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Sample records for import proteins karyopherin-alpha1

  1. Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import.

    Science.gov (United States)

    Choo, Hyo-Jung; Cutler, Alicia; Pavlath, Grace K

    2016-07-19

    Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self-renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical cotranscription factor for β-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. Stem Cells 2016.

  2. Import of ribosomal proteins into yeast mitochondria.

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    Woellhaf, Michael W; Hansen, Katja G; Garth, Christoph; Herrmann, Johannes M

    2014-12-01

    Mitochondrial ribosomes of baker's yeast contain at least 78 protein subunits. All but one of these proteins are nuclear-encoded, synthesized on cytosolic ribosomes, and imported into the matrix for biogenesis. The import of matrix proteins typically relies on N-terminal mitochondrial targeting sequences that form positively charged amphipathic helices. Interestingly, the N-terminal regions of many ribosomal proteins do not closely match the characteristics of matrix targeting sequences, suggesting that the import processes of these proteins might deviate to some extent from the general import route. So far, the biogenesis of only two ribosomal proteins, Mrpl32 and Mrp10, was studied experimentally and indeed showed surprising differences to the import of other preproteins. In this review article we summarize the current knowledge on the transport of proteins into the mitochondrial matrix, and thereby specifically focus on proteins of the mitochondrial ribosome.

  3. Mitochondrial Machineries for Protein Import and Assembly.

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    Wiedemann, Nils; Pfanner, Nikolaus

    2017-03-15

    Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  4. Import of proteins into isolated yeast mitochondria.

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    Peleh, Valentina; Ramesh, Ajay; Herrmann, Johannes M

    2015-01-01

    Mitochondria are essential organelles of eukaryotic cells. The vast majority of mitochondrial proteins is encoded within the nuclear genome and translocated into various mitochondrial compartments after translation in the cytosol as preproteins. Even in rather primitive eukaryotes like yeasts, there are 700-1,000 different proteins that need to be recognized in the cytosol, directed to the protein translocases in the two mitochondrial membranes and sorted to their appropriate mitochondrial subcompartment. In vitro reconstituted import systems have proved to be important tools to study these processes in detail. Using isolated mitochondria and radioactively labeled precursor proteins, it was possible to identify several import machineries and pathways consisting of a large number of components during the last few decades.

  5. Serotonylation of vascular proteins important to contraction.

    Directory of Open Access Journals (Sweden)

    Stephanie W Watts

    Full Text Available BACKGROUND: Serotonin (5-hydroxytryptamine, 5-HT was named for its source (sero- and ability to modify smooth muscle tone (tonin. The biological effects of 5-HT are believed to be carried out by stimulation of serotonin receptors at the plasma membrane. Serotonin has recently been shown to be synthesized in vascular smooth muscle and taken up from external sources, placing 5-HT inside the cell. The enzyme transglutaminase uses primary amines such as 5-HT to covalently modify proteins on glutamine residues. We tested the hypothesis that 5-HT is a substrate for transglutaminase in arterial vascular smooth muscle, with protein serotonylation having physiological function. METHODOLOGY/PRINCIPAL FINDINGS: The model was the rat aorta and cultured aortic smooth muscle cells. Western analysis demonstrated that transglutaminase II was present in vascular tissue, and transglutaminase activity was observed as a cystamine-inhibitable incorporation of the free amine pentylamine-biotin into arterial proteins. Serotonin-biotin was incorporated into alpha-actin, beta-actin, gamma-actin, myosin heavy chain and filamin A as shown through tandem mass spectrometry. Using antibodies directed against biotin or 5-HT, immunoprecipitation and immunocytochemistry confirmed serotonylation of smooth muscle alpha-actin. Importantly, the alpha-actin-dependent process of arterial isometric contraction to 5-HT was reduced by cystamine. CONCLUSIONS: 5-HT covalently modifies proteins integral to contractility and the cytoskeleton. These findings suggest new mechanisms of action for 5-HT in vascular smooth muscle and consideration for intracellular effects of primary amines.

  6. Importance of bestrophin-4 protein for vasomotion

    DEFF Research Database (Denmark)

    Matchkov, Vladimir; Larsen, Per; Bouzinova, Elena V.

    2008-01-01

    We have recently characterized a cGMP-dependent Ca2+-activated Cl- current (ICl,cGMP) with unique characteristics in smooth muscle cells (SMCs) and suggested that this could be important for vasomotion. In SMCs this current co-exists with the "classical" Ca2+-activated Cl- current. We hypothesized......). In this study we tested the hypotheses that ICl,cGMP is mediated by bestrophin-4 and that bestrophin-4 is important for vasomotion. Bestrophin-4 was downregulated with small interference (siRNA) in A7r5 cells and in rat mesenteric small arteries in vivo. siRNAs targeting different exons of bestrophin-4 were...... used. Non-related siRNA and bestrophin-4 siRNA with 2 changed nucleotides were used as the controls for non-specific siRNA action. Downregulation induced by siRNA was evident by reduced expression of bestrophin-4 mRNA and protein (by 82±6 % and by 54±4 % (n=3 and 6), respectively, for cell culture...

  7. Pathophysiological importance of aggregated damaged proteins.

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    Höhn, Annika; Jung, Tobias; Grune, Tilman

    2014-06-01

    Reactive oxygen species (ROS) are formed continuously in the organism even under physiological conditions. If the level of ROS in cells exceeds the cellular defense capacity, components such as RNA/DNA, lipids, and proteins are damaged and modified, thus affecting the functionality of organelles as well. Proteins are especially prominent targets of various modifications such as oxidation, glycation, or conjugation with products of lipid peroxidation, leading to the alteration of their biological function, nonspecific interactions, and the production of high-molecular-weight protein aggregates. To ensure the maintenance of cellular functions, two proteolytic systems are responsible for the removal of oxidized and modified proteins, especially the proteasome and organelles, mainly the autophagy-lysosomal systems. Furthermore, increased protein oxidation and oxidation-dependent impairment of proteolytic systems lead to an accumulation of oxidized proteins and finally to the formation of nondegradable protein aggregates. Accordingly, the cellular homeostasis cannot be maintained and the cellular metabolism is negatively affected. Here we address the current knowledge of protein aggregation during oxidative stress, aging, and disease.

  8. Ubiquitin in the peroxisomal protein import pathway.

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    Francisco, Tânia; Rodrigues, Tony A; Pinto, Manuel P; Carvalho, Andreia F; Azevedo, Jorge E; Grou, Cláudia P

    2014-03-01

    PEX5 is the shuttling receptor for newly synthesized peroxisomal matrix proteins. Alone, or with the help of an adaptor protein, this receptor binds peroxisomal matrix proteins in the cytosol and transports them to the peroxisomal membrane docking/translocation module (DTM). The interaction between cargo-loaded PEX5 and the DTM ultimately results in its insertion into the DTM with the concomitant translocation of the cargo protein across the organelle membrane. PEX5 is not consumed in this event; rather it is dislocated back into the cytosol so that it can promote additional rounds of protein transportation. Remarkably, the data collected in recent years indicate that dislocation is preceded by monoubiquitination of PEX5 at a conserved cysteine residue. This mandatory modification is not the only type of ubiquitination occurring at the DTM. Indeed, several findings suggest that defective receptors jamming the DTM are polyubiquitinated and targeted to the proteasome for degradation.

  9. Functional Importance of Mobile Ribosomal Proteins.

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    Chang, Kai-Chun; Wen, Jin-Der; Yang, Lee-Wei

    2015-01-01

    Although the dynamic motions and peptidyl transferase activity seem to be embedded in the rRNAs, the ribosome contains more than 50 ribosomal proteins (r-proteins), whose functions remain largely elusive. Also, the precise forms of some of these r-proteins, as being part of the ribosome, are not structurally solved due to their high flexibility, which hinders the efforts in their functional elucidation. Owing to recent advances in cryo-electron microscopy, single-molecule techniques, and theoretical modeling, much has been learned about the dynamics of these r-proteins. Surprisingly, allosteric regulations have been found in between spatially separated components as distant as those in the opposite sides of the ribosome. Here, we focus on the functional roles and intricate regulations of the mobile L1 and L12 stalks and L9 and S1 proteins. Conformational flexibility also enables versatile functions for r-proteins beyond translation. The arrangement of r-proteins may be under evolutionary pressure that fine-tunes mass distributions for optimal structural dynamics and catalytic activity of the ribosome.

  10. Protein import into plant mitochondria: signals, machinery, processing, and regulation.

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    Murcha, Monika W; Kmiec, Beata; Kubiszewski-Jakubiak, Szymon; Teixeira, Pedro F; Glaser, Elzbieta; Whelan, James

    2014-12-01

    The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis.

  11. Dynamic organization of the mitochondrial protein import machinery.

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    Straub, Sebastian P; Stiller, Sebastian B; Wiedemann, Nils; Pfanner, Nikolaus

    2016-11-01

    Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

  12. Mitochondrial protein import and human health and disease.

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    MacKenzie, James A; Payne, R Mark

    2007-05-01

    The targeting and assembly of nuclear-encoded mitochondrial proteins are essential processes because the energy supply of humans is dependent upon the proper functioning of mitochondria. Defective import of mitochondrial proteins can arise from mutations in the targeting signals within precursor proteins, from mutations that disrupt the proper functioning of the import machinery, or from deficiencies in the chaperones involved in the proper folding and assembly of proteins once they are imported. Defects in these steps of import have been shown to lead to oxidative stress, neurodegenerative diseases, and metabolic disorders. In addition, protein import into mitochondria has been found to be a dynamically regulated process that varies in response to conditions such as oxidative stress, aging, drug treatment, and exercise. This review focuses on how mitochondrial protein import affects human health and disease.

  13. Giardia mitosomal protein import machinery differentially recognizes mitochondrial targeting signals.

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    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Estraño, Carlos E

    2014-01-01

    Giardia lamblia mitosomes are believed to be vestigial mitochondria which lack a genome. Similar to higher eukaryotes, mitosomal proteins possess either N-terminal or internal mitosomal targeting sequences. To date, some components of the higher eukaryote archetypal mitochondrial protein import apparatus have been identified and characterized in Giardia mitosomes; therefore, it is expected that mitochondrial signals will be recognized by the mitosomal protein import system. To further determine the level of conservation of the Giardia mitosome protein import apparatus, we expressed mitochondrial proteins from higher eukaryotes in Giardia. These recombinant proteins include Tom20 and Tom22; two components of the mitochondrial protein import machinery. Our results indicate that N-terminal mitochondrial targeting sequence is recognized by the mitosomal protein import machinery; however, interestingly the internal mitochondrial targeting sequences of higher eukaryotes are not recognized by the mitosome. Our results indicate that Giardia mitosome protein transport machinery shows differential recognition of higher eukaryotic mitochondria transfer signals, suggesting a divergence of the transport system in G. lamblia. Therefore, our data support the hypothesis that the protein import machinery in Giardia lamblia mitosome is an incomplete vestigial derivative of mitochondria components.

  14. Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

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    Ruan, Linhao; Zhou, Chuankai; Jin, Erli; Kucharavy, Andrei; Zhang, Ying; Wen, Zhihui; Florens, Laurence; Li, Rong

    2017-03-16

    Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.

  15. The functional importance of co-evolving residues in proteins.

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    Sandler, Inga; Zigdon, Nitzan; Levy, Efrat; Aharoni, Amir

    2014-02-01

    Computational approaches for detecting co-evolution in proteins allow for the identification of protein-protein interaction networks in different organisms and the assignment of function to under-explored proteins. The detection of co-variation of amino acids within or between proteins, moreover, allows for the discovery of residue-residue contacts and highlights functional residues that can affect the binding affinity, catalytic activity, or substrate specificity of a protein. To explore the functional impact of co-evolutionary changes in proteins, a combined experimental and computational approach must be recruited. Here, we review recent studies that apply computational and experimental tools to obtain novel insight into the structure, function, and evolution of proteins. Specifically, we describe the application of co-evolutionary analysis for predicting high-resolution three-dimensional structures of proteins. In addition, we describe computational approaches followed by experimental analysis for identifying specificity-determining residues in proteins. Finally, we discuss studies addressing the importance of such residues in terms of the functional divergence of proteins, allowing proteins to evolve new functions while avoiding crosstalk with existing cellular pathways or forming reproductive barriers and hence promoting speciation.

  16. Solubilization of proteins: the importance of lysis buffer choice.

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    Peach, Mandy; Marsh, Noelle; Miskiewicz, Ewa I; MacPhee, Daniel J

    2015-01-01

    The efficient extraction of proteins of interest from cells and tissues is not always straightforward. Here we demonstrate the differences in extraction of the focal adhesion protein Kindlin-2 from choriocarcinoma cells using NP-40 and RIPA lysis buffer. Furthermore, we demonstrate the use of a more denaturing urea/thiourea lysis buffer for solubilization, by comparing its effectiveness for solubilization of small heat-shock proteins from smooth muscle with the often utilized RIPA lysis buffer. Overall, the results demonstrate the importance of establishing the optimal lysis buffer for specific protein solubilization within the experimental workflow.

  17. PRDM proteins: Important players in differentiation and disease

    DEFF Research Database (Denmark)

    Tonnesen, Cathrine Kolster Fog; Galli, Giorgio G; Lund, Anders H

    2012-01-01

    The PRDM family has recently spawned considerable interest as it has been implicated in fundamental aspects of cellular differentiation and exhibits expanding ties to human diseases. The PRDMs belong to the SET domain family of histone methyltransferases, however, enzymatic activity has been dete...... a more comprehensive understanding of the biological and biochemical functions of this important protein family....... suppressors or drivers of oncogenic processes. The molecular mechanisms have been delineated for only few PRDMs and little is known about functional redundancy within the family. Future studies should identify target genes of PRDM proteins and the protein complexes in which PRDM proteins reside to provide...

