions for all proteins of interest. Consequently, the use of computational methods has become more attractive. Here we address two questions. The first one is how to use protein aa sequence and physicochemical properties to characterize a family of proteins. The second one focuses on how to use transcription factor (TF) protein’s domains to enhance accuracy of predicting TF DNA binding sites (TFBSs). To address the first question, we developed a novel method using computational representation of proteins based on characteristics of different protein regions (N-terminal, M-region and C-terminal) and combined these with the properties of protein aa sequences. We show that this description provides important biological insight about characterization of the protein functional groups. Using feature selection techniques, we identified key properties of proteins that allow for very accurate characterization of different protein families. We demonstrated efficiency of our method in application to a number of antimicrobial peptide families. To address the second question we developed another novel method that uses a combination of aa properties of DNA binding domains of TFs and their TFBS properties to develop machine learning models for predicting TFBSs. Feature selection is used to identify the most relevant characteristics

  7. Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family

    DEFF Research Database (Denmark)

    Schjoldager, Katrine Ter-Borch Gram; Clausen, Henrik

    2012-01-01

    Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis...... and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3...

  8. A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

    Energy Technology Data Exchange (ETDEWEB)

    Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra, E-mail: rr228@georgetown.edu

    2015-05-15

    Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination.

  9. A novel method for monitoring functional lesion-specific recruitment of repair proteins in live cells

    International Nuclear Information System (INIS)

    Woodrick, Jordan; Gupta, Suhani; Khatkar, Pooja; Dave, Kalpana; Levashova, Darya; Choudhury, Sujata; Elias, Hadi; Saha, Tapas; Mueller, Susette; Roy, Rabindra

    2015-01-01

    Highlights: • A method of monitoring lesion-specific recruitment of proteins in vivo is described. • Recruitment of repair enzymes to abasic sites is monitored by co-localization. • Repair protein recruitment is consistent with known protein–protein relationships. • Cells demonstrated complete repair of abasic sites by 90 min. - Abstract: DNA–protein relationships have been studied by numerous methods, but a particular gap in methodology lies in the study of DNA adduct-specific interactions with proteins in vivo, which particularly affects the field of DNA repair. Using the repair of a well-characterized and ubiquitous adduct, the abasic (AP) site, as a model, we have developed a comprehensive method of monitoring DNA lesion-specific recruitment of proteins in vivo over time. We utilized a surrogate system in which a Cy3-labeled plasmid containing a single AP-site was transfected into cells, and the interaction of the labeled DNA with BER enzymes, including APE1, Polβ, LIG1, and FEN1, was monitored by immunofluorescent staining of the enzymes by Alexafluor-488-conjugated secondary antibody. The recruitment of enzymes was characterized by quantification of Cy3-Alexafluor-488 co-localization. To validate the microscopy-based method, repair of the transfected AP-site DNA was also quantified at various time points post-transfection using a real time PCR-based method. Notably, the recruitment time kinetics for each enzyme were consistent with AP-site repair time kinetics. This microscopy-based methodology is reliable in detecting the recruitment of proteins to specific DNA substrates and can be extended to study other in vivo DNA–protein relationships in any DNA sequence and in the context of any DNA structure in transfectable proliferating or quiescent cells. The method may be applied to a variety of disciplines of nucleic acid transaction pathways, including repair, replication, transcription, and recombination

  10. Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

    Science.gov (United States)

    McCarthy, Alex J; Lindsay, Jodi A

    2013-10-01

    Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights

  11. Adaptation in CRISPR-Cas Systems.

    Science.gov (United States)

    Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

    2016-03-17

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

    Science.gov (United States)

    Ponomarenko, Natalia A; Durova, Oxana M; Vorobiev, Ivan I; Belogurov, Alexey A; Kurkova, Inna N; Petrenko, Alexander G; Telegin, Georgy B; Suchkov, Sergey V; Kiselev, Sergey L; Lagarkova, Maria A; Govorun, Vadim M; Serebryakova, Marina V; Avalle, Bérangère; Tornatore, Pete; Karavanov, Alexander; Morse, Herbert C; Thomas, Daniel; Friboulet, Alain; Gabibov, Alexander G

    2006-01-10

    Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.

  13. Hidden Markov model-derived structural alphabet for proteins: the learning of protein local shapes captures sequence specificity.

    Science.gov (United States)

    Camproux, A C; Tufféry, P

    2005-08-05

    Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. We have recently set up a Hidden Markov Model to optimally compress protein three-dimensional conformations into a one-dimensional series of letters of a structural alphabet. Such a model learns simultaneously the shape of representative structural letters describing the local conformation and the logic of their connections, i.e. the transition matrix between the letters. Here, we move one step further and report some evidence that such a model of protein local architecture also captures some accurate amino acid features. All the letters have specific and distinct amino acid distributions. Moreover, we show that words of amino acids can have significant propensities for some letters. Perspectives point towards the prediction of the series of letters describing the structure of a protein from its amino acid sequence.

  14. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    KAUST Repository

    Martinez, N.

    2016-09-06

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  15. High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

    KAUST Repository

    Martinez, N.; Michoud, Gregoire; Cario, A.; Ollivier, J.; Franzetti, B.; Jebbar, M.; Oger, P.; Peters, J.

    2016-01-01

    Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

  16. Modular protein switches derived from antibody mimetic proteins.

    Science.gov (United States)

    Nicholes, N; Date, A; Beaujean, P; Hauk, P; Kanwar, M; Ostermeier, M

    2016-02-01

    Protein switches have potential applications as biosensors and selective protein therapeutics. Protein switches built by fusion of proteins with the prerequisite input and output functions are currently developed using an ad hoc process. A modular switch platform in which existing switches could be readily adapted to respond to any ligand would be advantageous. We investigated the feasibility of a modular protein switch platform based on fusions of the enzyme TEM-1 β-lactamase (BLA) with two different antibody mimetic proteins: designed ankyrin repeat proteins (DARPins) and monobodies. We created libraries of random insertions of the gene encoding BLA into genes encoding a DARPin or a monobody designed to bind maltose-binding protein (MBP). From these libraries, we used a genetic selection system for β-lactamase activity to identify genes that conferred MBP-dependent ampicillin resistance to Escherichia coli. Some of these selected genes encoded switch proteins whose enzymatic activity increased up to 14-fold in the presence of MBP. We next introduced mutations into the antibody mimetic domain of these switches that were known to cause binding to different ligands. To different degrees, introduction of the mutations resulted in switches with the desired specificity, illustrating the potential modularity of these platforms. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Strand-Specific Analysis of DNA Synthesis and Proteins Association with DNA Replication Forks in Budding Yeast.

    Science.gov (United States)

    Yu, Chuanhe; Gan, Haiyun; Zhang, Zhiguo

    2018-01-01

    DNA replication initiates at DNA replication origins after unwinding of double-strand DNA(dsDNA) by replicative helicase to generate single-stranded DNA (ssDNA) templates for the continuous synthesis of leading-strand and the discontinuous synthesis of lagging-strand. Therefore, methods capable of detecting strand-specific information will likely yield insight into the association of proteins at leading and lagging strand of DNA replication forks and the regulation of leading and lagging strand synthesis during DNA replication. The enrichment and Sequencing of Protein-Associated Nascent DNA (eSPAN), which measure the relative amounts of proteins at nascent leading and lagging strands of DNA replication forks, is a step-wise procedure involving the chromatin immunoprecipitation (ChIP) of a protein of interest followed by the enrichment of protein-associated nascent DNA through BrdU immunoprecipitation. The isolated ssDNA is then subjected to strand-specific sequencing. This method can detect whether a protein is enriched at leading or lagging strand of DNA replication forks. In addition to eSPAN, two other strand-specific methods, (ChIP-ssSeq), which detects potential protein-ssDNA binding and BrdU-IP-ssSeq, which can measure synthesis of both leading and lagging strand, were developed along the way. These methods can provide strand-specific and complementary information about the association of the target protein with DNA replication forks as well as synthesis of leading and lagging strands genome wide. Below, we describe the detailed eSPAN, ChIP-ssSeq, and BrdU-IP-ssSeq protocols.

  18. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  19. Concordance of gene expression in human protein complexes reveals tissue specificity and pathology

    DEFF Research Database (Denmark)

    Börnigen, Daniela; Pers, Tune Hannes; Thorrez, Lieven

    2013-01-01

    Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among prot...

  20. Fluorescent molecularly imprinted polymer thin films for specific protein detection prepared with dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline.

    Science.gov (United States)

    Inoue, Yuki; Kuwahara, Atsushi; Ohmori, Kohei; Sunayama, Hirobumi; Ooya, Tooru; Takeuchi, Toshifumi

    2013-10-15

    Protein-imprinted polymers, capable of specific transduction of protein binding events into fluorescent signal change, were designed and synthesized by using dansyl ethylenediamine-conjugated O-acryloyl L-hydroxyproline (Hyp-En-Dans). Human serum albumin (HSA) was used as a model target protein and HSA-imprinted polymers (HSA-IP) were prepared on glass substrates. Specific fluorescence change was observed for HSA binding on the imprinted polymer thin film, whereas a weaker response was observed for other proteins, including bovine serum albumin, chymotrypsin, lysozyme, and avidin. The binding specificity was found to derive from the rigid structure of the hydrogen-bondable pyrrolidine moiety. Compared with SPR measurements, the non-specific binding caused by the polymer matrix and/or randomly located fluorescent monomer residues that did not compose specific binding sites did not contribute to the observed fluorescence change. These results revealed that the proposed protein-imprinting technique using Hyp-En-Dans could provide a highly selective protein-sensing platform, in which only specific binding events would be detected by fluorescent measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates.

    Science.gov (United States)

    Sun, Yuanxia; Hayakawa, Shigeru; Ogawa, Masahiro; Izumori, Ken

    2005-12-28

    Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.

  2. Effect of Maillard induced glycation on protein hydrolysis by lysine/arginine and non-lysine/arginine specific proteases

    NARCIS (Netherlands)

    Deng, Y.; Wierenga, P.A.; Schols, H.A.; Sforza, S.; Gruppen, H.

    2017-01-01

    Enzymatic protein hydrolysis is sensitive to modifications of protein structure, e.g. Maillard reaction. In early stages of the reaction glycation takes place, modifying the protein primary structure. In later stages protein aggregation occurs. The specific effect of glycation on protein

  3. Intracellular localisation of proteins to specific cellular areas by nanocapsule mediated delivery.

    Science.gov (United States)

    Wang, Huabin; Chen, Ligang; Sun, Xianchao; Fu, Ailing

    2017-09-01

    Nanocapsules are promising carriers with great potential for intracellular protein transport. Although many studies have intended to improve cell uptake efficacy, there is an increasing interest in understanding of subcellular distribution of cargoes inside cells, which is essential for purposeful delivery of biomolecules into specific sites within cells. Herein, we interrogate the intracellular localisation of exogenous proteins, including fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) and green fluorescent protein (GFP), mediated by specially designed nanocapsules. The results show that the designed nanocapsules can deliver the two types of fluorescent proteins into different cellular destinations (cytosol, nucleus or the whole cell), depending on the composition of nanocapsules. Meanwhile, several impact factors that influence the distribution of proteins in cells have also been investigated, and the results suggest that the localisation of capsule-mediated proteins in cells is strongly affected by the surface properties of nanocapsules, the types of stabilisers and proteins, and environmental temperatures. The rational control of intracellular localised delivery of exogenous proteins as we demonstrated in this study might open new avenues to obtain desired magnitude of drug effects for modulating cell activity.

  4. Detection of site specific glycosylation in proteins using flow cytometry†

    Science.gov (United States)

    Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

    2009-01-01

    We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

  5. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

    Directory of Open Access Journals (Sweden)

    Shimpei Morimoto

    2018-03-01

    Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

  6. Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

    Directory of Open Access Journals (Sweden)

    Bryan D Moyer

    Full Text Available BACKGROUND: Using fungiform (FG and circumvallate (CV taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed. METHODOLOGY/PRINCIPAL FINDINGS: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive, sour cells (PKD2L1-positive, as well as other taste cell populations. Transmembrane protein 44 (TMEM44, a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1, a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1, a calcium-binding transmembrane protein; and anoctamin 7 (ANO7, a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B, a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release. CONCLUSIONS/SIGNIFICANCE: Identification of genes encoding multi-transmembrane domain proteins

  7. Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

    OpenAIRE

    Demangeat, Gerard; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, Coralie

    1992-01-01

    The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a sta...

  8. Full-Length Venom Protein cDNA Sequences from Venom-Derived mRNA: Exploring Compositional Variation and Adaptive Multigene Evolution.

    Science.gov (United States)

    Modahl, Cassandra M; Mackessy, Stephen P

    2016-06-01

    Envenomation of humans by snakes is a complex and continuously evolving medical emergency, and treatment is made that much more difficult by the diverse biochemical composition of many venoms. Venomous snakes and their venoms also provide models for the study of molecular evolutionary processes leading to adaptation and genotype-phenotype relationships. To compare venom complexity and protein sequences, venom gland transcriptomes are assembled, which usually requires the sacrifice of snakes for tissue. However, toxin transcripts are also present in venoms, offering the possibility of obtaining cDNA sequences directly from venom. This study provides evidence that unknown full-length venom protein transcripts can be obtained from the venoms of multiple species from all major venomous snake families. These unknown venom protein cDNAs are obtained by the use of primers designed from conserved signal peptide sequences within each venom protein superfamily. This technique was used to assemble a partial venom gland transcriptome for the Middle American Rattlesnake (Crotalus simus tzabcan) by amplifying sequences for phospholipases A2, serine proteases, C-lectins, and metalloproteinases from within venom. Phospholipase A2 sequences were also recovered from the venoms of several rattlesnakes and an elapid snake (Pseudechis porphyriacus), and three-finger toxin sequences were recovered from multiple rear-fanged snake species, demonstrating that the three major clades of advanced snakes (Elapidae, Viperidae, Colubridae) have stable mRNA present in their venoms. These cDNA sequences from venom were then used to explore potential activities derived from protein sequence similarities and evolutionary histories within these large multigene superfamilies. Venom-derived sequences can also be used to aid in characterizing venoms that lack proteomic profiles and identify sequence characteristics indicating specific envenomation profiles. This approach, requiring only venom, provides

  9. Gene duplication and adaptive evolution of digestive proteases in Drosophila arizonae female reproductive tracts.

    Directory of Open Access Journals (Sweden)

    Erin S Kelleher

    2007-08-01

    Full Text Available It frequently has been postulated that intersexual coevolution between the male ejaculate and the female reproductive tract is a driving force in the rapid evolution of reproductive proteins. The dearth of research on female tracts, however, presents a major obstacle to empirical tests of this hypothesis. Here, we employ a comparative EST approach to identify 241 candidate female reproductive proteins in Drosophila arizonae, a repleta group species in which physiological ejaculate-female coevolution has been documented. Thirty-one of these proteins exhibit elevated amino acid substitution rates, making them candidates for molecular coevolution with the male ejaculate. Strikingly, we also discovered 12 unique digestive proteases whose expression is specific to the D. arizonae lower female reproductive tract. These enzymes belong to classes most commonly found in the gastrointestinal tracts of a diverse array of organisms. We show that these proteases are associated with recent, lineage-specific gene duplications in the Drosophila repleta species group, and exhibit strong signatures of positive selection. Observation of adaptive evolution in several female reproductive tract proteins indicates they are active players in the evolution of reproductive tract interactions. Additionally, pervasive gene duplication, adaptive evolution, and rapid acquisition of a novel digestive function by the female reproductive tract points to a novel coevolutionary mechanism of ejaculate-female interaction.

  10. Improving protein fold recognition by extracting fold-specific features from predicted residue-residue contacts.

    Science.gov (United States)

    Zhu, Jianwei; Zhang, Haicang; Li, Shuai Cheng; Wang, Chao; Kong, Lupeng; Sun, Shiwei; Zheng, Wei-Mou; Bu, Dongbo

    2017-12-01

    Accurate recognition of protein fold types is a key step for template-based prediction of protein structures. The existing approaches to fold recognition mainly exploit the features derived from alignments of query protein against templates. These approaches have been shown to be successful for fold recognition at family level, but usually failed at superfamily/fold levels. To overcome this limitation, one of the key points is to explore more structurally informative features of proteins. Although residue-residue contacts carry abundant structural information, how to thoroughly exploit these information for fold recognition still remains a challenge. In this study, we present an approach (called DeepFR) to improve fold recognition at superfamily/fold levels. The basic idea of our approach is to extract fold-specific features from predicted residue-residue contacts of proteins using deep convolutional neural network (DCNN) technique. Based on these fold-specific features, we calculated similarity between query protein and templates, and then assigned query protein with fold type of the most similar template. DCNN has showed excellent performance in image feature extraction and image recognition; the rational underlying the application of DCNN for fold recognition is that contact likelihood maps are essentially analogy to images, as they both display compositional hierarchy. Experimental results on the LINDAHL dataset suggest that even using the extracted fold-specific features alone, our approach achieved success rate comparable to the state-of-the-art approaches. When further combining these features with traditional alignment-related features, the success rate of our approach increased to 92.3%, 82.5% and 78.8% at family, superfamily and fold levels, respectively, which is about 18% higher than the state-of-the-art approach at fold level, 6% higher at superfamily level and 1% higher at family level. An independent assessment on SCOP_TEST dataset showed consistent

  11. Delineation of the Pasteurellaceae-specific GbpA-family of glutathione-binding proteins

    Directory of Open Access Journals (Sweden)

    Vergauwen Bjorn

    2011-11-01

    Full Text Available Abstract Background The Gram-negative bacterium Haemophilus influenzae is a glutathione auxotroph and acquires the redox-active tripeptide by import. The dedicated glutathione transporter belongs to the ATP-binding cassette (ABC-transporter superfamily and displays more than 60% overall sequence identity with the well-studied dipeptide (Dpp permease of Escherichia coli. The solute binding protein (SBP that mediates glutathione transport in H. influenzae is a lipoprotein termed GbpA and is 54% identical to E. coli DppA, a well-studied member of family 5 SBP's. The discovery linking GbpA to glutathione import came rather unexpectedly as this import-priming SBP was previously annotated as a heme-binding protein (HbpA, and was thought to mediate heme acquisition. Nonetheless, although many SBP's have been implicated in more than one function, a prominent physiological role for GbpA and its partner permease in heme acquisition appears to be very unlikely. Here, we sought to characterize five representative GbpA homologs in an effort to delineate the novel GbpA-family of glutathione-specific family 5 SBPs and to further clarify their functional role in terms of ligand preferences. Results Lipoprotein and non-lipoprotein GbpA homologs were expressed in soluble form and substrate specificity was evaluated via a number of ligand binding assays. A physiologically insignificant affinity for hemin was observed for all five GbpA homologous test proteins. Three out of five test proteins were found to bind glutathione and some of its physiologically relevant derivatives with low- or submicromolar affinity. None of the tested SBP family 5 allocrites interacted with the remaining two GbpA test proteins. Structure-based sequence alignments and phylogenetic analysis show that the two binding-inert GbpA homologs clearly form a separate phylogenetic cluster. To elucidate a structure-function rationale for this phylogenetic differentiation, we determined the crystal

  12. Shift in the isoelectric-point of milk proteins as a consequence of adaptive divergence between the milks of mammalian species.

    LENUS (Irish Health Repository)

    Khaldi, Nora

    2011-07-29

    Abstract Background Milk proteins are required to proceed through a variety of conditions of radically varying pH, which are not identical across mammalian digestive systems. We wished to investigate if the shifts in these requirements have resulted in marked changes in the isoelectric point and charge of milk proteins during evolution. Results We investigated nine major milk proteins in 13 mammals. In comparison with a group of orthologous non-milk proteins, we found that 3 proteins κ-casein, lactadherin, and muc1 have undergone the highest change in isoelectric point during evolution. The pattern of non-synonymous substitutions indicate that selection has played a role in the isoelectric point shift, since residues that show significant evidence of positive selection are much more likely to be charged (p = 0.03 for κ-casein; p < 10-8 for muc1). However, this selection does not appear to be solely due to adaptation to the diversity of mammalian digestive systems, since striking changes are seen among species that resemble each other in terms of their digestion. Conclusion The changes in charge are most likely due to changes of other protein functions, rather than an adaptation to the different mammalian digestive systems. These functions may include differences in bioactive peptide releases in the gut between different mammals, which are known to be a major contributing factor in the functional and nutritional value of mammalian milk. This raises the question of whether bovine milk is optimal in terms of particular protein functions, for human nutrition and possibly disease resistance. This article was reviewed by Fyodor Kondrashov, David Liberles (nominated by David Ardell), and Christophe Lefevre (nominated by Mark Ragan).

  13. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms.

    Science.gov (United States)

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T; Texier, Yves; van Beersum, Sylvia E C; Horn, Nicola; Willer, Jason R; Mans, Dorus A; Dougherty, Gerard; Lamers, Ideke J C; Coene, Karlien L M; Arts, Heleen H; Betts, Matthew J; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J F; Marcelis, Carlo L; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A; Toedt, Grischa; van Dam, Teunis J P; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L; Blacque, Oliver E; Gibson, Toby J; Huynen, Martijn A; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E; Franco, Brunella; Giles, Rachel H; Ueffing, Marius; Russell, Robert B; Roepman, Ronald

    2016-05-13

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine.