  18. Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants.

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    Ling, Qihua; Jarvis, Paul

    2016-11-01

    Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with (35)S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

  19. Protein expression changes in breast cancer and their importance

    Directory of Open Access Journals (Sweden)

    Tuğba Semerci Sevimli

    2013-03-01

    Full Text Available Studies about nucleic acids have increased after thepublication of DNA’s three dimensional structure by Watsonand Crick. Nucleic acids are the heritable moleculeswhich contain codes for proteins. Proteins are the mostimportant elements in molecular world because they arethe basic structural and functional components of a livingorganism. Clarifying the celluler events that involve proteinsare important in many areas for example diagnosisand treatment determination of diseases or developmentof new drugs. Proteome that comes from a combinationof the terms protein and genome, is one of the importantfield in these days. The studies in this area have acceleratedand gained a different place especially with afterthe completion of human genome project. In synthesis ofa protein just only genetic information is not enough. Atthe same time the change or changes of a protein afterthe synthesis, the final version and transporting to finallocalization of it also important. Because having defects inmailing cells of breast cancer, the first targets of treatmentmust be proteins. In this way the studies on proteins areimportant to determine prognostic and diagnostic diseasemarkers and also significant for identifying new treatmentstrategies.Key words: Genom, proteom, breast cancer

  20. Factors of importance for a successful delivery system for proteins

    DEFF Research Database (Denmark)

    van de Weert, Marco; Jorgensen, Lene; Horn Moeller, Eva

    2005-01-01

    Protein pharmaceuticals have matured into an important class of drugs, now comprising one in three novel drugs introduced on the market. However, significant gains are still to be made in reducing the costs of production, ensuring proper pharmacokinetics and efficacy, increasing patient compliance...

  1. Interaction of actin and the chloroplast protein import apparatus.

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    Jouhet, Juliette; Gray, John C

    2009-07-10

    Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

  2. Therapeutically important proteins from in vitro plant tissue culture systems.

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    Doran, Pauline M

    2013-01-01

    Plant cells cultured in liquid medium in bioreactors are now being used commercially to produce biopharmaceutical proteins. The emergence of in vitro plant cell culture as a production vehicle reflects the importance of key biosafety and biocontainment concerns affecting the competitiveness of alternative systems such as mammalian cell culture and agriculture. Food plant species are particularly attractive as hosts for in vitro protein production: the risk of transgene escape and food chain contamination is eliminated using containment facilities, while regulatory approval for oral delivery of drugs may be easier than if non-edible species were used. As in whole plants, proteolysis in cultured plant cells can lead to significant degradation of foreign proteins after synthesis; however, substantial progress has been made to counter the destructive effects of proteases in plant systems. Although protein secretion into the culture medium is advantageous for product recovery and purification, measures are often required to minimise extracellular protease activity and product losses due to irreversible surface adsorption. Disposable plastic bioreactors, which are being used increasingly in mammalian cell bioprocessing, are also being adopted for plant cell culture to allow rapid scale-up and generation of saleable product. This review examines a range of technical and regulatory issues affecting the choice of industrial production platform for foreign proteins, and assesses progress in the development of in vitro plant systems for biopharmaceutical production.

  3. Small proteins: untapped area of potential biological importance.

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    Su, Mingming; Ling, Yunchao; Yu, Jun; Wu, Jiayan; Xiao, Jingfa

    2013-12-16

    Polypeptides containing ≤100 amino acid residues (AAs) are generally considered to be small proteins (SPs). Many studies have shown that some SPs are involved in important biological processes, including cell signaling, metabolism, and growth. SP generally has a simple domain and has an advantage to be used as model system to overcome folding speed limits in protein folding simulation and drug design. But SPs were once thought to be trivial molecules in biological processes compared to large proteins. Because of the constraints of experimental methods and bioinformatics analysis, many genome projects have used a length threshold of 100 amino acid residues to minimize erroneous predictions and SPs are relatively under-represented in earlier studies. The general protein discovery methods have potential problems to predict and validate SPs, and very few effective tools and algorithms were developed specially for SPs identification. In this review, we mainly consider the diverse strategies applied to SPs prediction and discuss the challenge for differentiate SP coding genes from artifacts. We also summarize current large-scale discovery of SPs in species at the genome level. In addition, we present an overview of SPs with regard to biological significance, structural application, and evolution characterization in an effort to gain insight into the significance of SPs.

  4. Importance of Atomic Contacts in Vibrational Energy Flow in Proteins.

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    Kondoh, Masato; Mizuno, Misao; Mizutani, Yasuhisa

    2016-06-02

    Vibrational energy flow in proteins was studied by monitoring the time-resolved anti-Stokes ultraviolet resonance Raman scattering of three myoglobin mutants in which a Trp residue substitutes a different amino acid residue near heme. The anti-Stokes Raman intensities of the Trp residue in the three mutants increased with similar rates after depositing excess vibrational energy at heme, despite the difference in distance between heme and each substituted Trp residue along the main chain of the protein. This indicates that vibrational energy is not transferred through the main chain of the protein but rather through atomic contacts between heme and the Trp residue. Distinct differences were observed in the amplitude of the band intensity change between the Trp residues at different positions, and the amplitude of the band intensity change exhibits a correlation with the extent of exposure of the Trp residue to solvent water. This correlation indicates that atomic contacts between an amino acid residue and solvent water play an important role in vibrational energy flow in a protein.

  5. Critical importance of RAB proteins for synaptic function.

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    Mignogna, Maria Lidia; D'Adamo, Patrizia

    2017-02-01

    Neurons are highly polarized cells that exhibit one of the more complex morphology and function. Neuronal intracellular trafficking plays a key role in dictating the directionality and specificity of vesicle formation, transport and fusion, allowing the transmission of information in sophisticate cellular network. Thus, the integrity of protein trafficking and spatial organization is especially important in neuronal cells. RAB proteins, small monomeric GTPases belonging to the RAS superfamily, spatially and temporally orchestrate specific vesicular trafficking steps. In this review we summarise the known roles of RAB GTPases involved in the maintenance of neuronal vesicular trafficking in the central nervous system. In particular, we discriminate the axonal pre-synaptic trafficking and dendritic post-synaptic trafficking, to better underlie how a correct orchestration of vesicle movement is necessary to maintain neuronal polarity and then, to permit an accurate architecture and functionality of synaptic activity.

  6. [The importance of ADAM family proteins in malignant tumors].

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    Walkiewicz, Katarzyna; Gętek, Monika; Muc-Wierzgoń, Małgorzata; Kokot, Teresa; Nowakowska-Zajdel, Ewa

    2016-02-11

    Increasing numbers of reports about the role of adamalysins (ADAM) in malignant tumors are being published. To date, more than 30 representatives of this group, out of which about 20 occur in humans, have been described. The ADAM family is a homogeneous group of proteins which regulate, from the stage of embryogenesis, a series of processes such as cell migration, adhesion, and cell fusion. Half of them have proteolytic activity and are involved in the degradation of the extracellular matrix and the disintegration of certain protein complexes, thereby regulating the bioavailability of various growth factors. Many of these functions have a direct role in the processes of carcinogenesis and promoting the growth of tumor, which affect some signaling pathways, including those related to insulin-like growth factors (IGF1, IGF2), vascular growth factor (VEGF), tumor necrosis factor α (TNFα) and the EGFR/HER pathway. Another branch of studies is the evaluation of the possibility of using members of ADAM family proteins in the diagnosis, especially in breast, colon and non- small cell lung cancer. The detection of concentrations of adamalysin in serum, urine and pleural aspirates might contribute to the development of methods of early diagnosis of cancer and monitoring the therapy. However, both the role of adamalysins in the development and progression of tumors and their importance as a diagnostic and predictive further research still need to be checked on large groups of patients.

  7. Modification of proteins by norepinephrine is important for vascular contraction

    Directory of Open Access Journals (Sweden)

    Kyle B Johnson

    2010-10-01

    Full Text Available Norepinephrine (NE is thought to mediate its effects through G-protein coupled receptors. However, previous studies have shown that norepinephrine and another primary amine, serotonin, also have the ability to exert effects in a receptor-independent manner. We hypothesized that the enzyme transglutaminase II (TG II has the ability modify proteins with NE and that this modification is physiologically relevant. As our model we used rat aortic and vena cava tissues, two tissues that depend on NE to modulate vascular tone. Immunohistochemical and immunocytochemical staining showed that NE and TG II are present in smooth muscle cells of these tissues. Western analysis shows aorta and vena cava homogenate proteins are recognized by an anti-NE antibody. NE and α-actin colocalize in cultured aorta and vena cava smooth muscle cells. Freshly dissociated smooth muscle cells from these vessels were able to take up NE-biotin. In isolated tissue baths, inhibition of TG II with cystamine (0.5 mM completely abolished NE-induced contraction in the aorta but only attenuated the receptor-independent contractant KCl (max contraction to 100 mM KCl in cystamine treated = 88.8 ± 7.5% of vehicle treated, p<0.05. In the vena cava, contraction to NE was abolished with 0.1 mM cystamine and KCl contraction was attenuated (max contraction to 100 mM KCl in cystamine treated = 54.8 ± 21.2% of vehicle treated, p<0.05. Taken together, these results show that vascular smooth muscle cells take up and utilize NE for the modification of proteins, and that this modification may play an important role in vascular contraction.

  8. Physiological Importance and Mechanisms of Protein Hydrolysate Absorption

    Science.gov (United States)

    Zhanghi, Brian M.; Matthews, James C.

    Understanding opportunities to maximize the efficient digestion and assimilation by production animals of plant- and animal-derived protein products is critical for farmers, nutritionists, and feed manufacturers to sustain and expand the affordable production of high quality animal products for human consumption. The challenge to nutritionists is to match gastrointestinal tract load to existing or ­inducible digestive and absorptive capacities. The challenge to feed manufacturers is to develop products that are efficient substrates for digestion, absorption, and/or both events. Ultimately, the efficient absorption of digesta proteins depends on the mediated passage (transport) of protein hydrosylate products as dipeptides and unbound amino acids across the lumen- and blood-facing membranes of intestinal absorptive cells. Data testing the relative efficiency of supplying protein as hydrolysates or specific dipeptides versus as free amino acids, and the response of animals in several physiological states to feeding of protein hydrolysates, are presented and reviewed in this chapter. Next, data describing the transport mechanisms responsible for absorbing protein hydrolysate digestion products, and the known and putative regulation of these mechanisms by their substrates (small peptides) and hormones are presented and reviewed. Several conclusions are drawn regarding the efficient use of protein hydrolysate-based diets for particular physiological states, the economically-practical application of which likely will depend on technological advances in the manufacture of protein hydrolysate products.

  9. The importance of lipid modified proteins in plants.

    Science.gov (United States)

    Hemsley, Piers A

    2015-01-01

    Membranes have long been known to act as more than physical barriers within and between plant cells. Trafficking of membrane proteins, signalling from and across membranes, organisation of membranes and transport through membranes are all essential processes for plant cellular function. These processes rely on a myriad array of proteins regulated in a variety of manners and are frequently required to be directly associated with membranes. For integral membrane proteins, the mode of membrane association is readily apparent, but many peripherally associated membrane proteins are outwardly soluble proteins. In these cases the proteins are frequently modified by the addition of lipids allowing direct interaction with the hydrophobic core of membranes. These modifications include N-myristoylation, S-acylation (palmitoylation), prenylation and GPI anchors but until recently little was truly known about their function in plants. New data suggest that these modifications are able to act as more than just membrane anchors, and dynamic S-acylation in particular is emerging as a means of regulating protein function in a similar manner to phosphorylation. This review discusses how these modifications occur, their impact on protein function, how they are regulated, recent advances in the field and technical approaches for studying these modifications.

  10. Important relationships between Rab and MICAL proteins in endocytic trafficking

    Institute of Scientific and Technical Information of China (English)

    Juliati; Rahajeng; Sai; Srinivas; Panapakkam; Giridharan; Naava; Naslavsky; Steve; Caplan

    2010-01-01

    The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.

  11. Proteins of the corneal stroma: importance in visual function.

    Science.gov (United States)

    Xuan, Meng; Wang, Shurong; Liu, Xin; He, Yuxi; Li, Ying; Zhang, Yan

    2016-04-01

    The human cornea, consisting of five layers, is the transparent tissue that refracts and transmits light to the lens and retina, providing about two thirds of the refractive power of the eye. The stroma layer comprises nearly 90 % of the thickness of the cornea and thus plays a pivotal role in normal visual function. The bulk of this layer is constituted by proteins in the extracellular martrix secreted by the corneal epithelial, stroma, and endothelial cells. Clinical research has shown that corneal stroma diseases are common and involve conditions such as infections, injuries, and genetic defects, which cause severe visual disturbances or even blindness. To improve our understanding of the basic molecular mechanisms involved in the physiological and pathological activities of the corneal stroma, its proteins have been brought into the limelight to determine their crucial and irreplaceable roles. The data presented in a previous study have demonstrated the presence of 1679 proteins in the stroma, and this data set has subsequently been perfected by utilizing a highly sensitive isobaric peptide-labeling approach. According to their manifestations, these proteins can be classified as a gel-like organic material composed of proteoglycans, enzymes, and hemocyanin-binding proteins and a network of filaments composed of collagen, elastin, keratin, vimentin, and interconnected filaments comprising fibronectin and laminin. The aim of this review is to describe some corneal stroma proteins by highlighting their major functions and valuable applications in ophthalmologic research toward the better characterization and treatment of eye diseases.

  12. Import of peroxisomal matrix proteins in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Gunkel, Katja

    2005-01-01

    Archaea, prokaryotes and eukaryotes form the three kingdoms of life. The smallest unit of life, which can exist independently, is a cell. Archaea and prokaryotes have a relatively very simple architecture. The cytoplasm (cellulars pace), containing all metabolites, proteins and genetic material (DNA

  13. Increasing importance of protein flexibility in designing biocatalytic processes

    Directory of Open Access Journals (Sweden)

    Joyeeta Mukherjee

    2015-06-01

    Full Text Available Enzymes require some flexibility for catalysis. Biotechnologists prefer stable enzymes but often this stabilization comes at the cost of reduced efficiency. Enzymes from thermophiles have low flexibility but poor catalytic rates. Enzymes from psychrophiles are less stable but show good catalytic rates at low temperature. In organic solvents enzymes perform poorly as the prior drying makes the enzyme molecules very rigid. Adding water or increasing reaction temperature improves flexibility and catalytic rates. In case of hydrolases, flexibility and enantioselectivity have interdependence. Understanding the complex role of protein flexibility in biocatalysis can help in designing biotechnological processes.

  14. Regulation of Chloroplast Protein Import by the Ubiquitin E3 Ligase SP1 Is Important for Stress Tolerance in Plants.

    Science.gov (United States)

    Ling, Qihua; Jarvis, Paul

    2015-10-05

    Chloroplasts are the organelles responsible for photosynthesis in plants [1, 2]. The chloroplast proteome comprises ∼3,000 different proteins, including components of the photosynthetic apparatus, which are highly abundant. Most chloroplast proteins are nucleus-encoded and imported following synthesis in the cytosol. Such import is mediated by multiprotein complexes in the envelope membranes that surround each organelle [3, 4]. The translocon at the outer envelope membrane of chloroplasts (TOC) mediates client protein recognition and early stages of import. The TOC apparatus is regulated by the ubiquitin-proteasome system (UPS) in a process controlled by the envelope-localized ubiquitin E3 ligase SUPPRESSOR OF PPI1 LOCUS1 (SP1) [5, 6]. Previous work showed that SP1-mediated regulation of chloroplast protein import contributes to the organellar proteome changes that occur during plant development (e.g., during de-etiolation). Here, we reveal a critical role for SP1 in plant responses to abiotic stress, which is a major and increasing cause of agricultural yield losses globally [7]. Arabidopsis plants lacking SP1 are hypersensitive to salt, osmotic, and oxidative stresses, whereas plants overexpressing SP1 are considerably more stress tolerant than wild-type. We present evidence that SP1 acts to deplete the TOC apparatus under stress conditions to limit the import of photosynthetic apparatus components, which may attenuate photosynthetic activity and reduce the potential for reactive oxygen species production and photo-oxidative damage. Our results indicate that chloroplast protein import is responsive to environmental cues, enabling dynamic regulation of the organellar proteome, and suggest new approaches for improving stress tolerance in crops.

  15. The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology.

    Science.gov (United States)

    Verroust, Pierre J; Birn, Henrik; Nielsen, Rikke; Kozyraki, Renata; Christensen, Erik Ilsø

    2002-09-01

    The molecular mechanisms controlling proximal tubule reabsorption of proteins have been much elucidated in recent years. Megalin and cubilin constitute two important endocytic receptor proteins involved in this process. Although structurally very different the two receptor proteins interact to mediate the reabsorption of a large number of filtered proteins, including carrier proteins important for transport and cellular uptake of several vitamins, lipids and other nutrients. Dysfunction of either protein results in tubular proteinuria and is associated with specific changes in vitamin metabolism due to the defective proximal tubular reabsorption of carrier proteins. Additional focus on the two receptors is attracted by the possible pathogenic role of excessive tubular protein uptake during conditions of increased filtration of proteins, and by recent findings implicating members of the low density lipoprotein-receptor family, which includes megalin, in the transduction of signals by association with cytoplasmic proteins.

  16. Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

    DEFF Research Database (Denmark)

    Esmarck, Birgitte; Olsen, Steen Schytte

    2001-01-01

    1. Age-associated loss of skeletal muscle mass and strength can partly be counteracted by resistance training, causing a net synthesis of muscular proteins. Protein synthesis is influenced synergistically by postexercise amino acid supplementation, but the importance of the timing of protein intake...... remains unresolved. 2. The study investigated the importance of immediate (P0) or delayed (P2) intake of an oral protein supplement upon muscle hypertrophy and strength over a period of resistance training in elderly males. 3. Thirteen men (age, 74 ± 1 years; body mass index (BMI), 25 ± 1 kg m−2 (means...... %, respectively (P important...

  17. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  18. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    Science.gov (United States)

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  19. Oxidation as an important factor of protein damage: Implications for Maillard reaction

    Indian Academy of Sciences (India)

    L Trnková; J Dršata; I Boušová

    2015-06-01

    Protein oxidation, the process caused especially by reactive oxygen and nitrogen species, is thought to play a major role in various oxidative processes within cells and is implicated in the development of many human diseases. This review provides a brief overview of the protein oxidation with the emphasis on the types of oxidation (oxidation of protein backbone and amino acid residues side chains, site-specific metal-catalysed protein oxidation), oxidation-dependent generation of protein hydroperoxides, carbonyl derivatives and protein–protein cross-linkages. Non-enzymatic glycoxidation (also known as Maillard reaction) as an important factor of protein damage, consequences of oxidative protein impairment and related diseases as well as means of monitoring and assessment of protein modifications are discussed.

  20. MPIC: a mitochondrial protein import components database for plant and non-plant species.

    Science.gov (United States)

    Murcha, Monika W; Narsai, Reena; Devenish, James; Kubiszewski-Jakubiak, Szymon; Whelan, James

    2015-01-01

    In the 2 billion years since the endosymbiotic event that gave rise to mitochondria, variations in mitochondrial protein import have evolved across different species. With the genomes of an increasing number of plant species sequenced, it is possible to gain novel insights into mitochondrial protein import pathways. We have generated the Mitochondrial Protein Import Components (MPIC) Database (DB; http://www.plantenergy.uwa.edu.au/applications/mpic) providing searchable information on the protein import apparatus of plant and non-plant mitochondria. An in silico analysis was carried out, comparing the mitochondrial protein import apparatus from 24 species representing various lineages from Saccharomyces cerevisiae (yeast) and algae to Homo sapiens (human) and higher plants, including Arabidopsis thaliana (Arabidopsis), Oryza sativa (rice) and other more recently sequenced plant species. Each of these species was extensively searched and manually assembled for analysis in the MPIC DB. The database presents an interactive diagram in a user-friendly manner, allowing users to select their import component of interest. The MPIC DB presents an extensive resource facilitating detailed investigation of the mitochondrial protein import machinery and allowing patterns of conservation and divergence to be recognized that would otherwise have been missed. To demonstrate the usefulness of the MPIC DB, we present a comparative analysis of the mitochondrial protein import machinery in plants and non-plant species, revealing plant-specific features that have evolved.

  1. Transient expression of a mitochondrial precursor protein - A new approach to study mitochondrial protein import in cells of higher eukaryotes

    NARCIS (Netherlands)

    Huckriede, A; Heikema, A; Wilschut, J; Agsteribbe, E

    1996-01-01

    In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly effici

  2. A network biology approach to understanding the importance of chameleon proteins in human physiology and pathology.

    Science.gov (United States)

    Bahramali, Golnaz; Goliaei, Bahram; Minuchehr, Zarrin; Marashi, Sayed-Amir

    2017-02-01

    Chameleon proteins are proteins which include sequences that can adopt α-helix-β-strand (HE-chameleon) or α-helix-coil (HC-chameleon) or β-strand-coil (CE-chameleon) structures to operate their crucial biological functions. In this study, using a network-based approach, we examined the chameleon proteins to give a better knowledge on these proteins. We focused on proteins with identical chameleon sequences with more than or equal to seven residues long in different PDB entries, which adopt HE-chameleon, HC-chameleon, and CE-chameleon structures in the same protein. One hundred and ninety-one human chameleon proteins were identified via our in-house program. Then, protein-protein interaction (PPI) networks, Gene ontology (GO) enrichment, disease network, and pathway enrichment analyses were performed for our derived data set. We discovered that there are chameleon sequences which reside in protein-protein interaction regions between two proteins critical for their dual function. Analysis of the PPI networks for chameleon proteins introduced five hub proteins, namely TP53, EGFR, HSP90AA1, PPARA, and HIF1A, which were presented in four PPI clusters. The outcomes demonstrate that the chameleon regions are in critical domains of these proteins and are important in the development and treatment of human cancers. The present report is the first network-based functional study of chameleon proteins using computational approaches and might provide a new perspective for understanding the mechanisms of diseases helping us in developing new medical therapies along with discovering new proteins with chameleon properties which are highly important in cancer.

  3. New functions of the chloroplast Preprotein and Amino acid Transporter (PRAT) family members in protein import.

    Science.gov (United States)

    Rossig, Claudia; Reinbothe, Christiane; Gray, John; Valdes, Oscar; von Wettstein, Diter; Reinbothe, Steffen

    2014-01-01

    Plant cells contain distinct compartments such as the nucleus, the endomembrane system comprising the endoplasmic reticulum and Golgi apparatus, peroxisomes, vacuoles, as well as mitochondria and chloroplasts. All of these compartments are surrounded by 1 or 2 limiting membranes and need to import proteins from the cytosol. Previous work led to the conclusion that mitochondria and chloroplasts use structurally different protein import machineries in their outer and inner membranes for the uptake of cytosolic precursor proteins. Our most recent data show that there is some unexpected overlap. Three members of the family of preprotein and amino acid transporters, PRAT, were identified in chloroplasts that mediate the uptake of transit sequence-less proteins into the inner plastid envelope membrane. By analogy, mitochondria contain with TIM22 a related PRAT protein that is involved in the import of transit sequence-less proteins into the inner mitochondrial membrane. Both mitochondria and chloroplasts thus make use of similar import mechanisms to deliver some of their proteins to their final place. Because single homologs of HP20- and HP30-like proteins are present in algae such as Chlamydomonas, Ostreococcus, and Volvox, which diverged from land plants approximately 1 billion years ago, it is likely that the discovered PRAT-mediated mechanism of protein translocation evolved concomitantly with the secondary endosymbiotic event that gave rise to green plants.

  4. Structural insights into the evolution of a non-biological protein: importance of surface residues in protein fold optimization.

    Directory of Open Access Journals (Sweden)

    Matthew D Smith

    Full Text Available Phylogenetic profiling of amino acid substitution patterns in proteins has led many to conclude that most structural information is carried by interior core residues that are solvent inaccessible. This conclusion is based on the observation that buried residues generally tolerate only conserved sequence changes, while surface residues allow more diverse chemical substitutions. This notion is now changing as it has become apparent that both core and surface residues play important roles in protein folding and stability. Unfortunately, the ability to identify specific mutations that will lead to enhanced stability remains a challenging problem. Here we discuss two mutations that emerged from an in vitro selection experiment designed to improve the folding stability of a non-biological ATP binding protein. These mutations alter two solvent accessible residues, and dramatically enhance the expression, solubility, thermal stability, and ligand binding affinity of the protein. The significance of both mutations was investigated individually and together, and the X-ray crystal structures of the parent sequence and double mutant protein were solved to a resolution limit of 2.8 and 1.65 A, respectively. Comparative structural analysis of the evolved protein to proteins found in nature reveals that our non-biological protein evolved certain structural features shared by many thermophilic proteins. This experimental result suggests that protein fold optimization by in vitro selection offers a viable approach to generating stable variants of many naturally occurring proteins whose structures and functions are otherwise difficult to study.

  5. Update on protein intake: importance of milk proteins for health status of the elderly.

    Science.gov (United States)

    Wolfe, Robert R

    2015-08-01

    Loss of lean body mass that occurs with aging is the primary endpoint with which sarcopenia is defined. Furthermore, loss of muscle mass is central to the development of many adverse health issues in the elderly. Consequently, the response of lean body mass to nutritional interventions, particularly to dietary protein, has been a commonly measured endpoint. However, increased protein intake has been associated with improved markers for cardiovascular health, improved bone health, management of weight and metabolic diseases, and reduced all-cause mortality. Strength, rather than lean body mass, may be a more accurate indicator of health, especially in the elderly. The recommended dietary allowance for protein has been set at 0.8 g/kg/day. Because the average protein intake in the United States is approximately 1.2 g/kg/day, it appears that the average protein intake is above the recommended dietary allowance but below the low end of the acceptable macronutrient distribution range recommended by expert committees of the National Academy of Sciences and below the dietary intake levels suggested by the US Department of Agriculture in the Dietary Guidelines.

  6. Update on protein intake: importance of milk proteins for health status of the elderly

    Science.gov (United States)

    2015-01-01

    Loss of lean body mass that occurs with aging is the primary endpoint with which sarcopenia is defined. Furthermore, loss of muscle mass is central to the development of many adverse health issues in the elderly. Consequently, the response of lean body mass to nutritional interventions, particularly to dietary protein, has been a commonly measured endpoint. However, increased protein intake has been associated with improved markers for cardiovascular health, improved bone health, management of weight and metabolic diseases, and reduced all-cause mortality. Strength, rather than lean body mass, may be a more accurate indicator of health, especially in the elderly. The recommended dietary allowance for protein has been set at 0.8 g/kg/day. Because the average protein intake in the United States is approximately 1.2 g/kg/day, it appears that the average protein intake is above the recommended dietary allowance but below the low end of the acceptable macronutrient distribution range recommended by expert committees of the National Academy of Sciences and below the dietary intake levels suggested by the US Department of Agriculture in the Dietary Guidelines. PMID:26175489

  7. Hypochlorous acid-mediated protein oxidation: how important are chloramine transfer reactions and protein tertiary structure?