  14. HaloTag protein-mediated specific labeling of living cells with quantum dots

    International Nuclear Information System (INIS)

    So, Min-kyung; Yao Hequan; Rao Jianghong

    2008-01-01

    Quantum dots emerge as an attractive alternative to small molecule fluorophores as fluorescent tags for in vivo cell labeling and imaging. This communication presents a method for specific labeling of live cells using quantum dots. The labeling is mediated by HaloTag protein expressed at the cell surface which forms a stable covalent adduct with its ligand (HaloTag ligand). The labeling can be performed in one single step with quantum dot conjugates that are functionalized with HaloTag ligand, or in two steps with biotinylated HaloTag ligand first and followed by streptavidin coated quantum dots. Live cell fluorescence imaging indicates that the labeling is specific and takes place at the cell surface. This HaloTag protein-mediated cell labeling method should facilitate the application of quantum dots for live cell imaging

  15. Adenovirus structural protein IIIa is involved in the serotype specificity of viral DNA packaging.

    Science.gov (United States)

    Ma, Hsin-Chieh; Hearing, Patrick

    2011-08-01

    The packaging of the adenovirus (Ad) genome into a capsid displays serotype specificity. This specificity has been attributed to viral packaging proteins, the IVa2 protein and the L1-52/55K protein. We previously found that the Ad17 L1-52/55K protein was not able to complement the growth of an Ad5 L1-52/55K mutant virus, whereas two other Ad17 packaging proteins, IVa2 and L4-22K, could complement the growth of Ad5 viruses with mutations in the respective genes. In this report, we investigated why the Ad17 L1-52/55K protein was not able to complement the Ad5 L1-52/55K mutant virus. We demonstrate that the Ad17 L1-52/55K protein binds to the Ad5 IVa2 protein in vitro and the Ad5 packaging domain in vivo, activities previously associated with packaging function. The Ad17 L1-52/55K protein also associates with empty Ad5 capsids. Interestingly, we find that the Ad17 L1-52/55K protein is able to complement the growth of an Ad5 L1-52/55K mutant virus in conjunction with the Ad17 structural protein IIIa. The same result was found with the L1-52/55K and IIIa proteins of several other Ad serotypes, including Ad3 and Ad4. The Ad17 IIIa protein associates with empty Ad5 capsids. Consistent with the complementation results, we find that the IIIa protein interacts with the L1-52/55K protein in vitro and associates with the viral packaging domain in vivo. These results underscore the complex nature of virus assembly and genome encapsidation and provide a new model for how the viral genome may tether to the empty capsid during the encapsidation process.

  16. Plant natriuretic peptides induce proteins diagnostic for an adaptive response to stress

    KAUST Repository

    Turek, Ilona; Marondedze, Claudius; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.

    2014-01-01

    In plants, structural and physiological evidence has suggested the presence of biologically active natriuretic peptides (PNPs). PNPs are secreted into the apoplast, are systemically mobile and elicit a range of responses signaling via cGMP. The PNP-dependent responses include tissue specific modifications of cation transport and changes in stomatal conductance and the photosynthetic rate. PNP also has a critical role in host defense responses. Surprisingly, PNP-homologs are produced by several plant pathogens during host colonization suppressing host defense responses. Here we show that a synthetic peptide representing the biologically active fragment of the Arabidopsis thaliana PNP (AtPNP-A) induces the production of reactive oxygen species in suspension-cultured A. thaliana (Col-0) cells. To identify proteins whose expression changes in an AtPNP-A dependent manner, we undertook a quantitative proteomic approach, employing tandem mass tag (TMT) labeling, to reveal temporal responses of suspension-cultured cells to 1 nM and 10 pM PNP at two different time-points post-treatment. Both concentrations yield a distinct differential proteome signature. Since only the higher (1 nM) concentration induces a ROS response, we conclude that the proteome response at the lower concentration reflects a ROS independent response. Furthermore, treatment with 1 nM PNP results in an over-representation of the gene ontology (GO) terms “oxidation-reduction process,” “translation” and “response to salt stress” and this is consistent with a role of AtPNP-A in the adaptation to environmental stress conditions.

  17. Plant natriuretic peptides induce proteins diagnostic for an adaptive response to stress

    KAUST Repository

    Turek, Ilona

    2014-11-26

    In plants, structural and physiological evidence has suggested the presence of biologically active natriuretic peptides (PNPs). PNPs are secreted into the apoplast, are systemically mobile and elicit a range of responses signaling via cGMP. The PNP-dependent responses include tissue specific modifications of cation transport and changes in stomatal conductance and the photosynthetic rate. PNP also has a critical role in host defense responses. Surprisingly, PNP-homologs are produced by several plant pathogens during host colonization suppressing host defense responses. Here we show that a synthetic peptide representing the biologically active fragment of the Arabidopsis thaliana PNP (AtPNP-A) induces the production of reactive oxygen species in suspension-cultured A. thaliana (Col-0) cells. To identify proteins whose expression changes in an AtPNP-A dependent manner, we undertook a quantitative proteomic approach, employing tandem mass tag (TMT) labeling, to reveal temporal responses of suspension-cultured cells to 1 nM and 10 pM PNP at two different time-points post-treatment. Both concentrations yield a distinct differential proteome signature. Since only the higher (1 nM) concentration induces a ROS response, we conclude that the proteome response at the lower concentration reflects a ROS independent response. Furthermore, treatment with 1 nM PNP results in an over-representation of the gene ontology (GO) terms “oxidation-reduction process,” “translation” and “response to salt stress” and this is consistent with a role of AtPNP-A in the adaptation to environmental stress conditions.

  18. Growth arrest specific protein (GAS) 6

    DEFF Research Database (Denmark)

    Haase, T N; Rasmussen, Morten; Jaksch, C A M

    2013-01-01

    using RNA microarray and quantitative PCR. The role of a differentially expressed gene, growth arrest specific protein 6 (GAS6), was evaluated in vitro using neonatal rat islets. Results The mRNA level of Gas6, known to be mitogenic in other tissues, was reduced in LP offspring. The mRNA content of Mafa...... was increased in LP offspring suggesting an early maturation of beta cells. When applied in vitro, GAS6 increased proliferation of neonatal pancreatic beta cells, while reducing glucose-stimulated insulin secretion without changing the total insulin content of the islets. In addition, GAS6 decreased the m......RNA content of Mafa. Conclusions/interpretation We propose a role for GAS6 in the regulation of pancreatic beta cells in the critical period around the time of birth. Our results support the hypothesis that the reduced beta cell mass seen in LP offspring is caused by a change in the intra-uterine environment...

  19. Detecting consistent patterns of directional adaptation using differential selection codon models.

    Science.gov (United States)

    Parto, Sahar; Lartillot, Nicolas

    2017-06-23

    Phylogenetic codon models are often used to characterize the selective regimes acting on protein-coding sequences. Recent methodological developments have led to models explicitly accounting for the interplay between mutation and selection, by modeling the amino acid fitness landscape along the sequence. However, thus far, most of these models have assumed that the fitness landscape is constant over time. Fluctuations of the fitness landscape may often be random or depend on complex and unknown factors. However, some organisms may be subject to systematic changes in selective pressure, resulting in reproducible molecular adaptations across independent lineages subject to similar conditions. Here, we introduce a codon-based differential selection model, which aims to detect and quantify the fine-grained consistent patterns of adaptation at the protein-coding level, as a function of external conditions experienced by the organism under investigation. The model parameterizes the global mutational pressure, as well as the site- and condition-specific amino acid selective preferences. This phylogenetic model is implemented in a Bayesian MCMC framework. After validation with simulations, we applied our method to a dataset of HIV sequences from patients with known HLA genetic background. Our differential selection model detects and characterizes differentially selected coding positions specifically associated with two different HLA alleles. Our differential selection model is able to identify consistent molecular adaptations as a function of repeated changes in the environment of the organism. These models can be applied to many other problems, ranging from viral adaptation to evolution of life-history strategies in plants or animals.

  20. Whole genome sequencing revealed host adaptation-focused genomic plasticity of pathogenic Leptospira

    Science.gov (United States)

    Xu, Yinghua; Zhu, Yongzhang; Wang, Yuezhu; Chang, Yung-Fu; Zhang, Ying; Jiang, Xiugao; Zhuang, Xuran; Zhu, Yongqiang; Zhang, Jinlong; Zeng, Lingbing; Yang, Minjun; Li, Shijun; Wang, Shengyue; Ye, Qiang; Xin, Xiaofang; Zhao, Guoping; Zheng, Huajun; Guo, Xiaokui; Wang, Junzhi

    2016-01-01

    Leptospirosis, caused by pathogenic Leptospira spp., has recently been recognized as an emerging infectious disease worldwide. Despite its severity and global importance, knowledge about the molecular pathogenesis and virulence evolution of Leptospira spp. remains limited. Here we sequenced and analyzed 102 isolates representing global sources. A high genomic variability were observed among different Leptospira species, which was attributed to massive gene gain and loss events allowing for adaptation to specific niche conditions and changing host environments. Horizontal gene transfer and gene duplication allowed the stepwise acquisition of virulence factors in pathogenic Leptospira evolved from a recent common ancestor. More importantly, the abundant expansion of specific virulence-related protein families, such as metalloproteases-associated paralogs, were exclusively identified in pathogenic species, reflecting the importance of these protein families in the pathogenesis of leptospirosis. Our observations also indicated that positive selection played a crucial role on this bacteria adaptation to hosts. These novel findings may lead to greater understanding of the global diversity and virulence evolution of Leptospira spp. PMID:26833181

  1. Evasion of adaptive and innate immune response mechanisms by γ-herpesviruses

    Science.gov (United States)

    Feng, Pinghui; Moses, Ashlee; Früh, Klaus

    2015-01-01

    γ-Herpesviral immune evasion mechanisms are optimized to support the acute, lytic and the longterm, latent phase of infection. During acute infection, specific immune modulatory proteins limit, but also exploit, the antiviral activities of cell intrinsic innate immune responses as well as those of innate and adaptive immune cells. During latent infection, a restricted gene expression program limits immune targeting and cis-acting mechanisms to reduce the antigen presentation as well as antigenicity of latency-associated proteins. Here, we will review recent progress in our understanding of γ-herpesviral immune evasion strategies. PMID:23735334

  2. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhang, Jun; Xia, Ning-Shao [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China); Miao, Ji, E-mail: jmiao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Life Sciences, Xiamen University, Xiamen, Fujian 361005 (China); Zhao, Qinjian, E-mail: qinjian_zhao@xmu.edu.cn [National Institute of Diagnostics and Vaccine Development in Infectious Diseases, Xiamen University, Xiamen, Fujian 361005 (China); School of Public Health, Xiamen University, Xiamen, Fujian 361005 (China)

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  3. The Polerovirus Minor Capsid Protein Determines Vector Specificity and Intestinal Tropism in the Aphid

    Science.gov (United States)

    Brault, Véronique; Périgon, Sophie; Reinbold, Catherine; Erdinger, Monique; Scheidecker, Danièle; Herrbach, Etienne; Richards, Ken; Ziegler-Graff, Véronique

    2005-01-01

    Aphid transmission of poleroviruses is highly specific, but the viral determinants governing this specificity are unknown. We used a gene exchange strategy between two poleroviruses with different vectors, Beet western yellows virus (BWYV) and Cucurbit aphid-borne yellows virus (CABYV), to analyze the role of the major and minor capsid proteins in vector specificity. Virus recombinants obtained by exchanging the sequence of the readthrough domain (RTD) between the two viruses replicated in plant protoplasts and in whole plants. The hybrid readthrough protein of chimeric viruses was incorporated into virions. Aphid transmission experiments using infected plants or purified virions revealed that vector specificity is driven by the nature of the RTD. BWYV and CABYV have specific intestinal sites in the vectors for endocytosis: the midgut for BWYV and both midgut and hindgut for CABYV. Localization of hybrid virions in aphids by transmission electron microscopy revealed that gut tropism is also determined by the viral origin of the RTD. PMID:16014930

  4. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein.

    Science.gov (United States)

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-11-30

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one of the mitosis-specific phosphorylation residues (i.e., Thr-622). However, the phosphorylation events at the remaining mitotic phosphorylation sites of TMAP have not been fully characterized in detail. Here, we report on generation and characterization of phosphorylated Thr-578- and phosphorylated Thr-596-specific antibodies. Using the antibodies, we show that phosphorylation of TMAP at Thr-578 and Thr-596 indeed occurs specifically during mitosis. Immunofluorescent staining using the antibodies shows that these residues become phosphorylated starting at prophase and then become rapidly dephosphorylated soon after initiation of anaphase. Subtle differences in the kinetics of phosphorylation between Thr-578 and Thr-596 imply that they may be under different mechanisms of phosphorylation during mitosis. Unlike the phosphorylation-deficient mutant form for Thr-622, the mutant in which both Thr-578 and Thr-596 had been mutated to alanines did not induce significant delay in progression of mitosis. These results show that the majority of mitosis-specific phosphorylation of TMAP is limited to pre-anaphase stages and suggest that the multiple phosphorylation may not act in concert but serve diverse functions.

  5. Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N

    2013-01-01

    To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a...... (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...... (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950...

  6. Expressing Adaptation Strategies Using Adaptation Patterns

    Science.gov (United States)

    Zemirline, N.; Bourda, Y.; Reynaud, C.

    2012-01-01

    Today, there is a real challenge to enable personalized access to information. Several systems have been proposed to address this challenge including Adaptive Hypermedia Systems (AHSs). However, the specification of adaptation strategies remains a difficult task for creators of such systems. In this paper, we consider the problem of the definition…

  7. Gene expression and adaptive noncoding changes during human evolution.

    Science.gov (United States)

    Babbitt, Courtney C; Haygood, Ralph; Nielsen, William J; Wray, Gregory A

    2017-06-05

    Despite evidence for adaptive changes in both gene expression and non-protein-coding, putatively regulatory regions of the genome during human evolution, the relationship between gene expression and adaptive changes in cis-regulatory regions remains unclear. Here we present new measurements of gene expression in five tissues of humans and chimpanzees, and use them to assess this relationship. We then compare our results with previous studies of adaptive noncoding changes, analyzing correlations at the level of gene ontology groups, in order to gain statistical power to detect correlations. Consistent with previous studies, we find little correlation between gene expression and adaptive noncoding changes at the level of individual genes; however, we do find significant correlations at the level of biological function ontology groups. The types of function include processes regulated by specific transcription factors, responses to genetic or chemical perturbations, and differentiation of cell types within the immune system. Among functional categories co-enriched with both differential expression and noncoding adaptation, prominent themes include cancer, particularly epithelial cancers, and neural development and function.

  8. Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

    Science.gov (United States)

    Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

    2016-09-16

    The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules.

    Science.gov (United States)

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-11-25

    Membrane-associated guanylate kinases (MAGUKs) are a large family of scaffold proteins that play essential roles in tissue developments, cell-cell communications, cell polarity control, and cellular signal transductions. Despite extensive studies over the past two decades, the functions of the signature guanylate kinase domain (GK) of MAGUKs are poorly understood. Here we show that the GK domain of DLG1/SAP97 binds to asymmetric cell division regulatory protein LGN in a phosphorylation-dependent manner. The structure of the DLG1 SH3-GK tandem in complex with a phospho-LGN peptide reveals that the GMP-binding site of GK has evolved into a specific pSer/pThr-binding pocket. Residues both N- and C-terminal to the pSer are also critical for the specific binding of the phospho-LGN peptide to GK. We further demonstrate that the previously reported GK domain-mediated interactions of DLGs with other targets, such as GKAP/DLGAP1/SAPAP1 and SPAR, are also phosphorylation dependent. Finally, we provide evidence that other MAGUK GKs also function as phospho-peptide-binding modules. The discovery of the phosphorylation-dependent MAGUK GK/target interactions indicates that MAGUK scaffold-mediated signalling complex organizations are dynamically regulated.

  10. [Genes of insecticidal crystal proteins with dual specificity in Bacillus thuringiensis strains, isolated in the Crimea territory].

    Science.gov (United States)

    Rymar, S Iu; Isakova, I A; Kuznietsova, L M; Kordium, V A

    2006-01-01

    The insecticidal crystal proteins of 15 B. thuringiensis strains, isolated in the Crimea territory that are toxical for some Lepidoptera and Colorado potato beetle larvae were identified by PAGE electrophoresis. Ten strains produced the crystal proteins with high molecular weight (> 120 kD). PCR with use of broad specificity primers and DNA of these B. thuringiensis strains as template demonstrated the specific PCR products (1000 bp). Amplified DNA fragments were cloned and sequenced. The nucleotide sequence analysis revealed that the genomes of ten strains of B. thuringiensis carried Cry1B genes, which are responsible for production of the insecticidal crystal proteins with dual specificity. The influence of the solubilization conditions on the structure and toxicity of Cry1B protein for Colorado potato beetle larvae was shown. The dual toxicity of studied B. thuringiensis strains is explained by the Cry1B genes presence in their genomes. These strains may be used to develop the broad specificity bioinsecticides.

  11. Purification and Initial Functions of Sex-Specific Storage Protein 2 in Bombyx mori.

    Science.gov (United States)

    Chen, Jianqing; Shu, Tejun; Chen, Jian; Ye, Man; Lv, Zhengbing; Nie, Zuoming; Gai, Qijing; Yu, Wei; Zhang, Yaozhou

    2015-08-01

    In this study, we identified a heat-resistant protein from the chrysalis stage of the silkworm which we named sex-specific storage protein 2 (SSP2). This protein was stable even at 80 °C, and has an amino acid sequence that is 90.65 % homologous to SP2. We utilized the heat-resistant characteristics of SSP2 to purify the protein and maintain its biological activity. In addition, using flow cytometry and the MTT assay, we found that SSP2 had anti-apoptotic effects on BmN cells, and that SSP2 could also inhibit cell apoptosis induced by chemical factors. These results suggest that SSP2 has a cell-protective function, and provides a basis for future work on the function of storage proteins in silkworm.

  12. Cth2 Protein Mediates Early Adaptation of Yeast Cells to Oxidative Stress Conditions.

    Directory of Open Access Journals (Sweden)

    Laia Castells-Roca

    Full Text Available Cth2 is an mRNA-binding protein that participates in remodeling yeast cell metabolism in iron starvation conditions by promoting decay of the targeted molecules, in order to avoid excess iron consumption. This study shows that in the absence of Cth2 immediate upregulation of expression of several of the iron regulon genes (involved in high affinity iron uptake and intracellular iron redistribution upon oxidative stress by hydroperoxide is more intense than in wild type conditions where Cth2 is present. The oxidative stress provokes a temporary increase in the levels of Cth2 (itself a member of the iron regulon. In such conditions Cth2 molecules accumulate at P bodies-like structures when the constitutive mRNA decay machinery is compromised. In addition, a null Δcth2 mutant shows defects, in comparison to CTH2 wild type cells, in exit from α factor-induced arrest at the G1 stage of the cell cycle when hydroperoxide treatment is applied. The cell cycle defects are rescued in conditions that compromise uptake of external iron into the cytosol. The observations support a role of Cth2 in modulating expression of diverse iron regulon genes, excluding those specifically involved in the reductive branch of the high-affinity transport. This would result in immediate adaptation of the yeast cells to an oxidative stress, by controlling uptake of oxidant-promoting iron cations.

  13. Specific oligobodies against ERK-2 that recognize both the native and the denatured state of the protein.

    Science.gov (United States)

    Bianchini, M; Radrizzani, M; Brocardo, M G; Reyes, G B; Gonzalez Solveyra, C; Santa-Coloma, T A

    2001-06-01

    Oligonucleotide aptamer(s), obtained by using the SELEX procedure, has been used as reagents to recognize different molecules with high affinity and specificity. However, until recently, it was not possible to obtain oligonucleotide-based reagents able to recognize proteins with high specificity in assays typical of antibodies, such as immunohistochemistry, Western blotting and immunoprecipitations. Here, we show the results obtained by applying the strategy of "target switching" to obtain specific polyclonal and monoclonal oligobodies against the protein ERK2. We were able to develop highly specific polyclonal oligobodies by using only one selection step with a temporary target and one selection step with the final target (ERK2). Since only two selection steps were required, these results demonstrate that it is possible to obtain specific reagents against a protein without a need for an "in vitro evolution" using many selection steps, or error-prone polymerases. After one additional selection step, the polyclonal oligobodies were cloned to obtain a highly specific monoclonal oligobody.

  14. Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT).

    Science.gov (United States)

    Bernat, Przemysław; Siewiera, Paulina; Soboń, Adrian; Długoński, Jerzy

    2014-09-01

    A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58%) and phosphatidylethanolamine (by 70%). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.

  15. Alteration of the carboxyl-terminal domain of Ada protein influences its inducibility, specificity, and strength as a transcriptional activator.

    OpenAIRE

    Shevell, D E; LeMotte, P K; Walker, G C

    1988-01-01

    The ada gene of Escherichia coli K-12 encodes the regulatory protein for the adaptive response to alkylating agents. A set of plasmids carrying ordered deletions from the 3' end of the ada gene were isolated and characterized. These ada deletions encode fusion proteins that derive their amino termini from ada and their carboxyl termini from the downstream vector sequence that occurs before an in-frame stop codon. Several of these ada deletions encode Ada derivatives that constitutively activa...