    Science.gov (United States)

    Pattison, David I; Hawkins, Clare L; Davies, Michael J

    2007-08-28

    Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

  8. The peroxisomal protein import machinery displays a preference for monomeric substrates.

    Science.gov (United States)

    Freitas, Marta O; Francisco, Tânia; Rodrigues, Tony A; Lismont, Celien; Domingues, Pedro; Pinto, Manuel P; Grou, Cláudia P; Fransen, Marc; Azevedo, Jorge E

    2015-04-01

    Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.

  9. Two putative protein kinase CK2 phosphorylation sites are important for Myf-5 activity

    DEFF Research Database (Denmark)

    Winter, B; Kautzner, I; Issinger, O G;

    1997-01-01

    Myf-5, a member of a family of muscle-specific transcription factors, is important for myogenic cell determination and differentiation. Here, we report that Myf-5 protein constitutes a substrate for phosphorylation in vitro by protein kinase CK2. We identified two potential phosphorylation sites ...

  10. Surface proteins of Staphylococcus aureus play an important role in experimental skin infection.

    Science.gov (United States)

    Kwiecinski, Jakub; Jin, Tao; Josefsson, Elisabet

    2014-12-01

    Staphylococcus aureus is the most common cause of skin infections that range from mild diseases up to life-threatening conditions. Mechanisms of S. aureus virulence in those infections remain poorly studied. To investigate the impact of S. aureus surface proteins on skin infection, we used mouse models of skin abscess formation and skin necrosis, induced by a subcutaneous injection of bacteria. In the skin abscess model, a sortase-deficient S. aureus strain lacking all of its cell-wall anchored proteins was less virulent than its wild-type strain. Also, strains specifically lacking protein A, fibronecting binding proteins, clumping factor A or surface protein SasF were impaired in their virulence. When a model of dermonecrosis was studied, the S. aureus surface proteins could not be shown to be involved. In summary, surface proteins play an important role in virulence of S. aureus skin abscess infections, but not in formation of skin necrosis.

  11. Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

    OpenAIRE

    Gnanasekar Munirathinam; Kalyanasundaram Ramaswamy

    2012-01-01

    Translationally controlled tumor protein (TCTP) lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO) motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into th...

  12. The invariant phenylalanine of precursor proteins discloses the importance of Omp85 for protein translocation into cyanelles

    Directory of Open Access Journals (Sweden)

    Schleiff Enrico

    2007-11-01

    Full Text Available Abstract Background Today it is widely accepted that plastids are of cyanobacterial origin. During their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. This process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. Such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. At present, most information on protein translocation is obtained by the analysis of land plants. However, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. Here, bacterial outer membrane proteins of the Omp85 family have recently been discussed as evolutionary seeds for the development of translocation systems. Results To further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. We document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an Omp85 homologue. Comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by Omp85. This finding is placed into the context of the evolutionary development of the plastid translocon. Conclusion The phenylalanine in the N-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. This amino acid appears to be optimized for specifically targeting the Omp85 protein without enforcing aggregation on the membrane surface

  13. Nuclear protein import is reduced in cells expressing nuclear envelopathy-causing lamin A mutants

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

    Lamins, which form the nuclear lamina, not only constitute an important determinant of nuclear architecture, but additionally play essential roles in many nuclear functions. Mutations in A-type lamins cause a wide range of human genetic disorders (laminopathies). The importance of lamin A (LaA) in the spatial arrangement of nuclear pore complexes (NPCs) prompted us to study the role of LaA mutants in nuclear protein transport. Two mutants, causing prenatal skin disease restrictive dermopathy (RD) and the premature aging disease Hutchinson Gilford progeria syndrome, were used for expression in HeLa cells to investigate their impact on the subcellular localization of NPC-associated proteins and nuclear protein import. Furthermore, dynamics of the LaA mutants within the nuclear lamina were studied. We observed affected localization of NPC-associated proteins, diminished lamina dynamics for both LaA mutants and reduced nuclear import of representative cargo molecules. Intriguingly, both LaA mutants displayed similar effects on nuclear morphology and functions, despite their differences in disease severity. Reduced nuclear protein import was also seen in RD fibroblasts and impaired lamina dynamics for the nucleoporin Nup153. Our data thus represent the first study of a direct link between LaA mutant expression and reduced nuclear protein import.

  14. An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins.

    Directory of Open Access Journals (Sweden)

    Swati Agrawal

    Full Text Available Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

  15. Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona

    Science.gov (United States)

    Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B.; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

    2017-01-01

    Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored—the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs. PMID:28052135

  16. Importance of native-state topology for determining the folding rate of two-state proteins.

    Science.gov (United States)

    Gromiha, M Michael

    2003-01-01

    Understanding the relationship between amino acid sequences and folding rate of proteins is a challenging task similar to protein folding problem. In this work, we have analyzed the relative importance of protein sequence and structure for predicting the protein folding rates in terms of amino acid properties and contact distances, respectively. We found that the parameters derived with protein sequence (physical-chemical, energetic, and conformational properties of amino acid residues) show very weak correlation (|r| proteins, indicating that the sequence information alone is not sufficient to understand the folding rates of two-state proteins. However, the maximum positive correlation obtained for the properties, number of medium-range contacts, and alpha-helical tendency reveals the importance of local interactions to initiate protein folding. On the other hand, a remarkable correlation (r varies from -0.74 to -0.88) has been obtained between structural parameters (contact order, long-range order, and total contact distance) and protein folding rates. Further, we found that the secondary structure content and solvent accessibility play a marginal role in determining the folding rates of two-state proteins. Multiple regression analysis carried out with the combination of three properties, beta-strand tendency, enthalpy change, and total contact distance improved the correlation to 0.92 with protein folding rates. The relative importance of existing methods along with multiple-regression model proposed in this work will be discussed. Our results demonstrate that the native-state topology is the major determinant for the folding rates of two-state proteins.

  17. Dispersion of Nanoparticles in Different Media Importantly Determines the Composition of Their Protein Corona.

    Science.gov (United States)

    Strojan, Klemen; Leonardi, Adrijana; Bregar, Vladimir B; Križaj, Igor; Svete, Jurij; Pavlin, Mojca

    2017-01-01

    Protein corona of nanoparticles (NPs), which forms when these particles come in to contact with protein-containing fluids, is considered as an overlooked factor in nanomedicine. Through numerous studies it has been becoming increasingly evident that it importantly dictates the interaction of NPs with their surroundings. Several factors that determine the compositions of NPs protein corona have been identified in recent years, but one has remained largely ignored-the composition of media used for dispersion of NPs. Here, we determined the effect of dispersion media on the composition of protein corona of polyacrylic acid-coated cobalt ferrite NPs (PAA NPs) and silica NPs. Our results confirmed some of the basic premises such as NPs type-dependent specificity of the protein corona. But more importantly, we demonstrated the effect of the dispersion media on the protein corona composition. The differences between constituents of the media used for dispersion of NPs, such as divalent ions and macromolecules were responsible for the differences in protein corona composition formed in the presence of fetal bovine serum (FBS). Our results suggest that the protein corona composition is a complex function of the constituents present in the media used for dispersion of NPs. Regardless of the dispersion media and FBS concentration, majority of proteins from either PAA NPs or silica NPs coronas were involved in the process of transport and hemostasis. Interestingly, corona of silica NPs contained three complement system related proteins: complement factor H, complement C3 and complement C4 while PAA NPs bound only one immune system related protein, α-2-glycoprotein. Importantly, relative abundance of complement C3 protein in corona of silica NPs was increased when NPs were dispersed in NaCl, which further implies the relevance of dispersion media used to prepare NPs.

  18. The child is father to the man: developmental roles for proteins of importance for neurodegenerative disease.

    Science.gov (United States)

    Rogers, Danny; Schor, Nina F

    2010-02-01

    Although Alzheimer's and Parkinson's diseases predominately affect elderly adults, the proteins that play a role in the pathogenesis of these diseases are expressed throughout life. In fact, many of the proteins hypothesized to be important in the progression of neurodegeneration play direct or indirect roles in the development of the central nervous system. The systems affected by these proteins include neural stem cell fate decisions, neuronal differentiation, cellular migration, protection from oxidative stress, and programmed cell death. Insights into the developmental roles of these proteins may ultimately impact the understanding of neurodegenerative diseases and lead to the discovery of novel treatments.

  19. Importance of post-translational modifications on the function of key haemostatic proteins.

    Science.gov (United States)

    Karlaftis, Vasiliki; Perera, Sachin; Monagle, Paul; Ignjatovic, Vera

    2016-01-01

    Post-translational modifications (PTMs) such as glycosylation and phosphorylation play an important role on the function of haemostatic proteins and are critical in the setting of disease. Such secondary level changes to haemostatic proteins have wide ranging effects on their ability to interact with other proteins. This review aimed to summarize the knowledge of the common PTMs associated with haemostatic proteins and the implications of such modifications on protein function. Haemostatic proteins that represent the main focus for studies specific to PTMs are von Willebrand factor, tissue factor, factor VIII, antithrombin and fibrinogen. These proteins are susceptible to PTMs by glycosylation, phosphorylation, sulphation, citrullination and nitration, respectively, with a significant impact on their function. During synthesis, vWF must undergo extensive PTMs, with N-linked glycosylation being the most common. Increased phosphorylation of tissue factor results in increased affinity for platelets to the vessel endothelium. Citrullination of antithrombin leads to an increased anticoagulant function of this protein and therefore an anticoagulant state that inhibits clot formation. On the contrary, nitration of fibrinogen has been shown to result in a prothrombotic state, whilst sulphation is required for the normal function of Factor VIII. From this review, it is evident that PTMs of haemostatic proteins as a change in protein structure at a secondary level greatly influences the behaviour of the protein at a tertiary level.

  20. Predicting important residues and interaction pathways in proteins using Gaussian Network Model: binding and stability of HLA proteins.

    Directory of Open Access Journals (Sweden)

    Turkan Haliloglu

    Full Text Available A statistical thermodynamics approach is proposed to determine structurally and functionally important residues in native proteins that are involved in energy exchange with a ligand and other residues along an interaction pathway. The structure-function relationships, ligand binding and allosteric activities of ten structures of HLA Class I proteins of the immune system are studied by the Gaussian Network Model. Five of these models are associated with inflammatory rheumatic disease and the remaining five are properly functioning. In the Gaussian Network Model, the protein structures are modeled as an elastic network where the inter-residue interactions are harmonic. Important residues and the interaction pathways in the proteins are identified by focusing on the largest eigenvalue of the residue interaction matrix. Predicted important residues match those known from previous experimental and clinical work. Graph perturbation is used to determine the response of the important residues along the interaction pathway. Differences in response patterns of the two sets of proteins are identified and their relations to disease are discussed.

  1. Unexpected diversity in Shisa-like proteins suggests the importance of their roles as transmembrane adaptors.

    Science.gov (United States)

    Pei, Jimin; Grishin, Nick V

    2012-03-01

    The Shisa family of single-transmembrane proteins is characterized by an N-terminal cysteine-rich domain and a proline-rich C-terminal region. Its founding member, Xenopus Shisa, promotes head development by antagonizing Wnt and FGF signaling. Recently, a mouse brain-specific Shisa protein CKAMP44 (Shisa9) was shown to play an important role in AMPA receptor desensitization. We used sequence similarity searches against protein, genome and EST databases to study the evolutionary origin and phylogenetic distribution of Shisa homologs. In addition to nine Shisa subfamilies in vertebrates, we detected distantly related Shisa homologs that possess an N-terminal domain with six conserved cysteines. These Shisa-like proteins include FAM159 and KIAA1644 mainly from vertebrates, and members from various bilaterian invertebrates and Porifera, suggesting their presence in the last common ancestor of Metazoa. Shisa-like genes have undergone large expansions in Branchiostoma floridae and Saccoglossus kowalevskii, and appear to have been lost in certain insects. Pattern-based searches against eukaryotic proteomes also uncovered several other families of predicted single-transmembrane proteins with a similar cysteine-rich domain. We refer to these proteins (Shisa/Shisa-like, WBP1/VOPP1, CX, DUF2650, TMEM92, and CYYR1) as STMC6 proteins (single-transmembrane proteins with conserved 6 cysteines). STMC6 genes are widespread in Metazoa, with the human genome containing 17 members. Frequent occurrences of PY motifs in STMC6 proteins suggest that most of them could interact with WW-domain-containing proteins, such as the NEDD4 family E3 ubiquitin ligases, and could play critical roles in protein degradation and sorting. STMC6 proteins are likely transmembrane adaptors that regulate membrane proteins such as cell surface receptors.

  2. FERM family proteins and their importance in cellular movements and wound healing (review).

    Science.gov (United States)

    Bosanquet, David C; Ye, Lin; Harding, Keith G; Jiang, Wen G

    2014-07-01

    Motility is a requirement for a number of biological processes, including embryonic development, neuronal development, immune responses, cancer progression and wound healing. Specific to wound healing is the migration of endothelial cells, fibroblasts and other key cellular players into the wound space. Aberrations in wound healing can result in either chronic wounds or abnormally healed wounds. The protein 4.1R, ezrin, radixin, moesin (FERM) superfamily consists of over 40 proteins all containing a three lobed N-terminal FERM domain which binds a variety of cell-membrane associated proteins and lipids. The C-terminal ends of these proteins typically contain an actin-binding domain (ABD). These proteins therefore mediate the linkage between the cell membrane and the actin cytoskeleton, and are involved in cellular movements and migration. Certain FERM proteins have been shown to promote cancer metastasis via this very mechanism. Herein we review the effects of a number of FERM proteins on wound healing and cancer. We show how these proteins typically aid wound healing through their effects on increasing cellular migration and movements, but also typically promote metastasis in cancer. We conclude that FERM proteins play important roles in cellular migration, with markedly different outcomes in the context of cancer and wound healing.