  16. Speed Controls in Translating Secretory Proteins in Eukaryotes - an Evolutionary Perspective

    Science.gov (United States)

    Mahlab, Shelly; Linial, Michal

    2014-01-01

    Protein translation is the most expensive operation in dividing cells from bacteria to humans. Therefore, managing the speed and allocation of resources is subject to tight control. From bacteria to humans, clusters of relatively rare tRNA codons at the N′-terminal of mRNAs have been implicated in attenuating the process of ribosome allocation, and consequently the translation rate in a broad range of organisms. The current interpretation of “slow” tRNA codons does not distinguish between protein translations mediated by free- or endoplasmic reticulum (ER)-bound ribosomes. We demonstrate that proteins translated by free- or ER-bound ribosomes exhibit different overall properties in terms of their translation efficiency and speed in yeast, fly, plant, worm, bovine and human. We note that only secreted or membranous proteins with a Signal peptide (SP) are specified by segments of “slow” tRNA at the N′-terminal, followed by abundant codons that are considered “fast.” Such profiles apply to 3100 proteins of the human proteome that are composed of secreted and signal peptide (SP)-assisted membranous proteins. Remarkably, the bulks of the proteins (12,000), or membranous proteins lacking SP (3400), do not have such a pattern. Alternation of “fast” and “slow” codons was found also in proteins that translocate to mitochondria through transit peptides (TP). The differential clusters of tRNA adapted codons is not restricted to the N′-terminal of transcripts. Specifically, Glycosylphosphatidylinositol (GPI)-anchored proteins are unified by clusters of low adapted tRNAs codons at the C′-termini. Furthermore, selection of amino acids types and specific codons was shown as the driving force which establishes the translation demands for the secretory proteome. We postulate that “hard-coded” signals within the secretory proteome assist the steps of protein maturation and folding. Specifically, “speed control” signals for delaying the translation

  17. Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal

    OpenAIRE

    Narayanan, Krishna; Chen, Chun-Jen; Maeda, Junko; Makino, Shinji

    2003-01-01

    For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into vi...

  18. Species-Specific Monoclonal Antibodies to Escherichia coli-Expressed p36 Cytosolic Protein of Mycoplasma hyopneumoniae

    Science.gov (United States)

    Caron, J.; Sawyer, N.; Moumen, B. Ben Abdel; Bouh, K. Cheikh Saad; Dea, S.

    2000-01-01

    The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an

  19. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  20. Optimization of photoactive protein Z for fast and efficient site-specific conjugation of native IgG.

    Science.gov (United States)

    Hui, James Z; Tsourkas, Andrew

    2014-09-17

    Antibody conjugates have been used in a variety of applications from immunoassays to drug conjugates. However, it is becoming increasingly clear that in order to maximize an antibody's antigen binding ability and to produce homogeneous antibody-conjugates, the conjugated molecule should be attached onto IgG site-specifically. We previously developed a facile method for the site-specific modification of full length, native IgGs by engineering a recombinant Protein Z that forms a covalent link to the Fc domain of IgG upon exposure to long wavelength UV light. To further improve the efficiency of Protein Z production and IgG conjugation, we constructed a panel of 13 different Protein Z variants with the UV-active amino acid benzoylphenylalanine (BPA) in different locations. By using this panel of Protein Z to cross-link a range of IgGs from different hosts, including human, mouse, and rat, we discovered two previously unknown Protein Z variants, L17BPA and K35BPA, that are capable of cross-linking many commonly used IgG isotypes with efficiencies ranging from 60% to 95% after only 1 h of UV exposure. When compared to existing site-specific methods, which often require cloning or enzymatic reactions, the Protein Z-based method described here, utilizing the L17BPA, K35BPA, and the previously described Q32BPA variants, represents a vastly more accessible and efficient approach that is compatible with nearly all native IgGs, thus making site-specific conjugation more accessible to the general research community.

  1. Nature of protein family signatures: insights from singular value analysis of position-specific scoring matrices.

    Directory of Open Access Journals (Sweden)

    Akira R Kinjo

    Full Text Available Position-specific scoring matrices (PSSMs are useful for detecting weak homology in protein sequence analysis, and they are thought to contain some essential signatures of the protein families. In order to elucidate what kind of ingredients constitute such family-specific signatures, we apply singular value decomposition to a set of PSSMs and examine the properties of dominant right and left singular vectors. The first right singular vectors were correlated with various amino acid indices including relative mutability, amino acid composition in protein interior, hydropathy, or turn propensity, depending on proteins. A significant correlation between the first left singular vector and a measure of site conservation was observed. It is shown that the contribution of the first singular component to the PSSMs act to disfavor potentially but falsely functionally important residues at conserved sites. The second right singular vectors were highly correlated with hydrophobicity scales, and the corresponding left singular vectors with contact numbers of protein structures. It is suggested that sequence alignment with a PSSM is essentially equivalent to threading supplemented with functional information. In addition, singular vectors may be useful for analyzing and annotating the characteristics of conserved sites in protein families.

  2. Host-specific adaptation of HIV-1 subtype B in the Japanese population.

    Science.gov (United States)

    Chikata, Takayuki; Carlson, Jonathan M; Tamura, Yoshiko; Borghan, Mohamed Ali; Naruto, Takuya; Hashimoto, Masao; Murakoshi, Hayato; Le, Anh Q; Mallal, Simon; John, Mina; Gatanaga, Hiroyuki; Oka, Shinichi; Brumme, Zabrina L; Takiguchi, Masafumi

    2014-05-01

    The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However, HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naive Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol, and Nef using phylogenetically corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with the plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate the pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-APs identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways in Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures. Cytotoxic T lymphocyte (CTL) escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele distributions differ among

  3. Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

    Science.gov (United States)

    Mehic, Denis; Bakiri, Latifa; Ghannadan, Minoo; Wagner, Erwin F; Tschachler, Erwin

    2005-01-01

    Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

  4. Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

    Science.gov (United States)

    Ouyang, Ruizhuo; Lei, Jianping; Ju, Huangxian

    2010-05-01

    This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 × 1018 g - 1, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

  5. Artificial receptor-functionalized nanoshell: facile preparation, fast separation and specific protein recognition

    Energy Technology Data Exchange (ETDEWEB)

    Ouyang, Ruizhuo; Lei Jianping; Ju Huangxian, E-mail: jpl@nju.edu.cn, E-mail: hxju@nju.edu.cn [Key Laboratory of Analytical Chemistry for Life Science (Education Ministry of China), Department of Chemistry, Nanjing University, Nanjing 210093 (China)

    2010-05-07

    This work combined molecular imprinting technology with superparamagnetic nanospheres as the core to prepare artificial receptor-functionalized magnetic nanoparticles for separation of homologous proteins. Using dopamine as a functional monomer, novel surface protein-imprinted superparamagnetic polydopamine (PDA) core-shell nanoparticles were successfully prepared in physiological conditions, which could maintain the natural structure of a protein template and achieved the development of molecularly imprinted polymers (MIPs) from one dimension to zero dimension for efficient recognition towards large biomolecules. The resultant nanoparticles could be used for convenient magnetic separation of homologous proteins with high specificity. The nanoparticles possessed good monodispersibility, uniform surface morphology and high saturation magnetization value. The bound amounts of template proteins measured by both indirect and direct methods were in good agreement. The maximum number of imprinted cavities on the surface of the bovine hemoglobin (Hb)-imprinted nanoshell was 2.21 x 10{sup 18} g{sup -1}, which well matched their maximum binding capacity toward bovine Hb. Both the simple method for preparation of MIPs and the magnetic nanospheres showed good application potential in fast separation, effective concentration and selective biosensing of large protein molecules.

  6. A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    McClure, B; Mou, B; Canevascini, S; Bernatzky, R

    1999-11-09

    Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

  7. Microdosimetric constraints on specific adaptation mechanisms to reduce DNA damage caused by ionising radiation

    International Nuclear Information System (INIS)

    Burkart, W.; Heusser, P.; Vijayalaxmi

    1990-01-01

    The protective effect of pre-exposure of lymphocytes to ionising radiation indicates the presence of 'adaptive repair' in mammalian cells. Microdosimetric considerations, however, raise some doubts on the advantage of such a cellular mechanism for specifically reducing the radiation damage caused by environmental exposures. Contrary to most chemicals which endanger the integrity of the mammalian genome, the local dose and dose rate from ionising radiation at the cellular level remain quite high, even at lowest exposures. A single electron or alpha particle passing through a cell nucleus already yields nuclear doses of up to about 3 mGy and 400 mGy, respectively. Macroscopic doses below these nuclear doses from a single event will only reduce the fraction of cell nuclei encountering the passage of a particle but not the dose or dose rate in the affected volume. At environmental doses in the range of 1 to 5 mGy per annum, the time between two consecutive hits in a specific cell nucleus is in the range of months to years. Very low concentrations of bleomycin, a drug with high affinity to DNA, also triggers an adaptive response. This points to a more general stress response mechanism which may benefit the cell even at environmental levels of radioactivity, e.g. by protecting the integrity of DNA from attacks by chemicals, by endogenous radicals, by acids from anoxia, etc. (author)

  8. Natural selection in avian protein-coding genes expressed in brain.

    Science.gov (United States)

    Axelsson, Erik; Hultin-Rosenberg, Lina; Brandström, Mikael; Zwahlén, Martin; Clayton, David F; Ellegren, Hans

    2008-06-01

    The evolution of birds from theropod dinosaurs took place approximately 150 million years ago, and was associated with a number of specific adaptations that are still evident among extant birds, including feathers, song and extravagant secondary sexual characteristics. Knowledge about the molecular evolutionary background to such adaptations is lacking. Here, we analyse the evolution of > 5000 protein-coding gene sequences expressed in zebra finch brain by comparison to orthologous sequences in chicken. Mean d(N)/d(S) is 0.085 and genes with their maximal expression in the eye and central nervous system have the lowest mean d(N)/d(S) value, while those expressed in digestive and reproductive tissues exhibit the highest. We find that fast-evolving genes (those which have higher than expected rate of nonsynonymous substitution, indicative of adaptive evolution) are enriched for biological functions such as fertilization, muscle contraction, defence response, response to stress, wounding and endogenous stimulus, and cell death. After alignment to mammalian orthologues, we identify a catalogue of 228 genes that show a significantly higher rate of protein evolution in the two bird lineages than in mammals. These accelerated bird genes, representing candidates for avian-specific adaptations, include genes implicated in vocal learning and other cognitive processes. Moreover, colouration genes evolve faster in birds than in mammals, which may have been driven by sexual selection for extravagant plumage characteristics.

  9. Specificity and sensitivity of noninvasive measurement of pulmonary vascular protein leak

    International Nuclear Information System (INIS)

    Dauber, I.M.; Pluss, W.T.; VanGrondelle, A.; Trow, R.S.; Weil, J.V.

    1985-01-01

    Noninvasive techniques employing external counting of radiolabeled protein have the potential for measuring pulmonary vascular protein permeability, but their specificity and sensitivity remain unclear. The authors tested the specificity and sensitivity of a double-radioisotope method by injecting radiolabeled albumin ( 131 I) and erythrocytes (/sup 99m/Tc) into anesthetized dogs and measuring the counts of each isotope for 150 min after injection with an external gamma probe fixed over the lung. They calculated the rate of increase of albumin counts measured by the probe (which reflects the rate at which protein leaks into the extravascular space). To assess permeability the authors normalized the rate of increase in albumin counts for changes in labeled erythrocyte signal to minimize influence of changes in vascular surface area and thus derived an albumin leak index. They measured the albumin leak index and gravimetric lung water during hydrostatic edema (acutely elevating left atrial pressure by left atrial balloon inflation: mean pulmonary arterial wedge pressure = 22.6 Torr) and in lung injury edema induced by high- (1.0 g/kg) and low-dose (0.25 g/kg) intravenous thiourea. To test specificity hydrostatic and high-dose thiourea edema were compared. The albumin leak index increased nearly fourfold from control after thiourea injury (27.2 +/- 2.3 x 10-4 vs. 7.6 +/- 0.9 x 10-4 min-1) but did not change from control levels after elevating left atrial pressure (8.9 +/- 1.2 x 10-4 min-1) despite comparable increases in gravimetric lung water. To test sensitivity the authors compared low-dose thiourea with controls. Following low-dose thiourea, the albumin leak index nearly doubled despite the absence of a measurable increase in lung water

  10. Profiling Humoral Immune Responses to Clostridium difficile-Specific Antigens by Protein Microarray Analysis.

    Science.gov (United States)

    Negm, Ola H; Hamed, Mohamed R; Dilnot, Elizabeth M; Shone, Clifford C; Marszalowska, Izabela; Lynch, Mark; Loscher, Christine E; Edwards, Laura J; Tighe, Patrick J; Wilcox, Mark H; Monaghan, Tanya M

    2015-09-01

    Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotype-specific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  11. Adaptive enhanced sampling with a path-variable for the simulation of protein folding and aggregation

    Science.gov (United States)

    Peter, Emanuel K.

    2017-12-01

    In this article, we present a novel adaptive enhanced sampling molecular dynamics (MD) method for the accelerated simulation of protein folding and aggregation. We introduce a path-variable L based on the un-biased momenta p and displacements dq for the definition of the bias s applied to the system and derive 3 algorithms: general adaptive bias MD, adaptive path-sampling, and a hybrid method which combines the first 2 methodologies. Through the analysis of the correlations between the bias and the un-biased gradient in the system, we find that the hybrid methodology leads to an improved force correlation and acceleration in the sampling of the phase space. We apply our method on SPC/E water, where we find a conservation of the average water structure. We then use our method to sample dialanine and the folding of TrpCage, where we find a good agreement with simulation data reported in the literature. Finally, we apply our methodologies on the initial stages of aggregation of a hexamer of Alzheimer's amyloid β fragment 25-35 (Aβ 25-35) and find that transitions within the hexameric aggregate are dominated by entropic barriers, while we speculate that especially the conformation entropy plays a major role in the formation of the fibril as a rate limiting factor.

  12. Design of application specific long period waveguide grating filters using adaptive particle swarm optimization algorithms

    International Nuclear Information System (INIS)

    Semwal, Girish; Rastogi, Vipul

    2014-01-01

    We present design optimization of wavelength filters based on long period waveguide gratings (LPWGs) using the adaptive particle swarm optimization (APSO) technique. We demonstrate optimization of the LPWG parameters for single-band, wide-band and dual-band rejection filters for testing the convergence of APSO algorithms. After convergence tests on the algorithms, the optimization technique has been implemented to design more complicated application specific filters such as erbium doped fiber amplifier (EDFA) amplified spontaneous emission (ASE) flattening, erbium doped waveguide amplifier (EDWA) gain flattening and pre-defined broadband rejection filters. The technique is useful for designing and optimizing the parameters of LPWGs to achieve complicated application specific spectra. (paper)

  13. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Science.gov (United States)

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum. PMID:25925056

  14. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    Directory of Open Access Journals (Sweden)

    Jae Eun Lee

    2015-06-01

    Full Text Available Two dimensional-fluorescence difference gel electrophoresis (2D DIGE is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2 non-pregnant were used in this study. The pre-electrophoretic labeling of pregnant and non-pregnant serum proteins were mixed with Cy3 and Cy5 fluorescent dyes, respectively, and an internal standard was labeled with Cy2. Labeled proteins with Cy2, Cy3, and Cy5 were separated together in a single gel, and then were detected by fluorescence image analyzer. The 2D DIGE method using fluorescence CyDye DIGE flour had higher sensitivity than conventional 2D gel electrophoresis, and showed reproducible results. Approximately 1,500 protein spots were detected by 2D DIGE. Several proteins showed a more than 1.5-fold up and down regulation between non-pregnant and pregnant serum proteins. The differentially expressed proteins were identified by MALDI-TOF mass spectrometer. A total 16 protein spots were detected to regulate differentially in the pregnant serum, among which 7 spots were up-regulated proteins such as conglutinin precursor, modified bovine fibrinogen and IgG1, and 6 spots were down-regulated proteins such as hemoglobin, complement component 3, bovine fibrinogen and IgG2a three spots were not identified. The identified proteins demonstrate that early pregnant bovine serum may have several pregnancy-specific proteins, and these could be a valuable information for the development of pregnancy-diagnostic markers in early pregnancy bovine serum.

  15. Signature proteins for the major clades of Cyanobacteria

    Directory of Open Access Journals (Sweden)

    Mathews Divya W

    2010-01-01

    Full Text Available Abstract Background The phylogeny and taxonomy of cyanobacteria is currently poorly understood due to paucity of reliable markers for identification and circumscription of its major clades. Results A combination of phylogenomic and protein signature based approaches was used to characterize the major clades of cyanobacteria. Phylogenetic trees were constructed for 44 cyanobacteria based on 44 conserved proteins. In parallel, Blastp searches were carried out on each ORF in the genomes of Synechococcus WH8102, Synechocystis PCC6803, Nostoc PCC7120, Synechococcus JA-3-3Ab, Prochlorococcus MIT9215 and Prochlor. marinus subsp. marinus CCMP1375 to identify proteins that are specific for various main clades of cyanobacteria. These studies have identified 39 proteins that are specific for all (or most cyanobacteria and large numbers of proteins for other cyanobacterial clades. The identified signature proteins include: (i 14 proteins for a deep branching clade (Clade A of Gloebacter violaceus and two diazotrophic Synechococcus strains (JA-3-3Ab and JA2-3-B'a; (ii 5 proteins that are present in all other cyanobacteria except those from Clade A; (iii 60 proteins that are specific for a clade (Clade C consisting of various marine unicellular cyanobacteria (viz. Synechococcus and Prochlorococcus; (iv 14 and 19 signature proteins that are specific for the Clade C Synechococcus and Prochlorococcus strains, respectively; (v 67 proteins that are specific for the Low B/A ecotype Prochlorococcus strains, containing lower ratio of chl b/a2 and adapted to growth at high light intensities; (vi 65 and 8 proteins that are specific for the Nostocales and Chroococcales orders, respectively; and (vii 22 and 9 proteins that are uniquely shared by various Nostocales and Oscillatoriales orders, or by these two orders and the Chroococcales, respectively. We also describe 3 conserved indels in flavoprotein, heme oxygenase and protochlorophyllide oxidoreductase proteins that

  16. Coupling genetics and proteomics to identify aphid proteins associated with vector-specific transmission of polerovirus (luteoviridae).

    Science.gov (United States)

    Yang, Xiaolong; Thannhauser, T W; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E; Gray, Stewart M

    2008-01-01

    Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F(2) progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F(2) genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

  17. Protein: MPA1 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available MPA1 TLR signaling molecules MAVS IPS1, KIAA1271, VISA VISA_(gene) Mitochondrial antiviral-signaling pr...otein CARD adapter inducing interferon beta, Interferon beta promoter stimulator protein... 1, Putative NF-kappa-B-activating protein 031N, Virus-induced-signaling adapter 9606 Homo sapiens Q7Z434 57506 2VGQ 57506 ...

  18. Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death.

    Science.gov (United States)

    Gorban, Alexander N; Tyukina, Tatiana A; Smirnova, Elena V; Pokidysheva, Lyudmila I

    2016-09-21

    In 1938, Selye proposed the notion of adaptation energy and published 'Experimental evidence supporting the conception of adaptation energy.' Adaptation of an animal to different factors appears as the spending of one resource. Adaptation energy is a hypothetical extensive quantity spent for adaptation. This term causes much debate when one takes it literally, as a physical quantity, i.e. a sort of energy. The controversial points of view impede the systematic use of the notion of adaptation energy despite experimental evidence. Nevertheless, the response to many harmful factors often has general non-specific form and we suggest that the mechanisms of physiological adaptation admit a very general and nonspecific description. We aim to demonstrate that Selye׳s adaptation energy is the cornerstone of the top-down approach to modelling of non-specific adaptation processes. We analyze Selye׳s axioms of adaptation energy together with Goldstone׳s modifications and propose a series of models for interpretation of these axioms. Adaptation energy is considered as an internal coordinate on the 'dominant path' in the model of adaptation. The phenomena of 'oscillating death' and 'oscillating remission' are predicted on the base of the dynamical models of adaptation. Natural selection plays a key role in the evolution of mechanisms of physiological adaptation. We use the fitness optimization approach to study of the distribution of resources for neutralization of harmful factors, during adaptation to a multifactor environment, and analyze the optimal strategies for different systems of factors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Recombinational DSBs-intersected genes converge on specific disease- and adaptability-related pathways.

    Science.gov (United States)

    Yang, Zhi-Kai; Luo, Hao; Zhang, Yanming; Wang, Baijing; Gao, Feng

    2018-05-03

    The budding yeast Saccharomyces cerevisiae is a model species powerful for studying the recombination of eukaryotes. Although many recombination studies have been performed for this species by experimental methods, the population genomic study based on bioinformatics analyses is urgently needed to greatly increase the range and accuracy of recombination detection. Here, we carry out the population genomic analysis of recombination in S. cerevisiae to reveal the potential rules between recombination and evolution in eukaryotes. By population genomic analysis, we discover significantly more and longer recombination events in clinical strains, which indicates that adverse environmental conditions create an obviously wider range of genetic combination in response to the selective pressure. Based on the analysis of recombinational DSBs-intersected genes (RDIGs), we find that RDIGs significantly converge on specific disease- and adaptability-related pathways, indicating that recombination plays a biologically key role in the repair of DSBs related to diseases and environmental adaptability, especially the human neurological disorders (NDs). By evolutionary analysis of RDIGs, we find that the RDIGs highly prevailing in populations of yeast tend to be more evolutionarily conserved, indicating the accurate repair of DSBs in these RDIGs is critical to ensure the eukaryotic survival or fitness. fgao@tju.edu.cn. Supplementary data are available at Bioinformatics online.

  20. Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

    Directory of Open Access Journals (Sweden)

    Amy eCurran

    2011-08-01

    Full Text Available The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs. While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16 and 34. Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ~70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites. Of these, 74 (27% were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

  1. Recent advances in covalent, site-specific protein immobilization [version 1; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Morten Meldal

    2016-09-01

    Full Text Available The properties of biosensors, biomedical implants, and other materials based on immobilized proteins greatly depend on the method employed to couple the protein molecules to their solid support. Covalent, site-specific immobilization strategies are robust and can provide the level of control that is desired in this kind of application. Recent advances include the use of enzymes, such as sortase A, to couple proteins in a site-specific manner to materials such as microbeads, glass, and hydrogels. Also, self-labeling tags such as the SNAP-tag can be employed. Last but not least, chemical approaches based on bioorthogonal reactions, like the azide–alkyne cycloaddition, have proven to be powerful tools. The lack of comparative studies and quantitative analysis of these immobilization methods hampers the selection process of the optimal strategy for a given application. However, besides immobilization efficiency, the freedom in selecting the site of conjugation and the size of the conjugation tag and the researcher’s expertise regarding molecular biology and/or chemical techniques will be determining factors in this regard.

  2. Evaluation of cross-cultural adaptation and measurement properties of breast cancer-specific quality-of-life questionnaires: a systematic review.

    Science.gov (United States)

    Oliveira, Indiara Soares; da Cunha Menezes Costa, Lucíola; Fagundes, Felipe Ribeiro Cabral; Cabral, Cristina Maria Nunes

    2015-05-01

    To assess the procedures of translation, cross-cultural adaptation, and measurement properties of breast cancer-specific quality-of-life questionnaires. Searches were conducted in the databases MEDLINE, EMBASE, CINAHL, and SciELO using the keywords: "Questionnaires," "Quality of life," and "Breast cancer." The studies were analyzed in terms of methodological quality according to the guidelines for the procedure of cross-cultural adaptation and the quality criteria for measurement properties of questionnaires. We found 24 eligible studies. Most of the articles assessed the translation and measurement properties of the instrument EORTC QLQ-BR23. Description about translation and cross-cultural adaptation was incomplete in 11 studies. Translation and back translation were the most tested phases, and synthesis of the translation was the most omitted phase in the articles. Information on assessing measurement properties was provided incompletely in 23 articles. Internal consistency was the most tested property in all of the eligible articles, but none of them provided information on agreement. Construct validity was adequately tested in only three studies that used the FACT-B and QLQ-BR23. Eight articles provided information on reliability; however, only four found positive classification. Responsiveness was tested in four articles, and ceiling and floor effects were tested in only three articles. None of the instruments showed fully adequate quality. There is limited evidence on cross-cultural adaptations and measurement properties; therefore, it is recommended that caution be exercised when using breast cancer-specific quality-of-life questionnaires that have been translated, adapted, and tested.

  3. Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

    Science.gov (United States)

    Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

    2006-12-21

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

  4. Salt-bridge energetics in halophilic proteins.

    Science.gov (United States)

    Nayek, Arnab; Sen Gupta, Parth Sarthi; Banerjee, Shyamashree; Mondal, Buddhadev; Bandyopadhyay, Amal K

    2014-01-01

    Halophilic proteins have greater abundance of acidic over basic and very low bulky hydrophobic residues. Classical electrostatic stabilization was suggested as the key determinant for halophilic adaptation of protein. However, contribution of specific electrostatic interactions (i.e. salt-bridges) to overall stability of halophilic proteins is yet to be understood. To understand this, we use Adaptive-Poison-Boltzmann-Solver Methods along with our home-built automation to workout net as well as associated component energy terms such as desolvation energy, bridge energy and background energy for 275 salt-bridges from 20 extremely halophilic proteins. We then perform extensive statistical analysis on general and energetic attributes on these salt-bridges. On average, 8 salt-bridges per 150 residues protein were observed which is almost twice than earlier report. Overall contributions of salt-bridges are -3.0 kcal mol-1. Majority (78%) of salt-bridges in our dataset are stable and conserved in nature. Although, average contributions of component energy terms are equal, their individual details vary greatly from one another indicating their sensitivity to local micro-environment. Notably, 35% of salt-bridges in our database are buried and stable. Greater desolvation penalty of these buried salt-bridges are counteracted by stable network salt-bridges apart from favorable equal contributions of bridge and background terms. Recruitment of extensive network salt-bridges (46%) with a net contribution of -5.0 kcal mol-1 per salt-bridge, seems to be a halophilic design wherein favorable average contribution of background term (-10 kcal mol-1) exceeds than that of bridge term (-7 kcal mol-1). Interiors of proteins from halophiles are seen to possess relatively higher abundance of charge and polar side chains than that of mesophiles which seems to be satisfied by cooperative network salt-bridges. Overall, our theoretical analyses provide insight into halophilic signature in its

  5. Bromodomain protein 4 discriminates tissue-specific super-enhancers containing disease-specific susceptibility loci in prostate and breast cancer

    DEFF Research Database (Denmark)

    Zuber, Verena; Bettella, Francesco; Witoelar, Aree

    2017-01-01

    progression. Although previous approaches have been tried to explain risk associated with SNPs in regulatory DNA elements, so far epigenetic readers such as bromodomain containing protein 4 (BRD4) and super-enhancers have not been used to annotate SNPs. In prostate cancer (PC), androgen receptor (AR) binding......Background: Epigenetic information can be used to identify clinically relevant genomic variants single nucleotide polymorphisms (SNPs) of functional importance in cancer development. Super-enhancers are cell-specific DNA elements, acting to determine tissue or cell identity and driving tumor...... the differential enrichment of SNPs mapping to specific categories of enhancers. We find that BRD4 is the key discriminant of tissue-specific enhancers, showing that it is more powerful than AR binding information to capture PC specific risk loci, and can be used with similar effect in breast cancer (BC...

  6. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    Science.gov (United States)

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  7. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

    Directory of Open Access Journals (Sweden)

    Daniel Bello-Gil

    Full Text Available We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

  8. Site-specific fluorescent labeling of nascent proteins on the translating ribosome.

    Science.gov (United States)

    Saraogi, Ishu; Zhang, Dawei; Chandrasekaran, Sandhya; Shan, Shu-ou

    2011-09-28

    As newly synthesized proteins emerge from the ribosome, they interact with a variety of cotranslational cellular machineries that facilitate their proper folding, maturation, and localization. These interactions are essential for proper function of the cell, and the ability to study these events is crucial to understanding cellular protein biogenesis. To this end, we have developed a highly efficient method to generate ribosome-nascent chain complexes (RNCs) site-specifically labeled with a fluorescent dye on the nascent polypeptide. The fluorescent RNC provides real-time, quantitative information on its cotranslational interaction with the signal recognition particle and will be a valuable tool in elucidating the role of the translating ribosome in numerous biochemical pathways.

  9. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

    Directory of Open Access Journals (Sweden)

    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  10. Specificity and sensitivity of binding proteins in the radioimmunoassay of cortisol

    International Nuclear Information System (INIS)

    Gijzen, A.H.J.

    1977-01-01

    A comparison concerning avidity towards cortisol and 10 other steroids was made between several binding proteins either in solution or bound to cellulose as so called ''solid phase'' reagent. Human blood cortisol binding protein (CBP, transcortin), and two distinctly different cortisol-binding rabbit antisera and the isolated immunoglobulins thereof were compared in their avidity to bind cortisol and several other steroids. The antisera were harvested from rabbits immunized with either cortisol-21-succinyl-albumin (CSA) or cortisol-3-oxim-albumin (COA). The latter antiserum, having the highest titre in cortisol titration, showed the greatest specificity and was most useful as a binding reagent in cortisol radioimmunoassay when used as a solid phase reagent. The determination of cortisol in micro samples of blood serum is possible without steroid extraction or serum protein denaturation and with only minor influence of steroid impurities in the sample to be analyzed. Affinity constants for all compared binding reagents and steroids are given

  11. Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

    Science.gov (United States)

    Zhu, Zhikai; Desaire, Heather

    2015-07-01

    Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

  12. Validation of a Cross-cultural Adaptation of the Hair Specific Skindex-29 Scale to Spanish.

    Science.gov (United States)

    Guerra-Tapia, A; Buendía-Eisman, A; Ferrando, J

    2018-06-01

    Female androgenetic alopecia (FAA) has considerable impact on quality of life. Our analysis of the clinical scales available in the literature to measure the impact of FAA led us to choose the Hair Specific Skindex 29 (HSS29) as the most appropriate for adaptation to Spanish as a tool for following patients in treatment for FAA. This tool assesses disease impact on quality of life over time so that treatments can be tailored to patients' needs. The HSS29 score reflects impact in 3 domains (emotions, symptoms, and functioning) on a scale of 0 (no effect) to 100 (maximum effect). The scale is useful in routine clinical practice, and patients can respond to all items in 5minutes. We followed recommended procedures to produce a cross-cultural adaptation of the scale. The process involved forward translation of the questionnaire to Spanish followed by back translation by 2 native speakers of the original language (English) and revision as needed after discussion and consensus by a committee of 3 expert dermatologists. The comprehensibility of the resulting translation was assessed in a test-retest step. Next, the psychometric properties, reliability, and construct validity were assessed. Sensitivity and specificity were evaluated with the area under the receiver operating characteristic (ROC) curve, reliability with Cronbach's α, and construct validity by factor analysis using a Varimax rotation. Face validity was also assessed during the process. The intraclass correlation coefficient (ICC) was calculated in the test-retest step. A total of 170 women with FAA and 30 control subjects completed the cross-culturally adapted Spanish questionnaire. A subgroup of 15 subjects responded a second time between 1 and 2 days after their first session (test-retest). Sensitivity and specificity were excellent according to the area under the ROC curve (0.98; 95% CI, 0.97-0.99), and high reliability was reflected by a Cronbach's α of 0.96. Factor analysis showed that the items were

  13. Poliovirus intrahost evolution is required to overcome tissue-specific innate immune responses.

    Science.gov (United States)

    Xiao, Yinghong; Dolan, Patrick Timothy; Goldstein, Elizabeth Faul; Li, Min; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

    2017-08-29

    RNA viruses, such as poliovirus, have a great evolutionary capacity, allowing them to quickly adapt and overcome challenges encountered during infection. Here we show that poliovirus infection in immune-competent mice requires adaptation to tissue-specific innate immune microenvironments. The ability of the virus to establish robust infection and virulence correlates with its evolutionary capacity. We further identify a region in the multi-functional poliovirus protein 2B as a hotspot for the accumulation of minor alleles that facilitate a more effective suppression of the interferon response. We propose that population genetic dynamics enables poliovirus spread between tissues through optimization of the genetic composition of low frequency variants, which together cooperate to circumvent tissue-specific challenges. Thus, intrahost virus evolution determines pathogenesis, allowing a dynamic regulation of viral functions required to overcome barriers to infection.RNA viruses, such as polioviruses, have a great evolutionary capacity and can adapt quickly during infection. Here, the authors show that poliovirus infection in mice requires adaptation to innate immune microenvironments encountered in different tissues.

  14. Design of Protein Multi-specificity Using an Independent Sequence Search Reduces the Barrier to Low Energy Sequences.

    Directory of Open Access Journals (Sweden)

    Alexander M Sevy

    2015-07-01

    Full Text Available Computational protein design has found great success in engineering proteins for thermodynamic stability, binding specificity, or enzymatic activity in a 'single state' design (SSD paradigm. Multi-specificity design (MSD, on the other hand, involves considering the stability of multiple protein states simultaneously. We have developed a novel MSD algorithm, which we refer to as REstrained CONvergence in multi-specificity design (RECON. The algorithm allows each state to adopt its own sequence throughout the design process rather than enforcing a single sequence on all states. Convergence to a single sequence is encouraged through an incrementally increasing convergence restraint for corresponding positions. Compared to MSD algorithms that enforce (constrain an identical sequence on all states the energy landscape is simplified, which accelerates the search drastically. As a result, RECON can readily be used in simulations with a flexible protein backbone. We have benchmarked RECON on two design tasks. First, we designed antibodies derived from a common germline gene against their diverse targets to assess recovery of the germline, polyspecific sequence. Second, we design "promiscuous", polyspecific proteins against all binding partners and measure recovery of the native sequence. We show that RECON is able to efficiently recover native-like, biologically relevant sequences in this diverse set of protein complexes.

  15. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G.; Ribeiro, José M. C.; Andersen, John F.

    2017-07-27

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes, Culex, and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary “long” D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  16. A mosquito hemolymph odorant-binding protein family member specifically binds juvenile hormone.

    Science.gov (United States)

    Kim, Il Hwan; Pham, Van; Jablonka, Willy; Goodman, Walter G; Ribeiro, José M C; Andersen, John F

    2017-09-15

    Juvenile hormone (JH) is a key regulator of insect development and reproduction. In adult mosquitoes, it is essential for maturation of the ovary and normal male reproductive behavior, but how JH distribution and activity is regulated after secretion is unclear. Here, we report a new type of specific JH-binding protein, given the name mosquito juvenile hormone-binding protein (mJHBP), which circulates in the hemolymph of pupal and adult Aedes aegypti males and females. mJHBP is a member of the odorant-binding protein (OBP) family, and orthologs are present in the genomes of Aedes , Culex , and Anopheles mosquito species. Using isothermal titration calorimetry, we show that mJHBP specifically binds JH II and JH III but not eicosanoids or JH derivatives. mJHBP was crystallized in the presence of JH III and found to have a double OBP domain structure reminiscent of salivary "long" D7 proteins of mosquitoes. We observed that a single JH III molecule is contained in the N-terminal domain binding pocket that is closed in an apparent conformational change by a C-terminal domain-derived α-helix. The electron density for the ligand indicated a high occupancy of the natural 10 R enantiomer of JH III. Of note, mJHBP is structurally unrelated to hemolymph JHBP from lepidopteran insects. A low level of expression of mJHBP in Ae. aegypti larvae suggests that it is primarily active during the adult stage where it could potentially influence the effects of JH on egg development, mating behavior, feeding, or other processes.

  17. Lack of adaptation to human tetherin in HIV-1 Group O and P

    Science.gov (United States)

    2011-01-01

    Background HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. Results We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. Conclusions The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread. PMID:21955466

  18. Lack of adaptation to human tetherin in HIV-1 Group O and P

    Directory of Open Access Journals (Sweden)

    Haworth Kevin G

    2011-09-01

    Full Text Available Abstract Background HIV-1 viruses are categorized into four distinct groups: M, N, O and P. Despite the same genomic organization, only the group M viruses are responsible for the world-wide pandemic of AIDS, suggesting better adaptation to human hosts. Previously, it has been reported that the group M Vpu protein is capable of both down-modulating CD4 and counteracting BST-2/tetherin restriction, while the group O Vpu cannot antagonize tetherin. This led us to investigate if group O, and the related group P viruses, possess functional anti-tetherin activities in Vpu or another viral protein, and to further map the residues required for group M Vpu to counteract human tetherin. Results We found a lack of activity against human tetherin for both the Vpu and Nef proteins from group O and P viruses. Furthermore, we found no evidence of anti-human tetherin activity in a fully infectious group O proviral clone, ruling out the possibility of an alternative anti-tetherin factor in this virus. Interestingly, an activity against primate tetherins was retained in the Nef proteins from both a group O and a group P virus. By making chimeras between a functional group M and non-functional group O Vpu protein, we were able to map the first 18 amino acids of group M Vpu as playing an essential role in the ability of the protein to antagonize human tetherin. We further demonstrated the importance of residue alanine-18 for the group M Vpu activity. This residue lies on a diagonal face of conserved alanines in the TM domain of the protein, and is necessary for specific Vpu-tetherin interactions. Conclusions The absence of human specific anti-tetherin activities in HIV-1 group O and P suggests a failure of these viruses to adapt to human hosts, which may have limited their spread.

  19. Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Bondarev, G.N.; Bikineeva, E.G.; Krasotskaya, G.I.; Noskin, L.A.

    2001-01-01

    The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time [ru

  20. Identification of brain-specific angiogenesis inhibitor 2 as an interaction partner of glutaminase interacting protein

    International Nuclear Information System (INIS)

    Zencir, Sevil; Ovee, Mohiuddin; Dobson, Melanie J.; Banerjee, Monimoy; Topcu, Zeki; Mohanty, Smita

    2011-01-01

    Highlights: → Brain-specific angiogenesis inhibitor 2 (BAI2) is a new partner protein for GIP. → BAI2 interaction with GIP was revealed by yeast two-hybrid assay. → Binding of BAI2 to GIP was characterized by NMR, CD and fluorescence. → BAI2 and GIP binding was mediated through the C-terminus of BAI2. -- Abstract: The vast majority of physiological processes in living cells are mediated by protein-protein interactions often specified by particular protein sequence motifs. PDZ domains, composed of 80-100 amino acid residues, are an important class of interaction motif. Among the PDZ-containing proteins, glutaminase interacting protein (GIP), also known as Tax Interacting Protein TIP-1, is unique in being composed almost exclusively of a single PDZ domain. GIP has important roles in cellular signaling, protein scaffolding and modulation of tumor growth and interacts with a number of physiological partner proteins, including Glutaminase L, β-Catenin, FAS, HTLV-1 Tax, HPV16 E6, Rhotekin and Kir 2.3. To identify the network of proteins that interact with GIP, a human fetal brain cDNA library was screened using a yeast two-hybrid assay with GIP as bait. We identified brain-specific angiogenesis inhibitor 2 (BAI2), a member of the adhesion-G protein-coupled receptors (GPCRs), as a new partner of GIP. BAI2 is expressed primarily in neurons, further expanding GIP cellular functions. The interaction between GIP and the carboxy-terminus of BAI2 was characterized using fluorescence, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy assays. These biophysical analyses support the interaction identified in the yeast two-hybrid assay. This is the first study reporting BAI2 as an interaction partner of GIP.

  1. SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

    Science.gov (United States)

    Bucholc, Maria; Ciesielski, Arkadiusz; Goch, Grażyna; Anielska-Mazur, Anna; Kulik, Anna; Krzywińska, Ewa; Dobrowolska, Grażyna

    2011-02-04

    SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.

  2. Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus.

    Science.gov (United States)

    Drappier, Melissa; Opperdoes, Fred R; Michiels, Thomas

    2017-07-15

    Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV. IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L

  3. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-05-18

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

  4. Association of Animal and Plant Protein Intake With All-Cause and Cause-Specific Mortality.

    Science.gov (United States)

    Song, Mingyang; Fung, Teresa T; Hu, Frank B; Willett, Walter C; Longo, Valter D; Chan, Andrew T; Giovannucci, Edward L

    2016-10-01

    Defining what represents a macronutritionally balanced diet remains an open question and a high priority in nutrition research. Although the amount of protein may have specific effects, from a broader dietary perspective, the choice of protein sources will inevitably influence other components of diet and may be a critical determinant for the health outcome. To examine the associations of animal and plant protein intake with the risk for mortality. This prospective cohort study of US health care professionals included 131 342 participants from the Nurses' Health Study (1980 to end of follow-up on June 1, 2012) and Health Professionals Follow-up Study (1986 to end of follow-up on January 31, 2012). Animal and plant protein intake was assessed by regularly updated validated food frequency questionnaires. Data were analyzed from June 20, 2014, to January 18, 2016. Hazard ratios (HRs) for all-cause and cause-specific mortality. Of the 131 342 participants, 85 013 were women (64.7%) and 46 329 were men (35.3%) (mean [SD] age, 49 [9] years). The median protein intake, as assessed by percentage of energy, was 14% for animal protein (5th-95th percentile, 9%-22%) and 4% for plant protein (5th-95th percentile, 2%-6%). After adjusting for major lifestyle and dietary risk factors, animal protein intake was not associated with all-cause mortality (HR, 1.02 per 10% energy increment; 95% CI, 0.98-1.05; P for trend = .33) but was associated with higher cardiovascular mortality (HR, 1.08 per 10% energy increment; 95% CI, 1.01-1.16; P for trend = .04). Plant protein was associated with lower all-cause mortality (HR, 0.90 per 3% energy increment; 95% CI, 0.86-0.95; P for trend animal protein of various origins with plant protein was associated with lower mortality. In particular, the HRs for all-cause mortality were 0.66 (95% CI, 0.59-0.75) when 3% of energy from plant protein was substituted for an equivalent amount of protein from processed red meat, 0.88 (95% CI

  5. Inter- and intra-specific differences in serum proteins of different species and subspecies of zebras.

    Science.gov (United States)

    Stratil, A; Cízová, D; Gábrisová, E; Pokorný, R

    1992-11-01

    1. Serum proteins of Equus grevyi, E. zebra hartmannae, E. burchelli boehmi, E. b. chapmanni and E. b. antiquorum were studied using starch-gel electrophoresis, 1-D polyacrylamide-gel electrophoresis, inhibitions of trypsin and chymotrypsin, immunoblotting, and specific staining for esterase. 2. Clear species-specific patterns were observed in albumin, transferrin, and for E. grevyi in protease inhibitor-1. Specific esterase was detected only in E. z. hartmannae. 3. Protein polymorphism was found in all studied species: E. grevyi--transferrin; E. z. hartmannae--protease inhibitor-1; E. b. boehmi--albumin, GC, transferrin, protease inhibitor-1, protease inhibitor-T; E. b. chapmanni--albumin, GC, transferrin, protease inhibitor-1; E. b. antiquorum--GC, transferrin, protease inhibitor-1. 4. Phenotype patterns of the polymorphic proteins were indicative of simple codominant inheritance. Further studies of polymorphism of protease inhibitor-2 and variability of protease inhibitor-X are needed. 5. alpha 1B glycoprotein in all zebra species was monomorphic. 6. The main transferrin components and alpha 1B glycoprotein of zebra (E. b. boehmi) were characterized for terminal sialic acid content.