  3. Proteomic analysis of the dysferlin protein complex unveils its importance for sarcolemmal maintenance and integrity.

    Directory of Open Access Journals (Sweden)

    Antoine de Morrée

    Full Text Available Dysferlin is critical for repair of muscle membranes after damage. Mutations in dysferlin lead to a progressive muscular dystrophy. Recent studies suggest additional roles for dysferlin. We set out to study dysferlin's protein-protein interactions to obtain comprehensive knowledge of dysferlin functionalities in a myogenic context. We developed a robust and reproducible method to isolate dysferlin protein complexes from cells and tissue. We analyzed the composition of these complexes in cultured myoblasts, myotubes and skeletal muscle tissue by mass spectrometry and subsequently inferred potential protein functions through bioinformatics analyses. Our data confirm previously reported interactions and support a function for dysferlin as a vesicle trafficking protein. In addition novel potential functionalities were uncovered, including phagocytosis and focal adhesion. Our data reveal that the dysferlin protein complex has a dynamic composition as a function of myogenic differentiation. We provide additional experimental evidence and show dysferlin localization to, and interaction with the focal adhesion protein vinculin at the sarcolemma. Finally, our studies reveal evidence for cross-talk between dysferlin and its protein family member myoferlin. Together our analyses show that dysferlin is not only a membrane repair protein but also important for muscle membrane maintenance and integrity.

  4. Suppression of a defect in mitochondrial protein import identifies cytosolic proteins required for viability of yeast cells lacking mitochondrial DNA.

    Science.gov (United States)

    Dunn, Cory D; Jensen, Robert E

    2003-01-01

    The TIM22 complex, required for the insertion of imported polytopic proteins into the mitochondrial inner membrane, contains the nonessential Tim18p subunit. To learn more about the function of Tim18p, we screened for high-copy suppressors of the inability of tim18Delta mutants to live without mitochondrial DNA (mtDNA). We identified several genes encoding cytosolic proteins, including CCT6, SSB1, ICY1, TIP41, and PBP1, which, when overproduced, rescue the mtDNA dependence of tim18Delta cells. Furthermore, these same plasmids rescue the petite-negative phenotype of cells lacking other components of the mitochondrial protein import machinery. Strikingly, disruption of the genes identified by the different suppressors produces cells that are unable to grow without mtDNA. We speculate that loss of mtDNA leads to a lowered inner membrane potential, and subtle changes in import efficiency can no longer be tolerated. Our results suggest that increased amounts of Cct6p, Ssb1p, Icy1p, Tip41p, and Pbp1p help overcome the problems resulting from a defect in protein import. PMID:14504216

  5. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  6. System wide analyses have underestimated protein abundances and the importance of transcription in mammals.

    Science.gov (United States)

    Li, Jingyi Jessica; Bickel, Peter J; Biggin, Mark D

    2014-01-01

    Large scale surveys in mammalian tissue culture cells suggest that the protein expressed at the median abundance is present at 8,000-16,000 molecules per cell and that differences in mRNA expression between genes explain only 10-40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances and the relative importance of transcription. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher. We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression. We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R(2) = 0.13). Based on this, our second strategy suggests that mRNA levels explain ∼81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller

  7. System wide analyses have underestimated protein abundances and the importance of transcription in mammals

    Directory of Open Access Journals (Sweden)

    Jingyi Jessica Li

    2014-02-01

    Full Text Available Large scale surveys in mammalian tissue culture cells suggest that the protein expressed at the median abundance is present at 8,000–16,000 molecules per cell and that differences in mRNA expression between genes explain only 10–40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances and the relative importance of transcription. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011, we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011, though because one major source of error could not be estimated the true percent contribution should be higher. We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression. We show that the variance in translation rates directly measured by ribosome profiling is only 9% of that inferred by Schwanhausser et al. (2011, and that the measured and inferred translation rates correlate poorly (R2 = 0.14. Based on this, our second strategy suggests that mRNA levels explain ∼84% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation

  8. The importance of adding EDTA for the nanopore analysis of proteins.

    Science.gov (United States)

    Krasniqi, Besnik; Lee, Jeremy S

    2012-06-01

    Nanopore analysis is a promising technique for studying the conformation of proteins and protein/protein interactions. Two proteins (bacterial thioredoxin and maltose binding protein) were subjected to nanopore analysis with α-hemolysin. Two types of events were observed; bumping events with a blockade current less than -40 pA and intercalation events with blockade currents between -40 pA and -100 pA. In potassium phosphate buffer, pH 7.8, both proteins gave intercalation events but the frequency of these events was significantly reduced in TRIS or HEPES buffers especially in the presence of 0.01 mM divalent metal ions. The frequency of events was restored by the addition of EDTA. For maltose binding protein, the frequency of intercalation events was also decreased in the presence of maltose but not lactose to which it does not bind. It is proposed that the events with large blockade currents represent transient intercalation of a loop or end of the protein into the pore and that divalent metal ions inhibit this process. The results demonstrate that the choice of buffer and the effects of metal ion contamination are important considerations in nanopore analysis.

  9. Karyopherin α 3 and karyopherin α 4 proteins mediate the nuclear import of methyl-CpG binding protein 2.

    Science.gov (United States)

    Baker, Steven Andrew; Lombardi, Laura Marie; Zoghbi, Huda Yahya

    2015-09-11

    Methyl-CpG binding protein 2 (MeCP2) is a nuclear protein with important roles in regulating chromatin structure and gene expression, and mutations in MECP2 cause Rett syndrome (RTT). Within the MeCP2 protein sequence, the nuclear localization signal (NLS) is reported to reside between amino acids 255-271, and certain RTT-causing mutations overlap with the MeCP2 NLS, suggesting that they may alter nuclear localization. One such mutation, R270X, is predicted to interfere with the localization of MeCP2, but recent in vivo studies have demonstrated that this mutant remains entirely nuclear. To clarify the mechanism of MeCP2 nuclear import, we isolated proteins that interact with the NLS and identified karyopherin α 3 (KPNA3 or Kap-α3) and karyopherin α 4 (KPNA4 or Kap-α4) as key binding partners of MeCP2. MeCP2-R270X did not interact with KPNA4, consistent with a requirement for an intact NLS in this interaction. However, this mutant retains binding to KPNA3, accounting for the normal localization of MeCP2-R270X to the nucleus. These data provide a mechanism for MeCP2 nuclear import and have implications for the design of therapeutics aimed at modulating the function of MeCP2 in RTT patients.

  10. Investigating the importance of Delaunay-based definition of atomic interactions in scoring of protein-protein docking results.

    Science.gov (United States)

    Jafari, Rahim; Sadeghi, Mehdi; Mirzaie, Mehdi

    2016-05-01

    The approaches taken to represent and describe structural features of the macromolecules are of major importance when developing computational methods for studying and predicting their structures and interactions. This study attempts to explore the significance of Delaunay tessellation for the definition of atomic interactions by evaluating its impact on the performance of scoring protein-protein docking prediction. Two sets of knowledge-based scoring potentials are extracted from a training dataset of native protein-protein complexes. The potential of the first set is derived using atomic interactions extracted from Delaunay tessellated structures. The potential of the second set is calculated conventionally, that is, using atom pairs whose interactions were determined by their separation distances. The scoring potentials were tested against two different docking decoy sets and their performances were compared. The results show that, if properly optimized, the Delaunay-based scoring potentials can achieve higher success rate than the usual scoring potentials. These results and the results of a previous study on the use of Delaunay-based potentials in protein fold recognition, all point to the fact that Delaunay tessellation of protein structure can provide a more realistic definition of atomic interaction, and therefore, if appropriately utilized, may be able to improve the accuracy of pair potentials.

  11. Defense-related Proteins from Chelidonium majus L. as Important Components of its Latex.

    Science.gov (United States)

    Nawrot, Robert

    2017-01-01

    The aim of this review is to cover most recent research on plant pathogenesis- and defenserelated proteins from latex-bearing medicinal plant Chelidonium majus (Papaveraceae) in the context of its importance for latex activity, function, pharmacological activities, and antiviral medicinal use. These results are compared with other latex-bearing plant species and recent research on proteins and chemical compounds contained in their latex. This is the first review, which clearly summarizes pathogenesisrelated (PR) protein families in latex-bearing plants pointing into their possible functions. The possible antiviral function of the latex by naming the abundant proteins present therein is also emphasized. Finally latex-borne defense system is hypothesized to constitute a novel type of preformed immediate defense response against viral, but also non-viral pathogens, and herbivores. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Importance of detecting multidrug resistance proteins in acute leukemia prognosis and therapy.

    Science.gov (United States)

    de Moraes, Ana Carolina Rabello; Maranho, Caroline Klein; Rauber, Gabriela Schneider; Santos-Silva, Maria Cláudia

    2013-01-01

    Multidrug resistance (MDR) is a multifactorial phenomenon and the role of these proteins in generating the MDR phenotype is controversial. With this in mind, this review compiled the current data on the role of ABCB1, ABCC1, and LRP proteins in the prognosis of hematologic neoplasms and their influence on the choice of therapy. Literature showed that the detection of these proteins, mainly ABCB1, is important in the AL prognosis. However, there is controversy regarding the methodology used for their detection. In summary, the expression and activity profiles of ABCB1, ABCC1, and LRP, proteins capable of promoting the efflux of a variety of chemotherapeutic agents from the cell cytoplasm represent one of the greatest causes of failure in AL treatment.

  13. A Crucial Role of the Mitochondrial Protein Import Receptor MOM19 for the Biogenesis of Mitochondria

    NARCIS (Netherlands)

    Harkness, Troy A.A.; Nargang, Frank E.; Klei, Ida van der; Neupert, Walter; Lill, Roland

    1994-01-01

    The novel genetic method of "sheltered RIP" (repeat induced point mutation) was used to generate a Neurospora crassa mutant in which MOM19, a component of the protein import machinery of the mitochondrial outer membrane, can be depleted. Deficiency in MOM19 resulted in a severe growth defect, but

  14. ISOLATION AND CHARACTERIZATION OF PEROXISOMAL PROTEIN IMPORT (PIM-) MUTANTS OF HANSENULA-POLYMORPHA

    NARCIS (Netherlands)

    WATERHAM, HR; TITORENKO, [No Value; VANDERKLEI, IJ; HARDER, W; VEENHUIS, M

    1992-01-01

    In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroxisomal matrix proteins. These mutants are characterized by the presence of few small intact per

  15. Tight Junction Proteins Claudin-1 and Occludin Are Important for Cutaneous Wound Healing.

    Science.gov (United States)

    Volksdorf, Thomas; Heilmann, Janina; Eming, Sabine A; Schawjinski, Kathrin; Zorn-Kruppa, Michaela; Ueck, Christopher; Vidal-Y-Sy, Sabine; Windhorst, Sabine; Jücker, Manfred; Moll, Ingrid; Brandner, Johanna M

    2017-06-01

    Tight junction (TJ) proteins are known to be involved in proliferation and differentiation. These processes are essential for normal skin wound healing. Here, we investigated the TJ proteins claudin-1 and occludin in ex vivo skin wound healing models and tissue samples of acute and chronic human wounds and observed major differences in localization/expression of these proteins, with chronic wounds often showing a loss of the proteins at the wound margins and/or in the regenerating epidermis. Knockdown experiments in primary human keratinocytes showed that decreased claudin-1 expression resulted in significantly impaired scratch wound healing, with delayed migration and reduced proliferation. Activation of AKT pathway was significantly attenuated after claudin-1 knockdown, and protein levels of extracellular signal-related kinase 1/2 were reduced. For occludin, down-regulation had no impact on wound healing in normal scratch assays, but after subjecting the cells to mechanical stress, which is normally present in wounds, wound healing was impaired. For both proteins we show that most of these actions are independent from the formation of barrier-forming TJ structures, thus demonstrating nonbarrier-related functions of TJ proteins in the skin. However, for claudin-1 effects on scratch wound healing were more pronounced when TJs could form. Together, our findings provide evidence for a role of claudin-1 and occludin in epidermal regeneration with potential clinical importance. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  16. Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins.

    Science.gov (United States)

    Strzalka, K; Ngernprasirtsiri, J; Watanabe, A; Akazawa, T

    1987-12-16

    Amyloplasts isolated from white-wild suspension-cultured cells of sycamore (Acer pseudoplatanus L.) are found to import and process the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach, but they lack the ability to form its holoenzyme due to the absence of both the large subunit and its binding-protein. They also import the precursor of the 33-kDa extrinsic protein (p33-kDa) of the O2-evolving complex of Photosystem II from spinach, but process is only to an intermediate form (i33-kDa). Chloroplasts from green-mutant cells of sycamore process p33-kDa to its mature form in this heterologous system. These results suggest that the thylakoid-associated protease responsible for the second processing step of p33-kDa is missing in amyloplasts, possibly due to the absence of thylakoid-membranes. In contrast, the apparent import of the precursor of the light-harvesting chlorophyll a/b-binding apoprotein (pLHCP) from spinach was not detected. Sycamore amyloplasts may lack the ability to import this particular thylakoid-protein, or rapidly degrade the imported molecules in the absence of thylakoid-membranes for their proper insertion.