  6. Vineland-II adaptive behavior profile of children with attention-deficit/hyperactivity disorder or specific learning disorders.

    Science.gov (United States)

    Balboni, Giulia; Incognito, Oriana; Belacchi, Carmen; Bonichini, Sabrina; Cubelli, Roberto

    2017-02-01

    The evaluation of adaptive behavior is informative in children with attention-deficit/hyperactivity disorder (ADHD) or specific learning disorders (SLD). However, the few investigations available have focused only on the gross level of domains of adaptive behavior. To investigate which item subsets of the Vineland-II can discriminate children with ADHD or SLD from peers with typical development. Student's t-tests, ROC analysis, logistic regression, and linear discriminant function analysis were used to compare 24 children with ADHD, 61 elementary students with SLD, and controls matched on age, sex, school level attended, and both parents' education level. Several item subsets that address not only ADHD core symptoms, but also understanding in social context and development of interpersonal relationships, allowed discrimination of children with ADHD from controls. The combination of four item subsets (Listening and attending, Expressing complex ideas, Social communication, and Following instructions) classified children with ADHD with both sensitivity and specificity of 87.5%. Only Reading skills, Writing skills, and Time and dates discriminated children with SLD from controls. Evaluation of Vineland-II scores at the level of item content categories is a useful procedure for an efficient clinical description. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

    Energy Technology Data Exchange (ETDEWEB)

    Monneau, Yoan R. [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Ishida, Yojiro [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Rossi, Paolo; Saio, Tomohide; Tzeng, Shiou-Ru [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States)

    2016-06-15

    A simple and cost effective method to independently and stereo-specifically incorporate [{sup 1}H,{sup 13}C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [{sup 1}H,{sup 13}C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [{sup 1}H,{sup 13}C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-{sup 13}C,2,3-{sup 2}H{sub 2},{sup 15}N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[{sup 13}CH{sub 3}]{sup γ2} labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for {sup 2}H-glycine to prevent scrambling.

  8. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    International Nuclear Information System (INIS)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro; Yamaguchi, Kazuo; Garcia, Andres J

    2011-01-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG 7 ). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni 2+ -ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG 7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII 7-10 ) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII 7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  9. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Energy Technology Data Exchange (ETDEWEB)

    Nakanishi, Jun; Nakayama, Hidekazu; Horiike, Yasuhiro [World Premier International (WPI) Research Center Initiative, International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science - NIMS (Japan); Yamaguchi, Kazuo [Department of Chemistry, Faculty of Science and Research Institute for Photofunctionalized Materials, Kanagawa University (Japan); Garcia, Andres J, E-mail: NAKANISHI.Jun@nims.go.jp [Institute for Bioengineering and Bioscience, Woodruff School of Mechanical Engineering, Georgia Institute of Technology (United States)

    2011-08-15

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG{sub 7}). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni{sup 2+}-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG{sub 7} underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII{sub 7-10}) to the irradiated regions. In contrast, when bovine serum albumin-a major serum protein-was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII{sub 7-10} was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  10. Differential protein expression and localization of CYP450 enzymes in three species of earthworm; is this a reflection of environmental adaptation?

    Science.gov (United States)

    Lu, Xiaoxu; Li, Yinsheng; Thunders, Michelle; Cavanagh, Jo; Matthew, Cory; Wang, Xiuhong; Zhou, Xinchu; Qiu, Jiangping

    2017-03-01

    Cytochrome P450 (CYP450) is a hemoprotein superfamily, among which CYP1, CYP2 and CYP3 play a major role in the metabolism of vast array of xenobiotics and endobiotics. This paper reports on three CYP enzyme variants (CYP1A2, CYP2E1 and CYP3A4) in three species of earthworm (Eisenia fetida, Metaphire guillelmi and Amynthas carnosus). The relative expression levels and localization of the three associated proteins were investigated at three life-cycle points (juvenile, sub-adult and adult), through comparison of anterior and posterior body tissue and between specific organs (body wall, intestine and reproductive tissues) using western blot analysis. This study confirmed the presence of CYP3A4, CYP1A2 and CYP2E1 in all three species of earthworm tested. The levels of expression varied with earthworm species, age, and body location. These differences in occurrence of the three CYP enzymes appeared to reflect the ecological niche (the spatial and temporal location and functional relationship of each individual or population in populations or communities), and the likelihood of contact with soil contaminants of the respective species. These results may help to explain why earthworms are capable of adapting to very different and extensively polluted soil environments and provide important data for subsequent ecotoxicology and ecological adaptability studies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Joint-Angle Specific Strength Adaptations Influence Improvements in Power in Highly Trained Athletes

    Directory of Open Access Journals (Sweden)

    Rhea Matthew R.

    2016-03-01

    Full Text Available Purpose. The purpose of this study was to examine the influence of training at different ranges of motion during the squat exercise on joint-angle specific strength adaptations. Methods. Twenty eight men were randomly assigned to one of three training groups, differing only in the depth of squats (quarter squat, half squat, and full squat performed in 16-week training intervention. Strength measures were conducted in the back squat pre-, mid-, and post-training at all three depths. Vertical jump and 40-yard sprint time were also measured. Results. Individuals in the quarter and full squat training groups improved significantly more at the specific depth at which they trained when compared to the other two groups (p < 0.05. Jump height and sprint speed improved in all groups (p < 0.05; however, the quarter squat had the greatest transfer to both outcomes. Conclusions. Consistently including quarter squats in workouts aimed at maximizing speed and jumping power can result in greater improvements.

  12. Sex-specific and blood meal-induced proteins of Anopheles gambiae midguts: analysis by two-dimensional gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Laurent-Winter C

    2003-02-01

    Full Text Available Abstract Background Anopheles gambiae is the main vector of Plasmodium falciparum in Africa. The mosquito midgut constitutes a barrier that the parasite must cross if it is to develop and be transmitted. Despite the central role of the mosquito midgut in the host/parasite interaction, little is known about its protein composition. Characterisation of An. gambiae midgut proteins may identify the proteins that render An. gambiae receptive to the malaria parasite. Methods We carried out two-dimensional gel electrophoresis of An. gambiae midgut proteins and compared protein profiles for midguts from males, sugar-fed females and females fed on human blood. Results Very few differences were detected between male and female mosquitoes for the approximately 375 silver-stained proteins. Male midguts contained ten proteins not detected in sugar-fed or blood-fed females, which are therefore probably involved in male-specific functions; conversely, female midguts contained twenty-three proteins absent from male midguts. Eight of these proteins were specific to sugar-fed females, and another ten, to blood-fed females. Conclusion Mass spectrometry analysis of the proteins found only in blood-fed female midguts, together with data from the recent sequencing of the An. gambiae genome, should make it possible to determine the role of these proteins in blood digestion or parasite receptivity.

  13. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    Science.gov (United States)

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

  14. The Effect of Salts in Promoting Specific and Competitive Interactions between Zinc Finger Proteins and Metals

    Science.gov (United States)

    Li, Gongyu; Yuan, Siming; Zheng, Shihui; Chen, Yuting; Zheng, Zhen; Liu, Yangzhong; Huang, Guangming

    2017-12-01

    Specific protein-metal interactions (PMIs) fulfill essential functions in cells and organic bodies, and activation of these functions in vivo are mostly modulated by the complex environmental factors, including pH value, small biomolecules, and salts. Specifically, the role of salts in promoting specific PMIs and their competition among various metals has remained untapped mainly due to the difficulty to distinguish nonspecific PMIs from specific PMIs by classic spectroscopic techniques. Herein, we report Hofmeister salts differentially promote the specific PMIs by combining nanoelectrospray ionization mass spectrometry and spectroscopic techniques (fluorescence measurement and circular dichroism). Furthermore, to explore the influence of salts in competitive binding between metalloproteins and various metals, we designed a series of competitive experiments and applied to a well-defined model system, the competitive binding of zinc (II) and arsenic (III) to holo-promyelocytic leukemia protein (PML). These experiments not only provided new insights at the molecular scale as complementary to previous NMR and spectroscopic results, but also deduced the relative binding ability between zinc finger proteins and metals at the molecular scale, which avoids the mass spectrometric titration-based determination of binding constants that is frequently affected and often degraded by variable solution conditions including salt contents. [Figure not available: see fulltext.

  15. CD4-specific designed ankyrin repeat proteins are novel potent HIV entry inhibitors with unique characteristics.

    Directory of Open Access Journals (Sweden)

    Andreas Schweizer

    2008-07-01

    Full Text Available Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins-high physical stability, specificity and low production costs-with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development.

  16. Next-Generation Sequencing for Binary Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Bernhard eSuter

    2015-12-01

    Full Text Available The yeast two-hybrid (Y2H system exploits host cell genetics in order to display binary protein-protein interactions (PPIs via defined and selectable phenotypes. Numerous improvements have been made to this method, adapting the screening principle for diverse applications, including drug discovery and the scale-up for proteome wide interaction screens in human and other organisms. Here we discuss a systematic workflow and analysis scheme for screening data generated by Y2H and related assays that includes high-throughput selection procedures, readout of comprehensive results via next-generation sequencing (NGS, and the interpretation of interaction data via quantitative statistics. The novel assays and tools will serve the broader scientific community to harness the power of NGS technology to address PPI networks in health and disease. We discuss examples of how this next-generation platform can be applied to address specific questions in diverse fields of biology and medicine.

  17. Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

    Science.gov (United States)

    Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

    2017-04-15

    RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. AptRank: an adaptive PageRank model for protein function prediction on   bi-relational graphs.

    Science.gov (United States)

    Jiang, Biaobin; Kloster, Kyle; Gleich, David F; Gribskov, Michael

    2017-06-15

    Diffusion-based network models are widely used for protein function prediction using protein network data and have been shown to outperform neighborhood-based and module-based methods. Recent studies have shown that integrating the hierarchical structure of the Gene Ontology (GO) data dramatically improves prediction accuracy. However, previous methods usually either used the GO hierarchy to refine the prediction results of multiple classifiers, or flattened the hierarchy into a function-function similarity kernel. No study has taken the GO hierarchy into account together with the protein network as a two-layer network model. We first construct a Bi-relational graph (Birg) model comprised of both protein-protein association and function-function hierarchical networks. We then propose two diffusion-based methods, BirgRank and AptRank, both of which use PageRank to diffuse information on this two-layer graph model. BirgRank is a direct application of traditional PageRank with fixed decay parameters. In contrast, AptRank utilizes an adaptive diffusion mechanism to improve the performance of BirgRank. We evaluate the ability of both methods to predict protein function on yeast, fly and human protein datasets, and compare with four previous methods: GeneMANIA, TMC, ProteinRank and clusDCA. We design four different validation strategies: missing function prediction, de novo function prediction, guided function prediction and newly discovered function prediction to comprehensively evaluate predictability of all six methods. We find that both BirgRank and AptRank outperform the previous methods, especially in missing function prediction when using only 10% of the data for training. The MATLAB code is available at https://github.rcac.purdue.edu/mgribsko/aptrank . gribskov@purdue.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  19. Highly specific salt bridges govern bacteriophage P22 icosahedral capsid assembly: identification of the site in coat protein responsible for interaction with scaffolding protein.

    Science.gov (United States)

    Cortines, Juliana R; Motwani, Tina; Vyas, Aashay A; Teschke, Carolyn M

    2014-05-01

    Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions. Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving

  20. Adaptation of Staphylococcus aureus to Airway Environments in Patients With Cystic Fibrosis by Upregulation of Superoxide Dismutase M and Iron-Scavenging Proteins.

    Science.gov (United States)

    Treffon, Janina; Block, Desiree; Moche, Martin; Reiss, Swantje; Fuchs, Stephan; Engelmann, Susanne; Becher, Dörte; Langhanki, Lars; Mellmann, Alexander; Peters, Georg; Kahl, Barbara C

    2018-04-11

    Adaptation of S. aureus to the hostile environment of CF airways resulted in changed abundance of proteins involved in energy metabolism, cellular processes, transport and binding, but most importantly in an iron-scavenging phenotype and increased activity of superoxide dismutase M.

  1. Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

    Science.gov (United States)

    Demangeat, G; Hemmer, O; Reinbolt, J; Mayo, M A; Fritsch, C

    1992-07-01

    The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

  2. CLAss-Specific Subspace Kernel Representations and Adaptive Margin Slack Minimization for Large Scale Classification.

    Science.gov (United States)

    Yu, Yinan; Diamantaras, Konstantinos I; McKelvey, Tomas; Kung, Sun-Yuan

    2018-02-01

    In kernel-based classification models, given limited computational power and storage capacity, operations over the full kernel matrix becomes prohibitive. In this paper, we propose a new supervised learning framework using kernel models for sequential data processing. The framework is based on two components that both aim at enhancing the classification capability with a subset selection scheme. The first part is a subspace projection technique in the reproducing kernel Hilbert space using a CLAss-specific Subspace Kernel representation for kernel approximation. In the second part, we propose a novel structural risk minimization algorithm called the adaptive margin slack minimization to iteratively improve the classification accuracy by an adaptive data selection. We motivate each part separately, and then integrate them into learning frameworks for large scale data. We propose two such frameworks: the memory efficient sequential processing for sequential data processing and the parallelized sequential processing for distributed computing with sequential data acquisition. We test our methods on several benchmark data sets and compared with the state-of-the-art techniques to verify the validity of the proposed techniques.

  3. Adenylate cyclase activity in fish gills in relation to salt adaptation

    International Nuclear Information System (INIS)

    Guibbolini, M.E.; Lahlou, B.

    1987-01-01

    The influence of salt adaptation on specific adenylate cyclase activity (measured by conversion of [α- 32 P] - ATP into [α- 32 P] - cAMP) was investigated in gill plasma membranes of rainbow trout (Salmo gairdneri) adapted to various salinities (deionized water, DW; fresh water, FW; 3/4 sea water, 3/4 SW; sea water, SW) and in sea water adapted- mullet (Mugil sp.). Basal activity declined by a factor of 2 in trout with increasing external salinity (pmoles cAMP/mg protein/10 min: 530 in DW, 440 in FW, 340 in 3/4 SW; 250 in SW) and was very low in SW adapted-mullet: 35. The Km for ATP was similar (0.5 mM) in both FW adapted- and SW adapted- trout in either the absence (basal activity) or in the presence of stimulating agents (isoproterenol; NaF) while the Vm varied. Analysis of stimulation ratios with respect to basal levels of the enzyme showed that hormones and pharmacological substances (isoproterenol, NaF) display a greater potency in high salt than in low salt adapted- fish gills. In contrast, salt adaptation did not have any effect on the regulation of adenylate cyclase by PGE 1 . These results are interpreted in relation to the general process of osmoregulation. 27 references, 6 figures

  4. Genome fluctuations in cyanobacteria reflect evolutionary, developmental and adaptive traits

    Science.gov (United States)

    2011-01-01

    Background Cyanobacteria belong to an ancient group of photosynthetic prokaryotes with pronounced variations in their cellular differentiation strategies, physiological capacities and choice of habitat. Sequencing efforts have shown that genomes within this phylum are equally diverse in terms of size and protein-coding capacity. To increase our understanding of genomic changes in the lineage, the genomes of 58 contemporary cyanobacteria were analysed for shared and unique orthologs. Results A total of 404 protein families, present in all cyanobacterial genomes, were identified. Two of these are unique to the phylum, corresponding to an AbrB family transcriptional regulator and a gene that escapes functional annotation although its genomic neighbourhood is conserved among the organisms examined. The evolution of cyanobacterial genome sizes involves a mix of gains and losses in the clade encompassing complex cyanobacteria, while a single event of reduction is evident in a clade dominated by unicellular cyanobacteria. Genome sizes and gene family copy numbers evolve at a higher rate in the former clade, and multi-copy genes were predominant in large genomes. Orthologs unique to cyanobacteria exhibiting specific characteristics, such as filament formation, heterocyst differentiation, diazotrophy and symbiotic competence, were also identified. An ancestral character reconstruction suggests that the most recent common ancestor of cyanobacteria had a genome size of approx. 4.5 Mbp and 1678 to 3291 protein-coding genes, 4%-6% of which are unique to cyanobacteria today. Conclusions The different rates of genome-size evolution and multi-copy gene abundance suggest two routes of genome development in the history of cyanobacteria. The expansion strategy is driven by gene-family enlargment and generates a broad adaptive potential; while the genome streamlining strategy imposes adaptations to highly specific niches, also reflected in their different functional capacities. A few

  5. Genome fluctuations in cyanobacteria reflect evolutionary, developmental and adaptive traits

    Directory of Open Access Journals (Sweden)

    Nylander Johan AA

    2011-06-01

    Full Text Available Abstract Background Cyanobacteria belong to an ancient group of photosynthetic prokaryotes with pronounced variations in their cellular differentiation strategies, physiological capacities and choice of habitat. Sequencing efforts have shown that genomes within this phylum are equally diverse in terms of size and protein-coding capacity. To increase our understanding of genomic changes in the lineage, the genomes of 58 contemporary cyanobacteria were analysed for shared and unique orthologs. Results A total of 404 protein families, present in all cyanobacterial genomes, were identified. Two of these are unique to the phylum, corresponding to an AbrB family transcriptional regulator and a gene that escapes functional annotation although its genomic neighbourhood is conserved among the organisms examined. The evolution of cyanobacterial genome sizes involves a mix of gains and losses in the clade encompassing complex cyanobacteria, while a single event of reduction is evident in a clade dominated by unicellular cyanobacteria. Genome sizes and gene family copy numbers evolve at a higher rate in the former clade, and multi-copy genes were predominant in large genomes. Orthologs unique to cyanobacteria exhibiting specific characteristics, such as filament formation, heterocyst differentiation, diazotrophy and symbiotic competence, were also identified. An ancestral character reconstruction suggests that the most recent common ancestor of cyanobacteria had a genome size of approx. 4.5 Mbp and 1678 to 3291 protein-coding genes, 4%-6% of which are unique to cyanobacteria today. Conclusions The different rates of genome-size evolution and multi-copy gene abundance suggest two routes of genome development in the history of cyanobacteria. The expansion strategy is driven by gene-family enlargment and generates a broad adaptive potential; while the genome streamlining strategy imposes adaptations to highly specific niches, also reflected in their different

  6. Diverse amino acid changes at specific positions in the N-terminal region of the coat protein allow Plum pox virus to adapt to new hosts.

    Science.gov (United States)

    Carbonell, Alberto; Maliogka, Varvara I; Pérez, José de Jesús; Salvador, Beatriz; León, David San; García, Juan Antonio; Simón-Mateo, Carmen

    2013-10-01

    Plum pox virus (PPV)-D and PPV-R are two isolates from strain D of PPV that differ in host specificity. Previous analyses of chimeras originating from PPV-R and PPV-D suggested that the N terminus of the coat protein (CP) includes host-specific pathogenicity determinants. Here, these determinants were mapped precisely by analyzing the infectivity in herbaceous and woody species of chimeras containing a fragment of the 3' region of PPV-D (including the region coding for the CP) in a PPV-R backbone. These chimeras were not infectious in Prunus persica, but systemically infected Nicotiana clevelandii and N. benthamiana when specific amino acids were modified or deleted in a short 30-amino-acid region of the N terminus of the CP. Most of these mutations did not reduce PPV fitness in Prunus spp. although others impaired systemic infection in this host. We propose a model in which the N terminus of the CP, highly relevant for virus systemic movement, is targeted by a host defense mechanism in Nicotiana spp. Mutations in this short region allow PPV to overcome the defense response in this host but can compromise the efficiency of PPV systemic movement in other hosts such as Prunus spp.

  7. Effects of UVB-induced oxidative stress on protein expression and specific protein oxidation in normal human epithelial keratinocytes: a proteomic approach

    Directory of Open Access Journals (Sweden)

    De Marco Federico

    2010-03-01

    Full Text Available Abstract Background The UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS, which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes. Results The expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized. Conclusions Despite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.

  8. Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (Luteoviridae)▿

    Science.gov (United States)

    Yang, Xiaolong; Thannhauser, T. W.; Burrows, Mary; Cox-Foster, Diana; Gildow, Fred E.; Gray, Stewart M.

    2008-01-01

    Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F2 progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission. PMID:17959668

  9. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation.

    Science.gov (United States)

    Henry, Romain; Bruneau, Emmanuelle; Gardan, Rozenn; Bertin, Stéphane; Fleuchot, Betty; Decaris, Bernard; Leblond-Bourget, Nathalie

    2011-10-07

    Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  10. The rgg0182 gene encodes a transcriptional regulator required for the full Streptococcus thermophilus LMG18311 thermal adaptation

    Directory of Open Access Journals (Sweden)

    Bertin Stéphane

    2011-10-01

    Full Text Available Abstract Background Streptococcus thermophilus is an important starter strain for the production of yogurt and cheeses. The analysis of sequenced genomes of four strains of S. thermophilus indicates that they contain several genes of the rgg familly potentially encoding transcriptional regulators. Some of the Rgg proteins are known to be involved in bacterial stress adaptation. Results In this study, we demonstrated that Streptococcus thermophilus thermal stress adaptation required the rgg0182 gene which transcription depends on the culture medium and the growth temperature. This gene encoded a protein showing similarity with members of the Rgg family transcriptional regulator. Our data confirmed that Rgg0182 is a transcriptional regulator controlling the expression of its neighboring genes as well as chaperones and proteases encoding genes. Therefore, analysis of a Δrgg0182 mutant revealed that this protein played a role in the heat shock adaptation of Streptococcus thermophilus LMG18311. Conclusions These data showed the importance of the Rgg0182 transcriptional regulator on the survival of S. thermophilus during dairy processes and more specifically during changes in temperature.