  17. Recurrent use of evolutionary importance for functional annotation of proteins based on local structural similarity.

    Science.gov (United States)

    Kristensen, David M; Chen, Brian Y; Fofanov, Viacheslav Y; Ward, R Matthew; Lisewski, Andreas Martin; Kimmel, Marek; Kavraki, Lydia E; Lichtarge, Olivier

    2006-06-01

    The annotation of protein function has not kept pace with the exponential growth of raw sequence and structure data. An emerging solution to this problem is to identify 3D motifs or templates in protein structures that are necessary and sufficient determinants of function. Here, we demonstrate the recurrent use of evolutionary trace information to construct such 3D templates for enzymes, search for them in other structures, and distinguish true from spurious matches. Serine protease templates built from evolutionarily important residues distinguish between proteases and other proteins nearly as well as the classic Ser-His-Asp catalytic triad. In 53 enzymes spanning 33 distinct functions, an automated pipeline identifies functionally related proteins with an average positive predictive power of 62%, including correct matches to proteins with the same function but with low sequence identity (the average identity for some templates is only 17%). Although these template building, searching, and match classification strategies are not yet optimized, their sequential implementation demonstrates a functional annotation pipeline which does not require experimental information, but only local molecular mimicry among a small number of evolutionarily important residues.

  18. Dimerization of TOC receptor GTPases and its implementation for the control of protein import into chloroplasts.

    Science.gov (United States)

    Aronsson, Henrik; Jarvis, Paul

    2011-06-01

    Pre-protein import into chloroplasts is facilitated by multiprotein translocon complexes in the envelope membranes. Major components of the TOC (translocon at the outer envelope membrane of chloroplasts) complex are the receptor proteins Toc33 and Toc159. These two receptors are related GTPases, and they are predicted to engage in homodimerization and/or heterodimerization. Although such dimerization has been studied extensively, its exact function in vivo remains elusive. In this issue of the Biochemical Journal, Oreb et al. present evidence that homodimerization of Toc33 prevents nucleotide exchange, thereby locking the receptor in the GDP-loaded state and preventing further activity. Pre-protein arrival is proposed to release this lock, through disruption of the dimer and subsequent nucleotide exchange. The Toc33-bound pre-protein is then able to progress to downstream steps in the translocation mechanism, with GTP hydrolysis defining another important control point as well as preparing the receptor for the next pre-protein client. These new results are discussed in the context of previous findings pertaining to TOC receptor dimerization and function.

  19. The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Pavel Dolezal

    2010-03-01

    Full Text Available Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM based search of the E. histolytica genome sequence to discover candidate (i mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.

  20. The essentials of protein import in the degenerate mitochondrion of Entamoeba histolytica.

    Science.gov (United States)

    Dolezal, Pavel; Dagley, Michael J; Kono, Maya; Wolynec, Peter; Likić, Vladimir A; Foo, Jung Hock; Sedinová, Miroslava; Tachezy, Jan; Bachmann, Anna; Bruchhaus, Iris; Lithgow, Trevor

    2010-03-19

    Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.

  1. Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

    Science.gov (United States)

    Jouhet, Juliette; Gray, John C

    2009-10-01

    Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

  2. Sumoylation of Human Translationally Controlled Tumor Protein Is Important for Its Nuclear Transport

    Directory of Open Access Journals (Sweden)

    Gnanasekar Munirathinam

    2012-01-01

    Full Text Available Translationally controlled tumor protein (TCTP lacks nuclear bipartite localization signal sequence; yet TCTP is present abundantly in the nucleus. At present it is not known how TCTP gets transported to the nucleus. Sequence analyses showed that all TCTPs described to date have putative small ubiquitin-like modifier (SUMO motifs. Since SUMO modification plays an important role in the nuclear transport of proteins, we evaluated whether SUMO motifs are important for transport of TCTP into the nucleus. We show that TCTP exists in sumoylated form in cytoplasm and nucleus of mammalian cells. Point mutation of lysine residue in the SUMO motif compromised the ability of TCTP to get sumoylated in vitro. When cells were transfected with FLAG-tagged mutated TCTP, nuclear transport of TCTP was inhibited confirming that sumoylation is critical for the nuclear transport of TCTP. Our previous studies demonstrated that TCTP can function as an antioxidant protein in the nucleus. When we mutated TCTP at the SUMO motif the antioxidant function of TCTP was compromised. Results presented in this study thus show that sumoylation plays an important role in the transport of TCTP into the nucleus where they function as antioxidant protein.

  3. Identification of a protein with antioxidant activity that is important for the protection against beer ageing.

    Science.gov (United States)

    Wu, Ming J; Clarke, Frank M; Rogers, Peter J; Young, Paul; Sales, Narelle; O'Doherty, Patrick J; Higgins, Vincent J

    2011-01-01

    This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

  4. Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

    Directory of Open Access Journals (Sweden)

    Vincent J. Higgins

    2011-09-01

    Full Text Available This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1. Further characterisation using diphenylpicrylhydrazyl (DPPH free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process.

  5. Increasing protein stability: Importance of ΔCp and the denatured state

    Science.gov (United States)

    Fu, Hailong; Grimsley, Gerald; Scholtz, J Martin; Pace, C Nick

    2010-01-01

    Increasing the conformational stability of proteins is an important goal for both basic research and industrial applications. In vitro selection has been used successfully to increase protein stability, but more often site-directed mutagenesis is used to optimize the various forces that contribute to protein stability. In previous studies, we showed that improving electrostatic interactions on the protein surface and improving the β-turn sequences were good general strategies for increasing protein stability, and used them to increase the stability of RNase Sa. By incorporating seven of these mutations in RNase Sa, we increased the stability by 5.3 kcal/mol. Adding one more mutation, D79F, gave a total increase in stability of 7.7 kcal/mol, and a melting temperature 28°C higher than the wild-type enzyme. Surprisingly, the D79F mutation lowers the change in heat capacity for folding, ΔCp, by 0.6 kcal/mol/K. This suggests that this mutation stabilizes structure in the denatured state ensemble. We made other mutants that give some insight into the structure present in the denatured state. Finally, the thermodynamics of folding of these stabilized variants of RNase Sa are compared with those observed for proteins from thermophiles. PMID:20340133

  6. Efficient quantification of the importance of contacts for the dynamical stability of proteins.

    Science.gov (United States)

    Hamacher, Kay

    2011-04-15

    Understanding the stability of the native state and the dynamics of a protein is of great importance for all areas of biomolecular design. The efficient estimation of the influence of individual contacts between amino acids in a protein structure is a first step in the reengineering of a particular protein for technological or pharmacological purposes. At the same time, the functional annotation of molecular evolution can be facilitated by such insight. Here, we use a recently suggested, information theoretical measure in biomolecular design - the Kullback-Leibler-divergence - to quantify and therefore rank residue-residue contacts within proteins according to their overall contribution to the molecular mechanics. We implement this protocol on the basis of a reduced molecular model, which allows us to use a well-known lemma of linear algebra to speed up the computation. The increase in computational performance is around 10(1)- to 10(4)-fold. We applied the method to two proteins to illustrate the protocol and its results. We found that our method can reliably identify key residues in the molecular mechanics and the protein fold in comparison to well-known properties in the serine protease inhibitor. We found significant correlations to experimental results, e.g., dissociation constants and Φ values.

  7. Identification of a Protein with Antioxidant Activity that is Important for the Protection against Beer Ageing

    Science.gov (United States)

    Wu, Ming J.; Clarke, Frank M.; Rogers, Peter J.; Young, Paul; Sales, Narelle; O’Doherty, Patrick J.; Higgins, Vincent J.

    2011-01-01

    This study was carried out with fresh Australian lager beer which was sampled directly off the production line, the same samples aged for 12 weeks at 30 °C, and the vintage beer which was kept at 20 °C for 5 years. Characteristic Australian lager flavour was maintained in the fresh and vintage beers but was lost in the aged beer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and free thiol group labelling analyses of beer proteins found that this flavour stability correlated with the presence of an unknown 10 kilodaltons (kDa) protein with a higher level of free thiols. The protein was purified by size-exclusion chromatography, then peptide sequencing and database matching identified it as the barley lipid transfer protein (LTP1). Further characterisation using diphenylpicrylhydrazyl (DPPH) free radical scavenging and a Saccharomyces cerevisiae-based antioxidant screening assay demonstrated that the LTP1 protein was active in DPPH reduction and antioxidant activity. The absence of free thiol in the aged beer indicates that the thiol functional groups within the LTP1 protein were saturated and suggests that it is important in the flavour stability of beer by maintaining reduction capacity during the ageing process. PMID:22016646

  8. Proteins Play Important Role in Intercellular Adhesion Affecting on Fruit Textural Quality

    DEFF Research Database (Denmark)

    Bahadur Adhikari, Khem; Shomer, Ilan

    2012-01-01

    Fruit textural quality is becoming a major quality parameter for export, postharvest preservation, handling and processing. The main determinant of textural quality is intercellular adhesion (ICA) as attributed by the cell wall (CW) and its components. The importance of CW protein in ICA......H 3.5 ( pKa) incubated fruits (~2 N). The protein bands at ~29 kDa, ~75 kDa, ~32 kDa and 87 kDa were exclusively or prominently found in ICA strengthened fruits (pH 3.5

  9. Republished: Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.

    Science.gov (United States)

    Valcz, Gabor; Sipos, Ferenc; Tulassay, Zsolt; Molnar, Bela; Yagi, Yukako

    2015-05-01

    Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy.

  10. Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.

    Science.gov (United States)

    Valcz, Gabor; Sipos, Ferenc; Tulassay, Zsolt; Molnar, Bela; Yagi, Yukako

    2014-12-01

    Carcinoma-associated fibroblast (CAF) as prominent cell type of the tumour microenvironment has complex interaction with both the cancer cells and other non-neoplastic surrounding cells. The CAF-derived regulators and extracellular matrix proteins can support cancer progression by providing a protective microenvironment for the cancer cells via reduction of chemotherapy sensitivity. On the other hand, these proteins may act as powerful prognostic markers as well as potential targets of anticancer therapy. In this review, we summarise the clinical importance of the major CAF-derived signals influencing tumour behaviour and determining the outcome of chemotherapy.

  11. Phylogenetic Gaussian process model for the inference of functionally important regions in protein tertiary structures.

    Science.gov (United States)

    Huang, Yi-Fei; Golding, G Brian

    2014-01-01

    A critical question in biology is the identification of functionally important amino acid sites in proteins. Because functionally important sites are under stronger purifying selection, site-specific substitution rates tend to be lower than usual at these sites. A large number of phylogenetic models have been developed to estimate site-specific substitution rates in proteins and the extraordinarily low substitution rates have been used as evidence of function. Most of the existing tools, e.g. Rate4Site, assume that site-specific substitution rates are independent across sites. However, site-specific substitution rates may be strongly correlated in the protein tertiary structure, since functionally important sites tend to be clustered together to form functional patches. We have developed a new model, GP4Rate, which incorporates the Gaussian process model with the standard phylogenetic model to identify slowly evolved regions in protein tertiary structures. GP4Rate uses the Gaussian process to define a nonparametric prior distribution of site-specific substitution rates, which naturally captures the spatial correlation of substitution rates. Simulations suggest that GP4Rate can potentially estimate site-specific substitution rates with a much higher accuracy than Rate4Site and tends to report slowly evolved regions rather than individual sites. In addition, GP4Rate can estimate the strength of the spatial correlation of substitution rates from the data. By applying GP4Rate to a set of mammalian B7-1 genes, we found a highly conserved region which coincides with experimental evidence. GP4Rate may be a useful tool for the in silico prediction of functionally important regions in the proteins with known structures.

  12. On the importance of polar interactions for complexes containing intrinsically disordered proteins.

    Science.gov (United States)

    Wong, Eric T C; Na, Dokyun; Gsponer, Jörg

    2013-01-01

    There is a growing recognition for the importance of proteins with large intrinsically disordered (ID) segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

  13. On the importance of polar interactions for complexes containing intrinsically disordered proteins.

    Directory of Open Access Journals (Sweden)

    Eric T C Wong

    Full Text Available There is a growing recognition for the importance of proteins with large intrinsically disordered (ID segments in cell signaling and regulation. ID segments in these proteins often harbor regions that mediate molecular recognition. Coupled folding and binding of the recognition regions has been proposed to confer high specificity to interactions involving ID segments. However, researchers recently questioned the origin of the interaction specificity of ID proteins because of the overrepresentation of hydrophobic residues in their interaction interfaces. Here, we focused on the role of polar and charged residues in interactions mediated by ID segments. Making use of the extended nature of most ID segments when in complex with globular proteins, we first identified large numbers of complexes between globular proteins and ID segments by using radius-of-gyration-based selection criteria. Consistent with previous studies, we found the interfaces of these complexes to be enriched in hydrophobic residues, and that these residues contribute significantly to the stability of the interaction interface. However, our analyses also show that polar interactions play a larger role in these complexes than in structured protein complexes. Computational alanine scanning and salt-bridge analysis indicate that interfaces in ID complexes are highly complementary with respect to electrostatics, more so than interfaces of globular proteins. Follow-up calculations of the electrostatic contributions to the free energy of binding uncovered significantly stronger Coulombic interactions in complexes harbouring ID segments than in structured protein complexes. However, they are counter-balanced by even higher polar-desolvation penalties. We propose that polar interactions are a key contributing factor to the observed high specificity of ID segment-mediated interactions.

  14. Cubilin is an albumin binding protein important for renal tubular albumin reabsorption.

    Science.gov (United States)

    Birn, H; Fyfe, J C; Jacobsen, C; Mounier, F; Verroust, P J; Orskov, H; Willnow, T E; Moestrup, S K; Christensen, E I

    2000-05-01

    Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show that previously identified low-molecular-weight renal albumin binding proteins are fragments of cubilin. In addition, we find that mice lacking the endocytic receptor megalin show altered urinary excretion, and reduced tubular reabsorption, of albumin. Because cubilin has been shown to colocalize and interact with megalin, we propose a mechanism of albumin reabsorption mediated by both of these proteins. This process may prove important for understanding interstitial renal inflammation and fibrosis caused by proximal tubule uptake of an increased load of filtered albumin.

  15. Timing of postexercise protein intake is important for muscle hypertrophy with resistance training in elderly humans

    DEFF Research Database (Denmark)

    Esmarck, Birgitte; Olsen, Steen Schytte

    2001-01-01

    remains unresolved. 2. The study investigated the importance of immediate (P0) or delayed (P2) intake of an oral protein supplement upon muscle hypertrophy and strength over a period of resistance training in elderly males. 3. Thirteen men (age, 74 ± 1 years; body mass index (BMI), 25 ± 1 kg m−2 (means...... ± S.E.M.)) completed a 12 week resistance training programme (3 times per week) receiving oral protein in liquid form (10 g protein, 7 g carbohydrate, 3 g fat) immediately after (P0) or 2 h after (P2) each training session. Muscle hypertrophy was evaluated by magnetic resonance imaging (MRI) and from...... for the development of hypertrophy in skeletal muscle of elderly men in response to resistance training....

  16. Molecular characterization and expression analysis of chloroplast protein import components in tomato (Solanum lycopersicum).

    Science.gov (United States)

    Yan, Jianmin; Campbell, James H; Glick, Bernard R; Smith, Matthew D; Liang, Yan

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and initial import into the organelle of thousands of nucleus-encoded proteins. These proteins are translated in the cytosol as precursor proteins with cleavable amino-terminal targeting sequences called transit peptides. The majority of the known Toc components that mediate chloroplast protein import were originally identified in pea, and more recently have been studied most extensively in Arabidopsis. With the completion of the tomato genome sequencing project, it is now possible to identify putative homologues of the chloroplast import components in tomato. In the work reported here, the Toc GTPase cDNAs from tomato were identified, cloned and analyzed. The analysis revealed that there are four Toc159 homologues (slToc159-1, -2, -3 and -4) and two Toc34 homologues (slToc34-1 and -2) in tomato, and it was shown that tomato Toc159 and Toc34 homologues share high sequence similarity with the comparable import apparatus components from Arabidopsis and pea. Thus, tomato is a valid model for further study of this system. The expression level of Toc complex components was also investigated in different tissues during tomato development. The two tomato Toc34 homologues are expressed at higher levels in non-photosynthetic tissues, whereas, the expression of two tomato Toc159 homologues, slToc159-1 and slToc159-4, were higher in photosynthetic tissues, and the expression patterns of slToc159-2 was not significantly different in photosynthetic and non-photosynthetic tissues, and slToc159-3 expression was limited to a few select tissues.

  17. Importance of translation and nonnucleolytic ago proteins for on-target RNA interference.

    Science.gov (United States)

    Wu, Ligang; Fan, Jihua; Belasco, Joel G

    2008-09-09

    In animals, both siRNAs and miRNAs are thought to diminish protein synthesis from transcripts that are perfectly complementary by directing endonucleolytic cleavage where they anneal, thereby triggering rapid degradation of the entire message [1-4]. By contrast, partially complementary messages are downregulated by a combination of translational repression and accelerated decay caused by rapid poly(A) tail removal [3, 5-12]. Here we present evidence that translational repression can also make a substantial contribution to the downregulation of fully complementary messages by RNA interference. Unlike mRNA destabilization, this inhibitory effect on translation is greater for perfectly complementary elements located in the 3' untranslated region rather than in the protein-coding region. In addition to known disparities in their endonucleolytic activity [13, 14], the four Ago proteins with which siRNAs associate in humans differ significantly in their capacity to direct translational repression. As a result, the relative effect of siRNA on targets that are fully versus partially complementary is influenced by the comparative abundance of the three nonnucleolytic Ago proteins, causing this on-target/off-target ratio to vary in a cell-type-dependent manner because of the dissimilar tissue distribution of these proteins. These findings have important implications for the efficacy and specificity of RNA interference.

  18. Presence of undeclared peanut protein in chocolate bars imported from Europe.

    Science.gov (United States)

    Vadas, Peter; Perelman, Boris

    2003-10-01

    Peanut allergens are both stable and potent and are capable of inducing anaphylactic reactions at low concentrations. Consequently, the consumption of peanuts remains the most common cause of food-induced anaphylactic death. Since accidental exposure to peanuts is a common cause of potentially fatal anaphylaxis in peanut-allergic individuals, we tested for the presence of peanut protein in chocolate bars produced in Europe and North America that did not list peanuts as an ingredient. Ninety-two chocolate bars, of which 32 were manufactured in North America and 60 were imported from Europe, were tested by the Veratox assay. None of the 32 North American chocolate products, including 19 with precautionary labeling, contained detectable peanut protein. In contrast, 30.8% of products from western Europe without precautionary labeling contained detectable levels of peanut protein. Sixty-two percent of products from eastern Europe without precautionary labeling contained detectable peanut protein at levels of up to 245 ppm. The absence of precautionary labeling and the absence of the declaration of "peanut" as an ingredient in chocolate bars made in eastern and central Europe were not found to guarantee that these products were actually free of contaminating peanut protein. In contrast, North American manufacturers have attained a consistent level of safety and reliability for peanut-allergic consumers.

  19. Nuclear importation of Mariner transposases among eukaryotes: motif requirements and homo-protein interactions.

    Directory of Open Access Journals (Sweden)

    Marie-Véronique Demattei

    Full Text Available Mariner-like elements (MLEs are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174, and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm.

  20. The requirement of matrix ATP for the import of precursor proteins into the mitochondrial matrix and intermembrane space

    NARCIS (Netherlands)

    Stuart, Rosemary A.; Gruhler, Albrecht; Klei, Ida van der; Guiard, Bernard; Koll, Hans; Neupert, Walter

    1994-01-01

    The role of ATP in the matrix for the import of precursor proteins into the various mitochondrial subcompartments was investigated by studying protein translocation at experimentally defined ATP levels. Proteins targeted to the matrix were neither imported or processed when matrix ATP was depleted.

  1. Determination of Signaling Pathways in Proteins through Network Theory: Importance of the Topology.

    Science.gov (United States)

    Ribeiro, Andre A S T; Ortiz, Vanessa

    2014-04-08

    Network theory methods are being increasingly applied to proteins to investigate complex biological phenomena. Residues that are important for signaling processes can be identified by their condition as critical nodes in a protein structure network. This analysis involves modeling the protein as a graph in which each residue is represented as a node and edges are drawn between nodes that are deemed connected. In this paper, we show that the results obtained from this type of network analysis (i.e., signaling pathways, key residues for signal transmission, etc.) are profoundly affected by the topology of the network, with normally used determination of network edges by geometrical cutoff schemes giving rise to substantial statistical errors. We propose a method of determining protein structure networks by calculating inter-residue interaction energies and show that it gives an accurate and reliable description of the signal-propagation properties of a known allosteric enzyme. We also show that including covalent interactions in the network topology is essential for accurate results to be obtained.

  2. The Fowler syndrome-associated protein FLVCR2 is an importer of heme.

    Science.gov (United States)

    Duffy, Simon P; Shing, Jennifer; Saraon, Punit; Berger, Lloyd C; Eiden, Maribeth V; Wilde, Andrew; Tailor, Chetankumar S

    2010-11-01

    Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.

  3. Border control: selectivity of chloroplast protein import and regulation at the TOC-complex.

    Science.gov (United States)

    Demarsy, Emilie; Lakshmanan, Ashok M; Kessler, Felix

    2014-01-01

    Plants have evolved complex and sophisticated molecular mechanisms to regulate their development and adapt to their surrounding environment. Particularly the development of their specific organelles, chloroplasts and other plastid-types, is finely tuned in accordance with the metabolic needs of the cell. The normal development and functioning of plastids require import of particular subsets of nuclear encoded proteins. Most preproteins contain a cleavable sequence at their N terminal (transit peptide) serving as a signal for targeting to the organelle and recognition by the translocation machinery TOC-TIC (translocon of outer membrane complex-translocon of inner membrane complex) spanning the dual membrane envelope. The plastid proteome needs constant remodeling in response to developmental and environmental factors. Therefore selective regulation of preprotein import plays a crucial role in plant development. In this review we describe the diversity of transit peptides and TOC receptor complexes, and summarize the current knowledge and potential directions for future research concerning regulation of the different Toc isoforms.

  4. Drep-2 is a novel synaptic protein important for learning and memory.

    Science.gov (United States)

    Andlauer, Till F M; Scholz-Kornehl, Sabrina; Tian, Rui; Kirchner, Marieluise; Babikir, Husam A; Depner, Harald; Loll, Bernhard; Quentin, Christine; Gupta, Varun K; Holt, Matthew G; Dipt, Shubham; Cressy, Michael; Wahl, Markus C; Fiala, André; Selbach, Matthias; Schwärzel, Martin; Sigrist, Stephan J

    2014-11-13

    CIDE-N domains mediate interactions between the DNase Dff40/CAD and its inhibitor Dff45/ICAD. In this study, we report that the CIDE-N protein Drep-2 is a novel synaptic protein important for learning and behavioral adaptation. Drep-2 was found at synapses throughout the Drosophila brain and was strongly enriched at mushroom body input synapses. It was required within Kenyon cells for normal olfactory short- and intermediate-term memory. Drep-2 colocalized with metabotropic glutamate receptors (mGluRs). Chronic pharmacological stimulation of mGluRs compensated for drep-2 learning deficits, and drep-2 and mGluR learning phenotypes behaved non-additively, suggesting that Drep 2 might be involved in effective mGluR signaling. In fact, Drosophila fragile X protein mutants, shown to benefit from attenuation of mGluR signaling, profited from the elimination of drep-2. Thus, Drep-2 is a novel regulatory synaptic factor, probably intersecting with metabotropic signaling and translational regulation.

  5. Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids.

    Directory of Open Access Journals (Sweden)

    Pitter F Huesgen

    Full Text Available In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

  6. An in silico analysis of the mitochondrial protein import apparatus of plants

    Directory of Open Access Journals (Sweden)

    Whelan James

    2010-11-01

    Full Text Available Abstract Background An in silico analysis of the mitochondrial protein import apparatus from a variety of species; including Chlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens, Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine if components differed within and between plant and non-plant species. Results The channel forming subunits of the outer membrane components Tom40 and Sam50 are conserved between plant groups and other eukaryotes. In contrast, the receptor component(s in green plants, particularly Tom20, (C. reinhardtii, C. variabilis, P. patens, S. moellendorffii, P. glauca, O. sativa and A. thaliana are specific to this lineage. Red algae contain a Tom22 receptor that is orthologous to yeast Tom22. Furthermore, plant mitochondrial receptors display differences between various plant lineages. These are evidenced by distinctive motifs in all plant Metaxins, which are absent in red algae, and the presence of the outer membrane receptor OM64 in Angiosperms (rice and Arabidopsis, but not in lycophytes (S. moellendorffii and gymnosperms (P. glauca. Furthermore, although the intermembrane space receptor Mia40 is conserved across a wide phylogenetic range, its function differs between lineages. In all plant lineages, Tim17 contains a C-terminal extension, which may act as a receptor component for the import of nucleic acids into plant mitochondria. Conclusions It is proposed that the observed functional divergences are due to the selective pressure to sort proteins between mitochondria and chloroplasts, resulting in differences in protein receptor components between plant groups and other organisms. Additionally, diversity of receptor components is observed within the plant kingdom. Even when receptor components are orthologous across plant and non-plant species, it appears that the functions of these have expanded or

  7. [Lipid droplet-associated proteins. Importance in steatosis, steatohepatitis and hepatocarcinogenesis].

    Science.gov (United States)

    Straub, B K

    2015-11-01

    Hepatocellular steatosis constitutes the most frequent liver disease in western countries and may progress to steatohepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). The lipid droplet (LD)-associated proteins perilipin, adipophilin, TIP47 ("tail interacting protein of 47 kDa"), S3-12 and myocardial LD protein (MLDP), so-called perilipins 1-5 (PAT family) govern formation, maintenance and degradation of LDs. A lack of perilipin in mice inhibits obesity and a lack of adipophilin or TIP47 inhibit the development of fatty liver disease. In long-term cell culture models as well as in liver biopsies of patients with different acute and chronic liver diseases, LD-associated proteins are sequentially recruited to LDs and regulated via peroxisome proliferator-activated receptor (PPAR) α and PPARγ as well as posttranscriptionally via alternative splicing, LD fusion and lipolysis. Whereas TIP47 and MLDP coat small newly formed LDs in acute microvesicular steatosis, adipophilin constitutes a robust general marker for LDs in many different cell types. Perilipin is important for the long-term storage of lipids in macrovesicular steatosis and controls lipolysis via hormone-dependent phosphorylation. During malignant transformation, increased formation of small LDs and overexpression of adipophilin, TIP47 and MLDP are detected, possibly as the expression of an altered tumor metabolism analogous to a Warburg effect. Adipophilin correlates positively with the proliferation rate of HCC cells. Cultured cells with downregulation of TIP47 or adipophilin via small interfering RNA (siRNA) or small hairpin RNA (shRNA) show less but larger LDs with reduced neutral fat content.