  11. Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni

    NARCIS (Netherlands)

    Silveira, da M.G.; Baumgärtner, M.; Rombouts, F.M.; Abee, T.

    2004-01-01

    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol

  12. Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

    Science.gov (United States)

    2013-01-01

    Background Lyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1). Results Robust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases. Conclusions Intra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues. PMID:24112474

  13. International Society of Sports Nutrition position stand: protein and exercise

    Directory of Open Access Journals (Sweden)

    Landis Jamie

    2007-09-01

    Full Text Available Abstract Position Statement The following seven points related to the intake of protein for healthy, exercising individuals constitute the position stand of the Society. They have been approved by the Research Committee of the Society. 1 Vast research supports the contention that individuals engaged in regular exercise training require more dietary protein than sedentary individuals. 2 Protein intakes of 1.4 – 2.0 g/kg/day for physically active individuals is not only safe, but may improve the training adaptations to exercise training. 3 When part of a balanced, nutrient-dense diet, protein intakes at this level are not detrimental to kidney function or bone metabolism in healthy, active persons. 4 While it is possible for physically active individuals to obtain their daily protein requirements through a varied, regular diet, supplemental protein in various forms are a practical way of ensuring adequate and quality protein intake for athletes. 5 Different types and quality of protein can affect amino acid bioavailability following protein supplementation. The superiority of one protein type over another in terms of optimizing recovery and/or training adaptations remains to be convincingly demonstrated. 6 Appropriately timed protein intake is an important component of an overall exercise training program, essential for proper recovery, immune function, and the growth and maintenance of lean body mass. 7 Under certain circumstances, specific amino acid supplements, such as branched-chain amino acids (BCAA's, may improve exercise performance and recovery from exercise.

  14. Use of recombinant purified protein derivative (PPD) antigens as specific skin test for tuberculosis.

    Science.gov (United States)

    Stavri, Henriette; Bucurenci, Nadia; Ulea, Irina; Costache, Adriana; Popa, Loredana; Popa, Mircea Ioan

    2012-11-01

    Purified protein derivative (PPD) is currently the only available skin test reagent used worldwide for the diagnosis of tuberculosis (TB). The aim of this study was to develop a Mycobacterium tuberculosis specific skin test reagent, without false positive results due to Bacillus Calmette-Guerin (BCG) vaccination using recombinant antigens. Proteins in PPD IC-65 were analyzed by tandem mass spectrometry and compared to proteins in M. tuberculosis culture filtrate; 54 proteins were found in common. Top candidates MPT64, ESAT 6, and CFP 10 were overexpressed in Escherichia coli expression strains and purified as recombinant proteins. To formulate optimal immunodiagnostic PPD cocktails, the antigens were evaluated by skin testing guinea pigs sensitized with M. tuberculosis H37Rv and BCG. For single antigens and a cocktail mixture of these antigens, best results were obtained using 3 μg/0.1 ml, equivalent to 105 TU (tuberculin units). Each animal was simultaneously tested with PPD IC-65, 2 TU/0.1 ml, as reference. Reactivity of the multi-antigen cocktail was greater than that of any single antigen. The skin test results were between 34.3 and 76.6 per cent the level of reactivity compared to that of the reference when single antigens were tested and 124 per cent the level of reactivity compared to the reference for the multi-antigen cocktail. Our results showed that this specific cocktail could represent a potential candidate for a new skin diagnostic test for TB.

  15. New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

    Energy Technology Data Exchange (ETDEWEB)

    Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

    2005-06-17

    Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

  16. Time-dependent changes in protein expression in rainbow trout muscle following hypoxia.

    Science.gov (United States)

    Wulff, Tune; Jokumsen, Alfred; Højrup, Peter; Jessen, Flemming

    2012-04-18

    Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Molecular signal for induction of the adaptive response to alkylating agents in E. coli

    Energy Technology Data Exchange (ETDEWEB)

    Lindahl, T.; Sedgwick, B.; Teo, I.; Kilpatrick, M.; McCarthy, T.; Hughes, S.

    1986-05-01

    Exposure of E. coli to simple alkylating agents such as methylnitrosourea causes the induction of at least three DNA repair functions, which are under the control of the ada gene. The intracellular signal for switching on the response has been identified as one of the two stereoisomers of methyl phosphotriesters generated in DNA by alkylation. The methyl group is transferred from the phosphotriester moiety to a specific cysteine residue within the regulatory Ada protein in a self-methylation reaction. This protein also corrects the mutagenic lesion O/sup 6/-methylguanine by transfer of its methyl group to a separate cysteine residue in the Ada protein. Methylation of the protein from repair of a DNA phosphotriester residue, but not from O/sup 6/-methylguanine, converts it to an activator of transcription of genes involved in the adaptive response. The activated form of the Ada protein enhances transcription by binding specifically to a sequence d(AAA--AAAGCGCA), located immediately upstream of the RNA polymerase binding site in relevant promoter regions.

  18. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

    Science.gov (United States)

    Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

    1997-07-01

    Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

  19. Distinct domains within the NITROGEN LIMITATION ADAPTATION protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway

    Science.gov (United States)

    The NITROGEN LIMITATION ADAPTATION (NLA) protein is a RING-type E3 ubiquitin ligase that plays an essential role in the regulation of nitrogen and phosphate homeostasis. NLA is localized to two distinct subcellular sites, the plasma membrane and nucleus, and contains four distinct domains: i) a RING...

  20. Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

    OpenAIRE

    Wang, Yen-Chih; Dozier, Jonathan K.; Beese, Lorena S.; Distefano, Mark D.

    2014-01-01

    Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequen...

  1. Common and metal-specific proteomic responses to cadmium and zinc in the metal tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn.

    Science.gov (United States)

    Chiapello, M; Martino, E; Perotto, S

    2015-05-01

    Although adaptive metal tolerance may arise in fungal populations in polluted soils, the mechanisms underlying metal-specific tolerance are poorly understood. Comparative proteomics is a powerful tool to identify variation in protein profiles caused by changing environmental conditions, and was used to investigate protein accumulation in a metal tolerant isolate of the ericoid mycorrhizal fungus Oidiodendron maius exposed to zinc and cadmium. Two-dimensional gel electrophoresis and shotgun proteomics followed by mass spectrometry lead to the identification of common and metal-specific proteins and pathways. Proteins selectively induced by cadmium exposure were molecular chaperons of the Hsp90 family, cytoskeletal proteins and components of the translation machinery. Zinc significantly up-regulated metabolic pathways related to energy production and carbohydrates metabolism, likely mirroring zinc adaptation of this fungal isolate. Common proteins induced by the two metal ions were the antioxidant enzyme Cu/Zn superoxide dismutase and ubiquitin. In mycelia exposed to zinc and cadmium, both proteomic techniques also identified agmatinase, an enzyme involved in polyamine biosynthesis. This novel finding suggests that, like plants, polyamines may have important functions in response to abiotic environmental stress in fungi. Genetic evidence also suggests that the biosynthesis of polyamines via an alternative metabolic pathway may be widespread in fungi.

  2. A user-specific human-machine interaction strategy for a prosthetic shank adapter

    Directory of Open Access Journals (Sweden)

    Stuhlenmiller Florian

    2017-09-01

    Full Text Available For people with lower limb amputation, a user-specific human-machine interaction with their prostheses is required to ensure safe and comfortable assistance. Especially during dynamic turning manoeuvres, users experience high loads at the stump, which decreases comfort and may lead to long-term tissue damage. Preliminary experiments with users wearing a configurable, passive torsional adaptor indicate increased comfort and safety achieved by adaptation of torsional stiffness and foot alignment. Moreover, the results show that the individual preference regarding both parameters depend on gait situation and individual preference. Hence, measured loads in the structure of the prosthesis and subjective feedback regarding comfort and safety during different turning motions are considered in a user-specific human-machine interaction strategy for a prosthetic shank adaptor. Therefore, the interrelations of gait parameters with optimal configuration are stored in an individual preference-setting matrix. Stiffness and foot alignment are actively adjusted to the optimal parameters by a parallel elastic actuator. Two subjects reported that they experienced appropriate variation of stiffness and foot alignment, a noticeable reduction of load at the stump and that they could turn with less effort.

  3. Human protein secretory pathway genes are expressed in a tissue-specific pattern to match processing demands of the secretome

    DEFF Research Database (Denmark)

    Feizi, Amir; Gatto, Francesco; Uhlén, Mathias

    2017-01-01

    Protein secretory pathway in eukaryal cells is responsible for delivering functional secretory proteins. The dysfunction of this pathway causes a range of important human diseases from congenital disorders to cancer. Despite the piled-up knowledge on the molecular biology and biochemistry level...... in specific gene families of the secretory pathway. We also inspected the potential functional link between detected extreme genes and the corresponding tissues enriched secretome. As a result, the detected extreme genes showed correlation with the enrichment of the nature and number of specific post......-translational modifications in each tissue's secretome. Our findings conciliate both the housekeeping and tissue-specific nature of the protein secretory pathway, which we attribute to a fine-tuned regulation of defined gene families to support the diversity of secreted proteins and their modifications....

  4. A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

    Science.gov (United States)

    Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

    2015-01-01

    An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

  5. Incorporating deep learning with convolutional neural networks and position specific scoring matrices for identifying electron transport proteins.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

    2017-09-05

    In several years, deep learning is a modern machine learning technique using in a variety of fields with state-of-the-art performance. Therefore, utilization of deep learning to enhance performance is also an important solution for current bioinformatics field. In this study, we try to use deep learning via convolutional neural networks and position specific scoring matrices to identify electron transport proteins, which is an important molecular function in transmembrane proteins. Our deep learning method can approach a precise model for identifying of electron transport proteins with achieved sensitivity of 80.3%, specificity of 94.4%, and accuracy of 92.3%, with MCC of 0.71 for independent dataset. The proposed technique can serve as a powerful tool for identifying electron transport proteins and can help biologists understand the function of the electron transport proteins. Moreover, this study provides a basis for further research that can enrich a field of applying deep learning in bioinformatics. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

    Energy Technology Data Exchange (ETDEWEB)

    Bielmann, Regula; Habann, Matthias; Eugster, Marcel R. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Lurz, Rudi [Max-Planck Institute for Molecular Genetics, 14195 Berlin (Germany); Calendar, Richard [Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3202 (United States); Klumpp, Jochen, E-mail: jochen.klumpp@hest.ethz.ch [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland); Loessner, Martin J. [Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich (Switzerland)

    2015-03-15

    Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wall teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.

  7. Positive Selection and Centrality in the Yeast and Fly Protein-Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Sandip Chakraborty

    2016-01-01

    Full Text Available Proteins within a molecular network are expected to be subject to different selective pressures depending on their relative hierarchical positions. However, it is not obvious what genes within a network should be more likely to evolve under positive selection. On one hand, only mutations at genes with a relatively high degree of control over adaptive phenotypes (such as those encoding highly connected proteins are expected to be “seen” by natural selection. On the other hand, a high degree of pleiotropy at these genes is expected to hinder adaptation. Previous analyses of the human protein-protein interaction network have shown that genes under long-term, recurrent positive selection (as inferred from interspecific comparisons tend to act at the periphery of the network. It is unknown, however, whether these trends apply to other organisms. Here, we show that long-term positive selection has preferentially targeted the periphery of the yeast interactome. Conversely, in flies, genes under positive selection encode significantly more connected and central proteins. These observations are not due to covariation of genes’ adaptability and centrality with confounding factors. Therefore, the distribution of proteins encoded by genes under recurrent positive selection across protein-protein interaction networks varies from one species to another.

  8. Protein profiles of Taenia solium cysts obtained from skeletal muscles and the central nervous system of pigs: Search for tissue-specific proteins.

    Science.gov (United States)

    Navarrete-Perea, José; Moguel, Bárbara; Bobes, Raúl José; Villalobos, Nelly; Carrero, Julio César; Sciutto, Edda; Soberón, Xavier; Laclette, Juan Pedro

    2017-01-01

    Taeniasis/cysticercosis caused by the tapeworm Taenia solium is a parasite disease transmitted among humans and pigs, the main intermediate host. The larvae/cysts can lodge in several tissues of the pig, i.e. skeletal muscles and different locations of the central nervous system. The molecular mechanisms associated to tissue preferences of the cysts remain poorly understood. The major public health concern about this zoonosis is due to the human infections by the larval form in the central nervous system, causing a highly pleomorphic and debilitating disease known as neurocysticercosis. This study was aimed to explore the 2DE protein maps of T. solium cysts obtained from skeletal muscles and central nervous system of naturally infected pigs. The gel images were analyzed through a combination of PDQuest™ and multivariate analysis. Results showed that differences in the protein patterns of cysts obtained from both tissues were remarkably discrete. Only 7 protein spots were found specifically associated to the skeletal muscle localization of the cysts; none was found significantly associated to the central nervous system. The use of distinct protein fractions of cysts allowed preliminary identification of several tissue-specific antigenic bands. The implications of these findings are discussed, as well as several strategies directed to achieve the complete characterization of this parasite's proteome, in order to extend our understanding of the molecular mechanisms underlying tissue localization of the cysts and to open avenues for the development of immunological tissue-specific diagnosis of the disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins

    Institute of Scientific and Technical Information of China (English)

    HSU Heng; Robert L. CHURCH

    2004-01-01

    During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a "CA" repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preliminarily identify trans factors which bind to specific cis elements of the LIM2 promoter and/or regulate transcription of LIM2. We conclude that developmental regulation and coordination of the MP 19 gene in ocular lens fiber cells is controlled by the presence of specific cis elements that bind regulatory trans factors that affect LIM2 gene expression. DNA methylation is one mechanism of controlling LIM2 gene expression during lens development.

  10. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    Directory of Open Access Journals (Sweden)

    Jun Nakanishi, Hidekazu Nakayama, Kazuo Yamaguchi, Andres J Garcia and Yasuhiro Horiike

    2011-01-01

    Full Text Available The development of methods for the off–on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs of three disulfide compounds containing (i a photocleavable poly(ethylene glycol (PEG, (ii nitrilotriacetic acid (NTA and (iii hepta(ethylene glycol (EG7. Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7–10 to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  11. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

    Science.gov (United States)

    Guilbert, Solenn M; Lambert, Herman; Rodrigue, Marc-Antoine; Fuchs, Margit; Landry, Jacques; Lavoie, Josée N

    2018-02-05

    BCL2-associated athanogene (BAG)-3 is viewed as a platform that would physically and functionally link distinct classes of molecular chaperones of the heat shock protein (HSP) family for the stabilization and clearance of damaged proteins. In this study, we show that HSPB8, a member of the small heat shock protein subfamily, cooperates with BAG3 to coordinate the sequestration of harmful proteins and the cellular adaptive response upon proteasome inhibition. Silencing of HSPB8, like depletion of BAG3, inhibited targeting of ubiquitinated proteins to the juxtanuclear aggresome, a mammalian system of spatial quality control. However, aggresome targeting was restored in BAG3-depleted cells by a mutant BAG3 defective in HSPB8 binding, uncoupling HSPB8 function from its binding to BAG3. Depletion of HSPB8 impaired formation of ubiquitinated microaggregates in an early phase and interfered with accurate modifications of the stress sensor p62/sequestosome (SQSTM)-1. This impairment correlated with decreased coupling of BAG3 to p62/SQSTM1 in response to stress, hindering Kelch-like ECH-associated protein (KEAP)-1 sequestration and stabilization of nuclear factor E2-related factor (Nrf)-2, an important arm of the antioxidant defense. Notably, the myopathy-associated mutation of BAG3 (P209L), which lies within the HSPB8-binding motif, deregulated the association between BAG3 and p62/SQSTM1 and the KEAP1-Nrf2 signaling axis. Together, our findings support a so-far-unrecognized role for the HSPB8-BAG3 connection in mounting of an efficient stress response, which may be involved in BAG3-related human diseases.-Guilbert, S. M., Lambert, H., Rodrigue, M.-A., Fuchs, M., Landry, J., Lavoie, J. N. HSPB8 and BAG3 cooperate to promote spatial sequestration of ubiquitinated proteins and coordinate the cellular adaptive response to proteasome insufficiency.

  12. 14-3-3 checkpoint regulatory proteins interact specifically with DNA repair protein human exonuclease 1 (hEXO1) via a semi-conserved motif

    DEFF Research Database (Denmark)

    Andersen, Sofie Dabros; Keijzers, Guido; Rampakakis, Emmanouil

    2012-01-01

    Human exonuclease 1 (hEXO1) acts directly in diverse DNA processing events, including replication, mismatch repair (MMR), and double strand break repair (DSBR), and it was also recently described to function as damage sensor and apoptosis inducer following DNA damage. In contrast, 14-3-3 proteins...... are specifically induced by replication inhibition leading to protein ubiquitination and degradation. We demonstrate direct and robust interaction between hEXO1 and six of the seven 14-3-3 isoforms in vitro, suggestive of a novel protein interaction network between DNA repair and cell cycle control. Binding...... and most likely a second unidentified binding motif. 14-3-3 associations do not appear to directly influence hEXO1 in vitro nuclease activity or in vitro DNA replication initiation. Moreover, specific phosphorylation variants, including hEXO1 S746A, are efficiently imported to the nucleus; to associate...

  13. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  14. Tissue specific phosphorylation of mitochondrial proteins isolated from rat liver, heart muscle, and skeletal muscle

    DEFF Research Database (Denmark)

    Bak, Steffen; León, Ileana R; Jensen, Ole Nørregaard

    2013-01-01

    -specific phosphorylation sites were identified in tissue-specific enzymes such as those encoded by HMGCS2, BDH1, PCK2, CPS1, and OTC in liver mitochondria, and CKMT2 and CPT1B in heart and skeletal muscle. Kinase prediction showed an important role for PKA and PKC in all tissues but also for proline-directed kinases......Phosphorylation of mitochondrial proteins in a variety of biological processes is increasingly being recognized and may contribute to the differences in function and energy demands observed in mitochondria from different tissues such as liver, heart, and skeletal muscle. Here, we used a combination...... of TiO2 phosphopeptide-enrichment, HILIC fractionation, and LC-MS/MS on isolated mitochondria to investigate the tissue-specific mitochondrial phosphoproteomes of rat liver, heart, and skeletal muscle. In total, we identified 899 phosphorylation sites in 354 different mitochondrial proteins including...

  15. Characterization of a new antifungal non-specific lipid transfer protein (nsLTP) from sugar beet leaves

    DEFF Research Database (Denmark)

    Kristensen, A K; Brunstedt, J; Madsen, M T

    2000-01-01

    A novel protein (IWF5) comprising 92 amino acids has been purified from the intercellular washing fluid of sugar beet leaves using cation exchange chromatography and reversed phase high performance liquid chromatography. Based on amino acid sequence homology, including the presence of eight...... cysteines at conserved positions, the protein can be classified as a member of the plant family of non-specific lipid transfer proteins (nsLTPs). The protein is 47% identical to IWF1, an antifungal nsLTP previously isolated from leaves of sugar beet. A potential site for N-linked glycosylation present...

  16. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    Science.gov (United States)

    Xie, Jianming [San Diego, CA; Wang, Lei [San Diego, CA; Wu, Ning [Boston, MA; Schultz, Peter G [La Jolla, CA

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  17. Site-Specific Protein Labeling Utilizing Lipoic Acid Ligase (LplA) and Bioorthogonal Inverse Electron Demand Diels-Alder Reaction.

    Science.gov (United States)

    Baalmann, Mathis; Best, Marcel; Wombacher, Richard

    2018-01-01

    Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplA W37V ) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif-a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DA inv ). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

  18. Discovering rules for protein-ligand specificity using support vector inductive logic programming.

    Science.gov (United States)

    Kelley, Lawrence A; Shrimpton, Paul J; Muggleton, Stephen H; Sternberg, Michael J E

    2009-09-01

    Structural genomics initiatives are rapidly generating vast numbers of protein structures. Comparative modelling is also capable of producing accurate structural models for many protein sequences. However, for many of the known structures, functions are not yet determined, and in many modelling tasks, an accurate structural model does not necessarily tell us about function. Thus, there is a pressing need for high-throughput methods for determining function from structure. The spatial arrangement of key amino acids in a folded protein, on the surface or buried in clefts, is often the determinants of its biological function. A central aim of molecular biology is to understand the relationship between such substructures or surfaces and biological function, leading both to function prediction and to function design. We present a new general method for discovering the features of binding pockets that confer specificity for particular ligands. Using a recently developed machine-learning technique which couples the rule-discovery approach of inductive logic programming with the statistical learning power of support vector machines, we are able to discriminate, with high precision (90%) and recall (86%) between pockets that bind FAD and those that bind NAD on a large benchmark set given only the geometry and composition of the backbone of the binding pocket without the use of docking. In addition, we learn rules governing this specificity which can feed into protein functional design protocols. An analysis of the rules found suggests that key features of the binding pocket may be tied to conformational freedom in the ligand. The representation is sufficiently general to be applicable to any discriminatory binding problem. All programs and data sets are freely available to non-commercial users at http://www.sbg.bio.ic.ac.uk/svilp_ligand/.

  19. Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

    Science.gov (United States)

    Taylor, Kathryne E.

    2015-01-01

    ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both

  20. Insights into the sequence parameters for halophilic adaptation.

    Science.gov (United States)

    Nath, Abhigyan

    2016-03-01

    The sequence parameters for halophilic adaptation are still not fully understood. To understand the molecular basis of protein hypersaline adaptation, a detailed analysis is carried out, and investigated the likely association of protein sequence attributes to halophilic adaptation. A two-stage strategy is implemented, where in the first stage a supervised machine learning classifier is build, giving an overall accuracy of 86 % on stratified tenfold cross validation and 90 % on blind testing set, which are better than the previously reported results. The second stage consists of statistical analysis of sequence features and possible extraction of halophilic molecular signatures. The results of this study showed that, halophilic proteins are characterized by lower average charge, lower K content, and lower S content. A statistically significant preference/avoidance list of sequence parameters is also reported giving insights into the molecular basis of halophilic adaptation. D, Q, E, H, P, T, V are significantly preferred while N, C, I, K, M, F, S are significantly avoided. Among amino acid physicochemical groups, small, polar, charged, acidic and hydrophilic groups are preferred over other groups. The halophilic proteins also showed a preference for higher average flexibility, higher average polarity and avoidance for higher average positive charge, average bulkiness and average hydrophobicity. Some interesting trends observed in dipeptide counts are also reported. Further a systematic statistical comparison is undertaken for gaining insights into the sequence feature distribution in different residue structural states. The current analysis may facilitate the understanding of the mechanism of halophilic adaptation clearer, which can be further used for rational design of halophilic proteins.

  1. Pharmacophore screening of the protein data bank for specific binding site chemistry.

    Science.gov (United States)

    Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

    2010-03-22

    A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

  2. Cellular plasticity enables adaptation to unforeseen cell-cycle rewiring challenges.

    Science.gov (United States)

    Katzir, Yair; Stolovicki, Elad; Stern, Shay; Braun, Erez

    2012-01-01

    The fundamental dynamics of the cell cycle, underlying cell growth and reproduction, were previously found to be robust under a wide range of environmental and internal perturbations. This property was commonly attributed to its network structure, which enables the coordinated interactions among hundreds of proteins. Despite significant advances in deciphering the components and autonomous interactions of this network, understanding the interfaces of the cell cycle with other major cellular processes is still lacking. To gain insight into these interfaces, we used the process of genome-rewiring in yeast by placing an essential metabolic gene HIS3 from the histidine biosynthesis pathway, under the exclusive regulation of different cell-cycle promoters. In a medium lacking histidine and under partial inhibition of the HIS3p, the rewired cells encountered an unforeseen multitasking challenge; the cell-cycle regulatory genes were required to regulate the essential histidine-pathway gene in concert with the other metabolic demands, while simultaneously driving the cell cycle through its proper temporal phases. We show here that chemostat cell populations with rewired cell-cycle promoters adapted within a short time to accommodate the inhibition of HIS3p and stabilized a new phenotypic state. Furthermore, a significant fraction of the population was able to adapt and grow into mature colonies on plates under such inhibiting conditions. The adapted state was shown to be stably inherited across generations. These adaptation dynamics were accompanied by a non-specific and irreproducible genome-wide transcriptional response. Adaptation of the cell-cycle attests to its multitasking capabilities and flexible interface with cellular metabolic processes and requirements. Similar adaptation features were found in our previous work when rewiring HIS3 to the GAL system and switching cells from galactose to glucose. Thus, at the basis of cellular plasticity is the emergence of a yet

  3. DNA binding specificity of the basic-helix-loop-helix protein MASH-1.

    Science.gov (United States)

    Meierhan, D; el-Ariss, C; Neuenschwander, M; Sieber, M; Stackhouse, J F; Allemann, R K

    1995-09-05

    Despite the high degree of sequence similarity in their basic-helix-loop-helix (BHLH) domains, MASH-1 and MyoD are involved in different biological processes. In order to define possible differences between the DNA binding specificities of these two proteins, we investigated the DNA binding properties of MASH-1 by circular dichroism spectroscopy and by electrophoretic mobility shift assays (EMSA). Upon binding to DNA, the BHLH domain of MASH-1 underwent a conformational change from a mainly unfolded to a largely alpha-helical form, and surprisingly, this change was independent of the specific DNA sequence. The same conformational transition could be induced by the addition of 20% 2,2,2-trifluoroethanol. The apparent dissociation constants (KD) of the complexes of full-length MASH-1 with various oligonucleotides were determined from half-saturation points in EMSAs. MASH-1 bound as a dimer to DNA sequences containing an E-box with high affinity KD = 1.4-4.1 x 10(-14) M2). However, the specificity of DNA binding was low. The dissociation constant for the complex between MASH-1 and the highest affinity E-box sequence (KD = 1.4 x 10(-14) M2) was only a factor of 10 smaller than for completely unrelated DNA sequences (KD = approximately 1 x 10(-13) M2). The DNA binding specificity of MASH-1 was not significantly increased by the formation of an heterodimer with the ubiquitous E12 protein. MASH-1 and MyoD displayed similar binding site preferences, suggesting that their different target gene specificities cannot be explained solely by differential DNA binding. An explanation for these findings is provided on the basis of the known crystal structure of the BHLH domain of MyoD.

  4. A Bystander Mechanism Explains the Specific Phenotype of a Broadly Expressed Misfolded Protein.

    Directory of Open Access Journals (Sweden)

    Lauren Klabonski

    2016-12-01

    Full Text Available Misfolded proteins in transgenic models of conformational diseases interfere with proteostasis machinery and compromise the function of many structurally and functionally unrelated metastable proteins. This collateral damage to cellular proteins has been termed 'bystander' mechanism. How a single misfolded protein overwhelms the proteostasis, and how broadly-expressed mutant proteins cause cell type-selective phenotypes in disease are open questions. We tested the gain-of-function mechanism of a R37C folding mutation in an endogenous IGF-like C.elegans protein DAF-28. DAF-28(R37C is broadly expressed, but only causes dysfunction in one specific neuron, ASI, leading to a distinct developmental phenotype. We find that this phenotype is caused by selective disruption of normal biogenesis of an unrelated endogenous protein, DAF-7/TGF-β. The combined deficiency of DAF-28 and DAF-7 biogenesis, but not of DAF-28 alone, explains the gain-of-function phenotype-deficient pro-growth signaling by the ASI neuron. Using functional, fluorescently-tagged protein, we find that, in animals with mutant DAF-28/IGF, the wild-type DAF-7/TGF-β is mislocalized to and accumulates in the proximal axon of the ASI neuron. Activation of two different branches of the unfolded protein response can modulate both the developmental phenotype and DAF-7 mislocalization in DAF-28(R37C animals, but appear to act through divergent mechanisms. Our finding that bystander targeting of TGF-β explains the phenotype caused by a folding mutation in an IGF-like protein suggests that, in conformational diseases, bystander misfolding may specify the distinct phenotypes caused by different folding mutations.

  5. Trypanosoma equiperdum Low Molecular Weight Proteins As Candidates for Specific Serological Diagnosis of Dourine

    Directory of Open Access Journals (Sweden)

    Mirella Luciani

    2018-03-01

    Full Text Available The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by Trypanosoma evansi. Moreover, T. equiperdum and T. evansi are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the T. equiperdum protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses specifically bind T. equiperdum low molecular weight proteins (from 16 to 35 kDa, which are not recognized by antibodies from uninfected horses. In this work, we tested other 615 sera (7 from naturally infected horses and 608 sera from healthy horses and donkeys: results confirmed the data obtained previously. In addition, six SDS-PAGE bands with molecular weight ranging from 10 to 37 kDa were analyzed by mass spectrometry, in order to identify immunogenic proteins that could be used as biomarkers for the diagnosis of dourine. A total of 167 proteins were identified. Among them, 37 were found unique for T. equiperdum. Twenty-four of them could represent possible candidate diagnostic antigens for the development of serological tests specific for T. equiperdum.

  6. Cross-cultural adaptation and initial validation of the Stroke-Specific Quality of Life Scale into the Yoruba language.

    Science.gov (United States)

    Akinpelu, Aderonke O; Odetunde, Marufat O; Odole, Adesola C

    2012-12-01

    Stroke-Specific Quality of Life 2.0 (SS-QoL 2.0) scale is used widely and has been cross-culturally adapted to many languages. This study aimed at the cross-cultural adaptation of SS-QoL 2.0 to Yoruba, the indigenous language of south-western Nigeria, and to carry out an initial investigation on its validity. English SS-QoL 2.0 was first adapted to Yoruba language by including Yoruba culture-specific examples in items SC4, UE2 and UE6. The adapted English version (AEV) was independently translated into Yoruba by two language experts who later agreed on a consensus translation, which was then back translated, subjected to an expert committee review and pretested; a cognitive debriefing interview was also carried out to generate the Yoruba translated version (YTV). Thirty-five stroke survivors completed the AEV and Yoruba version (YV) in English and Yoruba. The order of administration was randomized. Data were analysed using Spearman's rank order correlation and Wilcoxon's signed-rank test at a P value less than 0.05. The mean age of the participants (23 men, 12 women) was 58.5±11.3 years. The domain scores of the participants on AEV and YV did not differ significantly, except in the work/productivity domain. In both versions, the mean domain score of the participants was the highest in the language domain [22.6±3.8 (AEV) and 22.7±3.4 (YV)] and the lowest in the work domain [9.0±3.7 (AEV) and 8.0±3.3 (YTV)]. Domain scores on both versions correlated significantly (P<0.05). Participants' ratings of their current state and prestroke state correlated significantly (P<0.01) in all the general areas, except energy and mood. The YTV of SS-QoL 2.0 fulfilled the initial criteria for validity.

  7. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    International Nuclear Information System (INIS)

    Tang, Zhaohua; Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse; Lin, Ren-Jang; Murray, Johanne; Carr, Antony

    2012-01-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A) + RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G 2 phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  8. Interacting factors and cellular localization of SR protein-specific kinase Dsk1

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Zhaohua, E-mail: ztang@jsd.claremont.edu [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Luca, Maria; Taggart-Murphy, Laura; Portillio, Jessica; Chang, Cathey; Guven, Ayse [W.M. Keck Science Center, The Claremont Colleges, Claremont, CA 91711 (United States); Lin, Ren-Jang [Department of Molecular and Cellular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010 (United States); Murray, Johanne; Carr, Antony [Genome Damage and Stability Center, University of Sussex, Falmer, BN1 9RQ (United Kingdom)

    2012-10-01

    Schizosaccharomyces pombe Dsk1 is an SR protein-specific kinase (SRPK), whose homologs have been identified in every eukaryotic organism examined. Although discovered as a mitotic regulator with protein kinase activity toward SR splicing factors, it remains largely unknown about what and how Dsk1 contributes to cell cycle and pre-mRNA splicing. In this study, we investigated the Dsk1 function by determining interacting factors and cellular localization of the kinase. Consistent with its reported functions, we found that pre-mRNA processing and cell cycle factors are prominent among the proteins co-purified with Dsk1. The identification of these factors led us to find Rsd1 as a novel Dsk1 substrate, as well as the involvement of Dsk1 in cellular distribution of poly(A){sup +} RNA. In agreement with its role in nuclear events, we also found that Dsk1 is mainly localized in the nucleus during G{sub 2} phase and at mitosis. Furthermore, we revealed the oscillation of Dsk1 protein in a cell cycle-dependent manner. This paper marks the first comprehensive analysis of in vivo Dsk1-associated proteins in fission yeast. Our results reflect the conserved role of SRPK family in eukaryotic organisms, and provide information about how Dsk1 functions in pre-mRNA processing and cell-division cycle.

  9. Mammalian keratin associated proteins (KRTAPs) subgenomes: disentangling hair diversity and adaptation to terrestrial and aquatic environments.

    Science.gov (United States)

    Khan, Imran; Maldonado, Emanuel; Vasconcelos, Vítor; O'Brien, Stephen J; Johnson, Warren E; Antunes, Agostinho

    2014-09-10

    Adaptation of mammals to terrestrial life was facilitated by the unique vertebrate trait of body hair, which occurs in a range of morphological patterns. Keratin associated proteins (KRTAPs), the major structural hair shaft proteins, are largely responsible for hair variation. We exhaustively characterized the KRTAP gene family in 22 mammalian genomes, confirming the existence of 30 KRTAP subfamilies evolving at different rates with varying degrees of diversification and homogenization. Within the two major classes of KRTAPs, the high cysteine (HS) subfamily experienced strong concerted evolution, high rates of gene conversion/recombination and high GC content. In contrast, high glycine-tyrosine (HGT) KRTAPs showed evidence of positive selection and low rates of gene conversion/recombination. Species with more hair and of higher complexity tended to have more KRATP genes (gene expansion). The sloth, with long and coarse hair, had the most KRTAP genes (175 with 141 being intact). By contrast, the "hairless" dolphin had 35 KRTAPs and the highest pseudogenization rate (74% relative to the 19% mammalian average). Unique hair-related phenotypes, such as scales (armadillo) and spines (hedgehog), were correlated with changes in KRTAPs. Gene expression variation probably also influences hair diversification patterns, for example human have an identical KRTAP repertoire as apes, but much less hair. We hypothesize that differences in KRTAP gene repertoire and gene expression, together with distinct rates of gene conversion/recombination, pseudogenization and positive selection, are likely responsible for micro and macro-phenotypic hair diversification among mammals in response to adaptations to ecological pressures.

  10. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  11. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  12. Molecular mechanism of radioadaptive response: A cross-adaptive response for enhanced repair of DNA damage in adapted cells

    International Nuclear Information System (INIS)

    Takaji Ikushima

    1997-01-01

    The radioadaptive response (RAR) has been attributed to the induction of a repair mechanism by low doses of ionizing radiation, but the molecular nature of the mechanism is not yet elucidated. We have characterized RAR in a series of experiments in cultured Chinese hamster V79 cells. A 4-h interval is required for the full expression of RAR, which decays with the progression of cell proliferation. Treatments with inhibitors of poly(ADP-ribose) polymerase, protein- or RNA synthesis, and protein kinase C suppress the RAR expression. The RAR cross-reacts on clastogenic lesions induced by other physical and chemical DNA-damaging agents. The presence of newly synthesised proteins has been detected during the expression period. Experiments performed using single-cell gel electrophoresis provided more direct evidence for a faster and enhaced DNA repair rate in adapted cells. Here, using single-cell gel electrophoresis, a cross-adaptive response has been demonstrated for enhanced repair of DNA damage induced by neocarzinostatin in radio-adapted cells. (author)

  13. DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

    Science.gov (United States)

    Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

    2013-01-01

    Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

  14. Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization*

    Science.gov (United States)

    Müller, Daniel B.; Schubert, Olga T.; Röst, Hannes; Aebersold, Ruedi; Vorholt, Julia A.

    2016-01-01

    Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota. PMID:27457762

  15. Functional redundancy of division specific penicillin-binding proteins in Bacillus subtilis.

    Science.gov (United States)

    Sassine, Jad; Xu, Meizhu; Sidiq, Karzan R; Emmins, Robyn; Errington, Jeff; Daniel, Richard A

    2017-10-01

    Bacterial cell division involves the dynamic assembly of a diverse set of proteins that coordinate the invagination of the cell membrane and synthesis of cell wall material to create the new cell poles of the separated daughter cells. Penicillin-binding protein PBP 2B is a key cell division protein in Bacillus subtilis proposed to have a specific catalytic role in septal wall synthesis. Unexpectedly, we find that a catalytically inactive mutant of PBP 2B supports cell division, but in this background the normally dispensable PBP 3 becomes essential. Phenotypic analysis of pbpC mutants (encoding PBP 3) shows that PBP 2B has a crucial structural role in assembly of the division complex, independent of catalysis, and that its biochemical activity in septum formation can be provided by PBP 3. Bioinformatic analysis revealed a close sequence relationship between PBP 3 and Staphylococcus aureus PBP 2A, which is responsible for methicillin resistance. These findings suggest that mechanisms for rescuing cell division when the biochemical activity of PBP 2B is perturbed evolved prior to the clinical use of β-lactams. © 2017 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  16. Strategies for specifically directing metal functionalization of protein nanotubes: constructing protein coated silver nanowires

    International Nuclear Information System (INIS)

    Carreño-Fuentes, Liliana; Palomares, Laura A; Ramírez, Octavio T; Ascencio, Jorge A; Medina, Ariosto; Aguila, Sergio

    2013-01-01

    Biological molecules that self-assemble in the nanoscale range are useful multifunctional materials. Rotavirus VP6 protein self-assembles into tubular structures in the absence of other rotavirus proteins. Here, we present strategies for selectively directing metal functionalization to the lumen of VP6 nanotubes. The specific in situ metal reduction in the inner surface of nanotube walls was achieved by the simple modification of a method previously reported to functionalize the nanotube outer surface. Silver nanorods and nanowires as long as 1.5 μm were formed inside the nanotubes by coalescence of nanoparticles. Such one-dimensional structures were longer than others previously obtained using bioscaffolds. The interactions between silver ions and the nanotube were simulated to understand the conditions that allowed nanowire formation. Molecular docking showed that a naturally occurring arrangement of aspartate residues enabled the stabilization of silver ions on the internal surface of the VP6 nanotubes. This is the first time that such a spatial arrangement has been proposed for the nucleation of silver nanoparticles, opening the possibility of using such an array to direct functionalization of other biomolecules. These results demonstrate the natural capabilities of VP6 nanotubes to function as a versatile biotemplate for nanomaterials. (paper)

  17. The Evolution of Two-Component Systems in Bacteria RevealsDifferent Strategies for Niche Adaptation

    Energy Technology Data Exchange (ETDEWEB)

    Alm, Eric; Huang, Katherine; Arkin, Adam

    2006-09-13

    Two-component systems including histidine protein kinasesrepresent the primary signal transduction paradigm in prokaryoticorganisms. To understand how these systems adapt to allow organisms todetect niche-specific signals, we analyzed the phylogenetic distributionof nearly 5000 histidine protein kinases from 207 sequenced prokaryoticgenomes. We found that many genomes carry a large repertoire of recentlyevolved signaling genes, which may reflect selective pressure to adapt tonew environmental conditions. Both lineage-specific gene family expansionand horizontal gene transfer play major roles in the introduction of newhistidine kinases into genomes; however, there are differences in howthese two evolutionary forces act. Genes imported via horizontal transferare more likely to retain their original functionality as inferred from asimilar complement of signaling domains, while gene family expansionaccompanied by domain shuffling appears to be a major source of novelgenetic diversity. Family expansion is the dominantsource of newhistidine kinase genes in the genomes most enriched in signalingproteins, and detailed analysis reveals that divergence in domainstructure and changes in expression patterns are hallmarks of recentexpansions. Finally, while these two modes of gene acquisition arewidespread across bacterial taxa, there are clear species-specificpreferences for which mode is used.

  18. Evidence of a New Role for the High-Osmolarity Glycerol Mitogen-Activated Protein Kinase Pathway in Yeast: Regulating Adaptation to Citric Acid Stress†

    OpenAIRE

    Lawrence, Clare L.; Botting, Catherine H.; Antrobus, Robin; Coote, Peter J.

    2004-01-01

    Screening the Saccharomyces cerevisiae disruptome, profiling transcripts, and determining changes in protein expression have identified an important new role for the high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway in the regulation of adaptation to citric acid stress. Deletion of HOG1, SSK1, PBS2, PTC2, PTP2, and PTP3 resulted in sensitivity to citric acid. Furthermore, citric acid resulted in the dual phosphorylation, and thus activation, of Hog1p. Despite mino...

  19. Genetic regulation by amino acids of specific membrane protein biosynthesis in isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Chiles, T.C.; Handlogten, M.E.; Kilberg, M.S.

    1986-01-01

    Rat Hepatocytes in primary culture were incubated in amino acid-free (AAF) medium or amino acid-supplemented (AAS) medium for 2-6 hr. The effect of amino acid starvation on the synthesis of specific membrane proteins was monitored by including 3 H-leucine during the incubation. A crude plasma membrane fraction was prepared and then analyzed by 2-D gel electrophoresis followed by fluorography. Amino acid deprivation caused an induction of the synthesis of 5 of the 30 proteins studied. The ratio (AAF/-AAS) of cpm incorporated into the remaining 25 proteins was 0.8 +/- 0.2, whereas the ratio for the 5 proteins that showed amino acid-dependent synthesis ranged from 1.5 to 2.5. The presence of 4 μM actinomycin in the AAF medium completely blocked the starvation-induced synthesis of the 5 proteins tested, but did not alter significantly the ratio of cpm incorporated into the other 25 proteins. Binding studies involving ConA suggested a plasma membrane location for the 5 proteins. The molecular weight values of the starvation-induced proteins are 70, 66, 66, 67, and 45kD. Surface-labelling of intact cells and preparation of antibodies against the 5 proteins will be used to establish the subcellular location and to describe the amino acid-dependent synthesis of each in more detail

  20. Adaptive compressive learning for prediction of protein-protein interactions from primary sequence.

    Science.gov (United States)

    Zhang, Ya-Nan; Pan, Xiao-Yong; Huang, Yan; Shen, Hong-Bin

    2011-08-21

    Protein-protein interactions (PPIs) play an important role in biological processes. Although much effort has been devoted to the identification of novel PPIs by integrating experimental biological knowledge, there are still many difficulties because of lacking enough protein structural and functional information. It is highly desired to develop methods based only on amino acid sequences for predicting PPIs. However, sequence-based predictors are often struggling with the high-dimensionality causing over-fitting and high computational complexity problems, as well as the redundancy of sequential feature vectors. In this paper, a novel computational approach based on compressed sensing theory is proposed to predict yeast Saccharomyces cerevisiae PPIs from primary sequence and has achieved promising results. The key advantage of the proposed compressed sensing algorithm is that it can compress the original high-dimensional protein sequential feature vector into a much lower but more condensed space taking the sparsity property of the original signal into account. What makes compressed sensing much more attractive in protein sequence analysis is its compressed signal can be reconstructed from far fewer measurements than what is usually considered necessary in traditional Nyquist sampling theory. Experimental results demonstrate that proposed compressed sensing method is powerful for analyzing noisy biological data and reducing redundancy in feature vectors. The proposed method represents a new strategy of dealing with high-dimensional protein discrete model and has great potentiality to be extended to deal with many other complicated biological systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. A global analysis of protein expression profiles in Sinorhizobium meliloti: discovery of new genes for nodule occupancy and stress adaptation.

    Science.gov (United States)

    Djordjevic, Michael A; Chen, Han Cai; Natera, Siria; Van Noorden, Giel; Menzel, Christian; Taylor, Scott; Renard, Clotilde; Geiger, Otto; Weiller, Georg F

    2003-06-01

    A proteomic examination of Sinorhizobium meliloti strain 1021 was undertaken using a combination of 2-D gel electrophoresis, peptide mass fingerprinting, and bioinformatics. Our goal was to identify (i) putative symbiosis- or nutrient-stress-specific proteins, (ii) the biochemical pathways active under different conditions, (iii) potential new genes, and (iv) the extent of posttranslational modifications of S. meliloti proteins. In total, we identified the protein products of 810 genes (13.1% of the genome's coding capacity). The 810 genes generated 1,180 gene products, with chromosomal genes accounting for 78% of the gene products identified (18.8% of the chromosome's coding capacity). The activity of 53 metabolic pathways was inferred from bioinformatic analysis of proteins with assigned Enzyme Commission numbers. Of the remaining proteins that did not encode enzymes, ABC-type transporters composed 12.7% and regulatory proteins 3.4% of the total. Proteins with up to seven transmembrane domains were identified in membrane preparations. A total of 27 putative nodule-specific proteins and 35 nutrient-stress-specific proteins were identified and used as a basis to define genes and describe processes occurring in S. meliloti cells in nodules and under stress. Several nodule proteins from the plant host were present in the nodule bacteria preparations. We also identified seven potentially novel proteins not predicted from the DNA sequence. Post-translational modifications such as N-terminal processing could be inferred from the data. The posttranslational addition of UMP to the key regulator of nitrogen metabolism, PII, was demonstrated. This work demonstrates the utility of combining mass spectrometry with protein arraying or separation techniques to identify candidate genes involved in important biological processes and niche occupations that may be intransigent to other methods of gene expression profiling.

  2. An adaptive signal-processing approach to online adaptive tutoring.

    Science.gov (United States)

    Bergeron, Bryan; Cline, Andrew

    2011-01-01

    Conventional intelligent or adaptive tutoring online systems rely on domain-specific models of learner behavior based on rules, deep domain knowledge, and other resource-intensive methods. We have developed and studied a domain-independent methodology of adaptive tutoring based on domain-independent signal-processing approaches that obviate the need for the construction of explicit expert and student models. A key advantage of our method over conventional approaches is a lower barrier to entry for educators who want to develop adaptive online learning materials.

  3. Proteomic Analysis of Bovine Pregnancy-specific Serum Proteins by 2D Fluorescence Difference Gel Electrophoresis

    OpenAIRE

    Lee, Jae Eun; Lee, Jae Young; Kim, Hong Rye; Shin, Hyun Young; Lin, Tao; Jin, Dong Il

    2015-01-01

    Two dimensional-fluorescence difference gel electrophoresis (2D DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples. The purpose of this study was to investigate bovine pregnancy-specific proteins in the proteome between bovine pregnant and non-pregnant serum using DIGE technique. Serums of 2 pregnant Holstein dairy cattle at day 21 after artificial insemination and those of 2...

  4. Specific metabolomics adaptations define a differential regional vulnerability in the adult human cerebral cortex

    Directory of Open Access Journals (Sweden)

    Rosanna Cabré

    2016-12-01

    Full Text Available Brain neurons offer diverse responses to stresses and detrimental factors during development and aging, and as a result of both neurodegenerative and neuropsychiatric disorders. This multiplicity of responses can be ascribed to the great diversity among neuronal populations. Here we have determined the metabolomic profile of three healthy adult human brain regions—entorhinal cortex, hippocampus, and frontal cortex—using mass spectrometry-based technologies. Our results show the existence of a lessened energy demand, mitochondrial stress, and lower one-carbon metabolism (particularly restricted to the methionine cycle specifically in frontal cortex. These findings, along with the better antioxidant capacity and lower mTOR signaling also seen in frontal cortex, suggest that this brain region is especially resistant to stress compared to the entorhinal cortex and hippocampus, which are more vulnerable regions. Globally, our results show the presence of specific metabolomics adaptations in three mature, healthy human brain regions, confirming the existence of cross-regional differences in cell vulnerability in the human cerebral cortex.

  5. High-throughput bioscreening system utilizing high-performance affinity magnetic carriers exhibiting minimal non-specific protein binding

    International Nuclear Information System (INIS)

    Hanyu, Naohiro; Nishio, Kosuke; Hatakeyama, Mamoru; Yasuno, Hiroshi; Tanaka, Toshiyuki; Tada, Masaru; Nakagawa, Takashi; Sandhu, Adarsh; Abe, Masanori; Handa, Hiroshi

    2009-01-01

    For affinity purification of drug target protein we have developed magnetic carriers, narrow in size distribution (184±9 nm), which exhibit minimal non-specific binding of unwanted proteins. The carriers were highly dispersed in aqueous solutions and highly resistant to organic solvents, which enabled immobilization of various hydrophobic chemicals as probes on the carrier surfaces. Utilizing the carriers we have automated the process of separation and purification of the target proteins that had been done by manual operation previously.

  6. C-terminal sequences of hsp70 and hsp90 as non-specific anchors for tetratricopeptide repeat (TPR) proteins.

    Science.gov (United States)

    Ramsey, Andrew J; Russell, Lance C; Chinkers, Michael

    2009-10-12

    Steroid-hormone-receptor maturation is a multi-step process that involves several TPR (tetratricopeptide repeat) proteins that bind to the maturation complex via the C-termini of hsp70 (heat-shock protein 70) and hsp90 (heat-shock protein 90). We produced a random T7 peptide library to investigate the roles played by the C-termini of the two heat-shock proteins in the TPR-hsp interactions. Surprisingly, phages with the MEEVD sequence, found at the C-terminus of hsp90, were not recovered from our biopanning experiments. However, two groups of phages were isolated that bound relatively tightly to HsPP5 (Homo sapiens protein phosphatase 5) TPR. Multiple copies of phages with a C-terminal sequence of LFG were isolated. These phages bound specifically to the TPR domain of HsPP5, although mutation studies produced no evidence that they bound to the domain's hsp90-binding groove. However, the most abundant family obtained in the initial screen had an aspartate residue at the C-terminus. Two members of this family with a C-terminal sequence of VD appeared to bind with approximately the same affinity as the hsp90 C-12 control. A second generation pseudo-random phage library produced a large number of phages with an LD C-terminus. These sequences acted as hsp70 analogues and had relatively low affinities for hsp90-specific TPR domains. Unfortunately, we failed to identify residues near hsp90's C-terminus that impart binding specificity to individual hsp90-TPR interactions. The results suggest that the C-terminal sequences of hsp70 and hsp90 act primarily as non-specific anchors for TPR proteins.

  7. PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer’s amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE

    Directory of Open Access Journals (Sweden)

    Lahiri Debomoy K

    2013-01-01

    Full Text Available Abstract Background Alzheimer’s disease (AD is intimately tied to amyloid-β (Aβ peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE, at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones. Results EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2, nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF, and specificity protein 1 (SP1. These sites crossed a known single nucleotide polymorphism (SNP. EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis. Conclusions We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also

  8. Design principle of TVO's final repository and preliminary adaptation to site specific conditions

    International Nuclear Information System (INIS)

    Salo, J-P.; Reikkola, R.

    1995-01-01

    Teollisuuden Voima Oy (TVO) is responsible for the management of spent fuel produced by the Olkiluoto power plant. TVO's current programme of spent fuel management is based on the guidelines and time schedule set by the Finnish Government. TVO has studied a final disposal concept in which the spent fuel bundles are encapsulated in copper canisters and emplaced in Finnish bedrock. According to the plan the final repository for spent fuel will be in operation by 2020. TVO's updated technical plans for the disposal of spent fuel together with a performance analysis (TVO-92) were submitted to the authorities in 1992. The paper describes the design principle of TVO's final repository and preliminary adaptation of the repository to site specific conditions. (author). 10 refs., 5 figs

  9. Electrochemical Characterization of Escherichia coli Adaptive Response Protein AidB

    Directory of Open Access Journals (Sweden)

    Sean J. Elliott

    2012-12-01

    Full Text Available When exposed to known DNA-damaging alkylating agents, Escherichia coli cells increase production of four DNA repair enzymes: Ada, AlkA, AlkB, and AidB. The role of three enzymes (Ada, AlkA, and AlkB in repairing DNA lesions has been well characterized, while the function of AidB is poorly understood. AidB has a distinct cofactor that is potentially related to the elusive role of AidB in adaptive response: a redox active flavin adenine dinucleotide (FAD. In this study, we report the thermodynamic redox properties of the AidB flavin for the first time, both for free protein and in the presence of potential substrates. We find that the midpoint reduction potential of the AidB flavin is within a biologically relevant window for redox chemistry at −181 mV, that AidB significantly stabilizes the flavin semiquinone, and that small molecule binding perturbs the observed reduction potential. Our electrochemical results combined with structural analysis allow for fresh comparisons between AidB and the homologous acyl-coenzyme A dehydrogenase (ACAD family of enzymes. AidB exhibits several discrepancies from ACADs that suggest a novel catalytic mechanism distinct from that of the ACAD family enzymes.

  10. Diet-induced obesity alters protein synthesis: Tissue-specific effects in fasted vs. fed mice

    OpenAIRE

    Anderson, Stephanie R.; Gilge, Danielle A.; Steiber, Alison L.; Previs, Stephen F.

    2008-01-01

    The influence of obesity on protein dynamics is not clearly understood. We have designed experiments to test the hypothesis that obesity impairs the stimulation of tissue-specific protein synthesis following nutrient ingestion. C57BL/6J mice were randomized into two groups: group 1 (control, n = 16) were fed a low-fat, high-carbohydrate diet and group 2 (experimental, n = 16) were fed a high-fat, low-carbohydrate diet ad libitum for 9 weeks. On the experiment day, all mice were fasted for 6 h...

  11. Molecular dynamics investigations of BioH protein substrate specificity for biotin synthesis.

    Science.gov (United States)

    Xue, Qiao; Cui, Ying-Lu; Zheng, Qing-Chuan; Zhang, Hong-Xing

    2016-05-01

    BioH, an enzyme of biotin synthesis, plays an important role in fatty acid synthesis which assembles the pimelate moiety. Pimeloyl-acyl carrier protein (ACP) methyl ester, which is long known to be a biotin precursor, is the physiological substrate of BioH. Azelayl methyl ester, which has a longer chain than pimeloyl methyl ester, conjugated to ACP is also indeed accepted by BioH with very low rate of hydrolysis. To date, the substrate specificity for BioH and the molecular origin for the experimentally observed rate changes of hydrolysis by the chain elongation have remained elusive. To this end, we have investigated chain elongation effects on the structures by using the fully atomistic molecular dynamics simulations combined with binding free energy calculations. The results indicate that the substrate specificity is determined by BioH together with ACP. The added two methylenes would increase the structural flexibility by protein motions at the interface of ACP and BioH, instead of making steric clashes with the side chains of the BioH hydrophobic cavity. On the other hand, the slower hydrolysis of azelayl substrate is suggested to be associated with the loose of contacts between BioH and ACP, and with the lost electrostatic interactions of two ionic/hydrogen bonding networks at the interface of the two proteins. The present study provides important insights into the structure-function relationships of the complex of BioH with pimeloyl-ACP methyl ester, which could contribute to further understanding about the mechanism of the biotin synthetic pathway, including the catalytic role of BioH.

  12. Vaccine platforms combining circumsporozoite protein and potent immune modulators, rEA or EAT-2, paradoxically result in opposing immune responses.

    Directory of Open Access Journals (Sweden)

    Nathaniel J Schuldt

    Full Text Available Malaria greatly impacts the health and wellbeing of over half of the world's population. Promising malaria vaccine candidates have attempted to induce adaptive immune responses to Circumsporozoite (CS protein. Despite the inclusion of potent adjuvants, these vaccines have limited protective efficacy. Conventional recombinant adenovirus (rAd based vaccines expressing CS protein can induce CS protein specific immune responses, but these are essentially equivalent to those generated after use of the CS protein subunit based vaccines. In this study we combined the use of rAds expressing CS protein along with rAds expressing novel innate immune response modulating proteins in an attempt to significantly improve the induction of CS protein specific cell mediated immune (CMI responses.BALB/cJ mice were co-vaccinated with a rAd vectors expressing CS protein simultaneous with a rAd expressing either TLR agonist (rEA or SLAM receptors adaptor protein (EAT-2. Paradoxically, expression of the TLR agonist uncovered a potent immunosuppressive activity inherent to the combined expression of the CS protein and rEA. Fortunately, use of the rAd vaccine expressing EAT-2 circumvented CS protein's suppressive activity, and generated a fivefold increase in the number of CS protein responsive, IFNγ secreting splenocytes, as well as increased the breadth of T cells responsive to peptides present in the CS protein. These improvements were positively correlated with the induction of a fourfold improvement in CS protein specific CTL functional activity in vivo.Our results emphasize the need for caution when incorporating CS protein into malaria vaccine platforms expressing or containing other immunostimulatory compounds, as the immunological outcomes may be unanticipated and/or counter-productive. However, expressing the SLAM receptors derived signaling adaptor EAT-2 at the same time of vaccination with CS protein can overcome these concerns, as well as significantly

  13. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    Science.gov (United States)

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (Pmutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other

  14. Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators

    Directory of Open Access Journals (Sweden)

    Shucai eWang

    2016-03-01

    Full Text Available OVATE FAMILY PROTEINS (OFPs are a class of proteins with a conserved OVATE domain. OVATE protein was first identified in tomato as a key regulator of fruit shape. OFPs are plant-specific proteins that are widely distributed in the plant kingdom including mosses and lycophytes. Transcriptional activity analysis of Arabidopsis OFPs (AtOFPs in protoplasts suggests that they act as transcription repressors. Functional characterization of OFPs from different plant species including Arabidopsis, rice, tomato, pepper and banana suggests that OFPs regulate multiple aspects of plant growth and development, which is likely achieved by interacting with different types of transcription factors including the KNOX and BELL classes, and/or directly regulating the expression of target genes such as Gibberellin 20 oxidase (GA20ox. Here, we examine how OVATE was originally identified, summarize recent progress in elucidation of the roles of OFPs in regulating plant growth and development, and describe possible mechanisms underpinning this regulation. Finally, we review potential new research directions that could shed additional light on the functional biology of OFPs in plants.

  15. Multisensory and Modality-Specific Influences on Adaptation to Optical Prisms

    Directory of Open Access Journals (Sweden)

    Elena Calzolari

    2017-11-01

    Full Text Available Visuo-motor adaptation to optical prisms displacing the visual scene (prism adaptation, PA is a method used for investigating visuo-motor plasticity in healthy individuals and, in clinical settings, for the rehabilitation of unilateral spatial neglect. In the standard paradigm, the adaptation phase involves repeated pointings to visual targets, while wearing optical prisms displacing the visual scene laterally. Here we explored differences in PA, and its aftereffects (AEs, as related to the sensory modality of the target. Visual, auditory, and multisensory – audio-visual – targets in the adaptation phase were used, while participants wore prisms displacing the visual field rightward by 10°. Proprioceptive, visual, visual-proprioceptive, auditory-proprioceptive straight-ahead shifts were measured. Pointing to auditory and to audio-visual targets in the adaptation phase produces proprioceptive, visual-proprioceptive, and auditory-proprioceptive AEs, as the typical visual targets did. This finding reveals that cross-modal plasticity effects involve both the auditory and the visual modality, and their interactions (Experiment 1. Even a shortened PA phase, requiring only 24 pointings to visual and audio-visual targets (Experiment 2, is sufficient to bring about AEs, as compared to the standard 92-pointings procedure. Finally, pointings to auditory targets cause AEs, although PA with a reduced number of pointings (24 to auditory targets brings about smaller AEs, as compared to the 92-pointings procedure (Experiment 3. Together, results from the three experiments extend to the auditory modality the sensorimotor plasticity underlying the typical AEs produced by PA to visual targets. Importantly, PA to auditory targets appears characterized by less accurate pointings and error correction, suggesting that the auditory component of the PA process may be less central to the building up of the AEs, than the sensorimotor pointing activity per se. These

  16. Small but Crucial: The Novel Small Heat Shock Protein Hsp21 Mediates Stress Adaptation and Virulence in Candida albicans

    Science.gov (United States)

    Mayer, François L.; Wilson, Duncan; Jacobsen, Ilse D.; Miramón, Pedro; Slesiona, Silvia; Bohovych, Iryna M.; Brown, Alistair J. P.; Hube, Bernhard

    2012-01-01

    Small heat shock proteins (sHsps) have multiple cellular functions. However, the biological function of sHsps in pathogenic microorganisms is largely unknown. In the present study we identified and characterized the novel sHsp Hsp21 of the human fungal pathogen Candida albicans. Using a reverse genetics approach we demonstrate the importance of Hsp21 for resistance of C. albicans to specific stresses, including thermal and oxidative stress. Furthermore, a hsp21Δ/Δ mutant was defective in invasive growth and formed significantly shorter filaments compared to the wild type under various filament-inducing conditions. Although adhesion to and invasion into human-derived endothelial and oral epithelial cells was unaltered, the hsp21Δ/Δ mutant exhibited a strongly reduced capacity to damage both cell lines. Furthermore, Hsp21 was required for resisting killing by human neutrophils. Measurements of intracellular levels of stress protective molecules demonstrated that Hsp21 is involved in both glycerol and glycogen regulation and plays a major role in trehalose homeostasis in response to elevated temperatures. Mutants defective in trehalose and, to a lesser extent, glycerol synthesis phenocopied HSP21 deletion in terms of increased susceptibility to environmental stress, strongly impaired capacity to damage epithelial cells and increased sensitivity to the killing activities of human primary neutrophils. Via systematic analysis of the three main C. albicans stress-responsive kinases (Mkc1, Cek1, Hog1) under a range of stressors, we demonstrate Hsp21-dependent phosphorylation of Cek1 in response to elevated temperatures. Finally, the hsp21Δ/Δ mutant displayed strongly attenuated virulence in two in vivo infection models. Taken together, Hsp21 mediates adaptation to specific stresses via fine-tuning homeostasis of compatible solutes and activation of the Cek1 pathway, and is crucial for multiple stages of C. albicans pathogenicity. Hsp21 therefore represents the first

  17. Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

    Science.gov (United States)

    Canova, Marc J.; Molle, Virginie

    2014-01-01

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

  18. Bacterial serine/threonine protein kinases in host-pathogen interactions.

    Science.gov (United States)

    Canova, Marc J; Molle, Virginie

    2014-04-04

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

  19. Suppressor of cytokine signaling 1 interacts with oncogenic lymphocyte-specific protein tyrosine kinase.

    Science.gov (United States)

    Venkitachalam, Srividya; Chueh, Fu-Yu; Leong, King-Fu; Pabich, Samantha; Yu, Chao-Lan

    2011-03-01

    Lymphocyte-specific protein tyrosine kinase (Lck) plays a key role in T cell signal transduction and is tightly regulated by phosphorylation and dephosphorylation. Lck can function as an oncoprotein when overexpressed or constantly activated by mutations. Our previous studies showed that Lck-induced cellular transformation could be suppressed by enforced expression of suppressor of cytokine signaling 1 (SOCS1), a SOCS family member involved in the negative feedback control of cytokine signaling. We observed attenuated Lck kinase activity in SOCS1-expressing cells, suggesting an important role of SOCS in regulating Lck functions. It remains largely unknown whether and how SOCS proteins interact with the oncogenic Lck kinase. Here, we report that among four SOCS family proteins, SOCS1, SOCS2, SOCS3 and CIS (cytokine-inducible SH2 domain containing protein), SOCS1 has the highest affinity in binding to the oncogenic Lck kinase. We identified the positive regulatory phosphotyrosine 394 residue in the kinase domain as the key interacting determinant in Lck. Additionally, the Lck kinase domain alone is sufficient to bind SOCS1. While the SH2 domain in SOCS1 is important in its association with the oncogenic Lck kinase, other functional domains may also contribute to overall binding affinity. These findings provide important mechanistic insights into the role of SOCS proteins as tumor suppressors in cells transformed by oncogenic protein tyrosine kinases.

  20. Combining substrate specificity analysis with support vector classifiers reveals feruloyl esterase as a phylogenetically informative protein group

    DEFF Research Database (Denmark)

    Olivares Hernandez, Roberto; Sunner, Hampus; Frisvad, Jens Christian

    2010-01-01

    Background Our understanding of how fungi evolved to develop a variety of ecological niches, is limited but of fundamental biological importance. Specifically, the evolution of enzymes affects how well species can adapt to new environmental conditions. Feruloyl esterases (FAEs) are enzymes able...