  8. Processing of the glycosomal matrix-protein import receptor PEX5 of Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Gualdrón-López, Melisa [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium); Michels, Paul A.M., E-mail: paul.michels@uclouvain.be [Research Unit for Tropical Diseases, de Duve Institute, Université catholique de Louvain, Brussels (Belgium)

    2013-02-01

    Highlights: ► Most eukaryotic cells have a single gene for the peroxin PEX5. ► PEX5 is sensitive to in vitro proteolysis in distantly related organisms. ► TbPEX5 undergoes N-terminal truncation in vitro and possibly in vivo. ► Truncated TbPEX5 is still capable of binding PTS1-containing proteins. ► PEX5 truncation is physiologically relevant or an evolutionary conserved artifact. -- Abstract: Glycolysis in kinetoplastid protists such as Trypanosoma brucei is compartmentalized in peroxisome-like organelles called glycosomes. Glycosomal matrix-protein import involves a cytosolic receptor, PEX5, which recognizes the peroxisomal-targeting signal type 1 (PTS1) present at the C-terminus of the majority of matrix proteins. PEX5 appears generally susceptible to in vitro proteolytic processing. On western blots of T. brucei, two PEX5 forms are detected with apparent M{sub r} of 100 kDa and 72 kDa. 5′-RACE-PCR showed that TbPEX5 is encoded by a unique transcript that can be translated into a protein of maximally 72 kDa. However, recombinant PEX5 migrates aberrantly in SDS–PAGE with an apparent M{sub r} of 100 kDa, similarly as observed for the native peroxin. In vitro protease susceptibility analysis of native and {sup 35}S-labelled PEX5 showed truncation of the 100 kDa form at the N-terminal side by unknown parasite proteases, giving rise to the 72 kDa form which remains functional for PTS1 binding. The relevance of these observations is discussed.

  9. Importance of a Conserved Lys/Arg Residue for Ligand/PDZ Domain Interactions as Examined by Protein Semisynthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren W; Moran, Griffin E; Sereikaité, Vita

    2016-01-01

    drug targets for diseases (in the brain in particular), so understanding the molecular details of PDZ domain interactions is of fundamental importance. PDZ domains bind to a protein partner at either a C-terminal peptide or internal peptide motifs. Here, we examined the importance of a conserved Lys......PDZ domains are ubiquitous small protein domains that are mediators of numerous protein-protein interactions, and play a pivotal role in protein trafficking, synaptic transmission, and the assembly of signaling-transduction complexes. In recent years, PDZ domains have emerged as novel and exciting...

  10. Gravity Plays an Important Role in Muscle Development and the Differentiation of Contractile Protein Phenotype

    Science.gov (United States)

    Adams, Gregory A.; Haddad, Fadia; Baldwin, Kenneth M.

    2003-01-01

    Several muscles in the body exist mainly to work against gravity. Whether gravity is important in the development of these muscles is not known. By examining the basic proteins that compose muscle, questions about the role of gravity in muscle development can be answered. Myosin heavy chains (MHCs) are a family of proteins critically important for muscle contraction. Several types of MHCs exist (e.g., neonatal, slow, fast), and each type is produced by a particular gene. Neonatal MHCs are produced early in life. Slow MHCs are important in antigravity muscles, and fast MHCs are found in fast-twitch power muscles. The gene that is turned on or expressed will determine which MHC is produced. Early in development, antigravity skeletal muscles (muscles that work against gravity) normally produce a combination of the neonatal/embryonic MHCs. The expression of these primitive MHCs is repressed early in development; and the adult slow and fast MHC genes become fully expressed. We tested the hypothesis that weightbearing activity is critical for inducing the normal expression of the slow MHC gene typically expressed in adult antigravity muscles. Also, we hypothesized that thyroid hormone, but not opposition to gravity, is necessary for expressing the adult fast IIb MHC gene essential for high-intensity muscle performance. Groups of normal thyroid and thyroid-deficient neonatal rats were studied after their return from the 16-day Neurolab mission and compared to matched controls. The results suggest: (1) Weightlessness impaired body and limb skeletal muscle growth in both normal and thyroid-deficient animals. Antigravity muscles were impaired more than those used primarily for locomotion andor nonweightbearing activity. (2) Systemic and muscle expression of insulin-like growth factor-I (IGF-I), an important body and tissue growth factor, was depressed in flight animals. (3) Normal slow, type I MHC gene expression was markedly repressed in the normal thyroid flight group. (4

  11. Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    David Gau

    Full Text Available Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1, an important regulator of actin polymerization in cells.We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK, an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS, GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1.We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89, a novel site. Consistent with PKA's ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with

  12. Altered mitochondrial morphology and defective protein import reveal novel roles for Bax and/or Bak in skeletal muscle.

    Science.gov (United States)

    Zhang, Yuan; Iqbal, Sobia; O'Leary, Michael F N; Menzies, Keir J; Saleem, Ayesha; Ding, Shuzhe; Hood, David A

    2013-09-01

    The function Bax and/or Bak in constituting a gateway for mitochondrial apoptosis in response to apoptotic stimuli has been unequivocally demonstrated. However, recent work has suggested that Bax/Bak may have unrecognized nonapoptotic functions related to mitochondrial function in nonstressful environments. Wild-type (WT) and Bax/Bak double knockout (DKO) mice were used to determine alternative roles for Bax and Bak in mitochondrial morphology and protein import in skeletal muscle. The absence of Bax and/or Bak altered mitochondrial dynamics by regulating protein components of the organelle fission and fusion machinery. Moreover, DKO mice exhibited defective mitochondrial protein import, both into the matrix and outer membrane compartments, which was consistent with our observations of impaired membrane potential and attenuated expression of protein import machinery (PIM) components in intermyofibrillar mitochondria. Furthermore, the cytosolic chaperones heat-shock protein 90 (Hsp90) and binding immunoglobulin protein (BiP) were markedly increased with the deletion of Bax/Bak, indicating that the cytosolic environment related to protein folding may be changed in DKO mice. Interestingly, endurance training fully restored the deficiency of protein import in DKO mice, likely via the upregulation of PIM components and through improved cytosolic chaperone protein expression. Thus our results emphasize novel roles for Bax and/or Bak in mitochondrial function and provide evidence, for the first time, of a curative function of exercise training in ameliorating a condition of defective mitochondrial protein import.

  13. Homeodomain-interacting protein kinase 2 plays an important role in normal terminal erythroid differentiation.

    Science.gov (United States)

    Hattangadi, Shilpa M; Burke, Karly A; Lodish, Harvey F

    2010-06-10

    Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic development because Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis, whereas the single knockouts do not. These serine-threonine kinases phosphorylate and consequently modify the functions of several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferation (explained in part by impaired cell-cycle progression as well as increased apoptosis) and terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesis, such as alpha-globin and mitoferrin 1, demonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.

  14. Enterococcal surface protein Esp is important for biofilm formation of Enterococcus faecium E1162.

    Science.gov (United States)

    Heikens, Esther; Bonten, Marc J M; Willems, Rob J L

    2007-11-01

    Enterococci have emerged as important nosocomial pathogens with resistance to multiple antibiotics. Adhesion to abiotic materials and biofilm formation on medical devices are considered important virulence properties. A single clonal lineage of Enterococcus faecium, complex 17 (CC17), appears to be a successful nosocomial pathogen, and most CC17 isolates harbor the enterococcal surface protein gene, esp. In this study, we constructed an esp insertion-deletion mutant in a clinical E. faecium CC17 isolate. In addition, initial adherence and biofilm assays were performed. Compared to the wild-type strain, the esp insertion-deletion mutant no longer produced Esp on the cell surface and had significantly lower initial adherence to polystyrene and significantly less biofilm formation, resulting in levels of biofilm comparable to those of an esp-negative isolate. Capacities for initial adherence and biofilm formation were restored in the insertion-deletion mutant by in trans complementation with esp. These results identify Esp as the first documented determinant in E. faecium CC17 with an important role in biofilm formation, which is an essential factor in infection pathogenesis.

  15. Expression of IAP family proteins and its clinical importance in breast cancer patients.

    Science.gov (United States)

    Pluta, P; Jeziorski, A; Cebula-Obrzut, A Pluta B; Wierzbowska, A; Piekarski, J; Smolewski, P

    2015-01-01

    Inhibitor of apoptosis (IAP) family proteins is involved in mechanisms of resistance to apoptosis in various cancer cells. The aim of this study was to assess the expression of selected IAP proteins such as XIAP, cIAP-1, cIAP-2 and survivin in breast cancer patients and evaluates their relationship with the prognostic and predictive factors and their impact to overall survival (OS) and progression free survival (PFS). The study was conducted with the use of tissue samples prospectively collected from 92 previously untreated female breast cancer patients. The control encompassed 10 fibroadenoma patients. The expression of XIAP, cIAP-1, cIAP-2 and survivin was assessed using flow multicolor cytometry. XIAP expression was present in 99 % of the breast cancer patients (91/92) with the median expression 13.65% (range 1-66.8%). Expression of XIAP in breast cancer was significantly higher compared to the control group (p=0.006). Median expression of cIAP-1, cIAP-2 and survivin in the study group was 25.95% (range 0.8-83.7%), 16.7% (range 1-53.2%) and 4.6% (range 0-43%) respectively. In the rank Spearman test, strong correlations (pproteins and survival. However, low expression of XIAP in breast cancer showed trend to longer PFS (p=0.08). XIAP, cIAP-1 cIAP-2 and survivin participate in antiapoptotic mechanisms in breast cancer and XIAP and survivin seem to have the most significant prognostic importance. Further studies are needed to establish more complete prognostic and predictive values of IAP family proteins in breast cancer patients.

  16. Have We Overlooked the Importance of Serine/Threonine Protein Phosphatases in Pancreatic Beta-Cells? Role Played by Protein Phosphatase 2A in Insulin Secretion

    Directory of Open Access Journals (Sweden)

    Esser V

    2005-07-01

    Full Text Available Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.

  17. Using phylogenomic patterns and gene ontology to identify proteins of importance in plant evolution.

    Science.gov (United States)

    Cibrián-Jaramillo, Angélica; De la Torre-Bárcena, Jose E; Lee, Ernest K; Katari, Manpreet S; Little, Damon P; Stevenson, Dennis W; Martienssen, Rob; Coruzzi, Gloria M; DeSalle, Rob

    2010-07-12

    We use measures of congruence on a combined expressed sequenced tag genome phylogeny to identify proteins that have potential significance in the evolution of seed plants. Relevant proteins are identified based on the direction of partitioned branch and hidden support on the hypothesis obtained on a 16-species tree, constructed from 2,557 concatenated orthologous genes. We provide a general method for detecting genes or groups of genes that may be under selection in directions that are in agreement with the phylogenetic pattern. Gene partitioning methods and estimates of the degree and direction of support of individual gene partitions to the overall data set are used. Using this approach, we correlate positive branch support of specific genes for key branches in the seed plant phylogeny. In addition to basic metabolic functions, such as photosynthesis or hormones, genes involved in posttranscriptional regulation by small RNAs were significantly overrepresented in key nodes of the phylogeny of seed plants. Two genes in our matrix are of critical importance as they are involved in RNA-dependent regulation, essential during embryo and leaf development. These are Argonaute and the RNA-dependent RNA polymerase 6 found to be overrepresented in the angiosperm clade. We use these genes as examples of our phylogenomics approach and show that identifying partitions or genes in this way provides a platform to explain some of the more interesting organismal differences among species, and in particular, in the evolution of plants.

  18. Induction of apoptosis by ribosome inactivating proteins: importance of N-glycosidase activity.

    Science.gov (United States)

    Das, Mrinal Kumar; Sharma, Radhey Shyam; Mishra, Vandana

    2012-03-01

    Apoptotic cell death is a fundamental process in the development and physiological homeostasis of multicellular organisms. It is associated with control of cell numbers in tissues and organs during development, with cell turnover, and with response to infection. Molecules that trigger this process in continuously proliferating cancer cells can be used as chemotherapeutic agents. Ribosome inactivating proteins (RIPs) that inhibit translation in a cell by depurinating (N-glycosidase activity) the 28S rRNA are known to serve as apoptosis inducers. However, the role of depurination activity of the RIPs in apoptosis induction is still controversial. Presently, there are three different hypotheses which propose that depurination is: (1) essential, (2) essential but not the sole factor, or (3) not essential for apoptosis induction. This article reviews various experimental outcomes on the importance of N-glycosidase activity of RIPs in the induction of apoptosis.

  19. Protein transport in organelles: The Toc complex way of preprotein import.

    Science.gov (United States)

    Agne, Birgit; Kessler, Felix

    2009-03-01

    Most of the estimated 1000 or so chloroplast proteins are synthesized as cytosolic preproteins with N-terminal cleavable targeting sequences (transit peptide). Translocon complexes at the outer (Toc) and inner chloroplast envelope membrane (Tic) concertedly facilitate post-translational import of preproteins into the chloroplast. Three components, the Toc34 and Toc159 GTPases together with the Toc75 channel, form the core of the Toc complex. The two GTPases act as GTP-dependent receptors at the chloroplast surface and promote insertion of the preprotein across the Toc75 channel. Additional factors guide preproteins to the Toc complex or support their stable ATP-dependent binding to the chloroplast. This minireview describes the components of the Toc complex and their function during the initial steps of preprotein translocation across the chloroplast envelope.

  20. Plant i - AAA protease controls the turnover of the essential mitochondrial protein import component.

    Science.gov (United States)

    Opalińska, Magdalena; Parys, Katarzyna; Murcha, Monika W; Jańska, Hanna

    2017-03-06

    Mitochondria are multifunctional organelles that play a central role in energy metabolism. Due to life-essential functions of these organelles, mitochondrial content, quality, and dynamics are tightly controlled. Across the species, highly conserved ATP - dependent proteases prevent malfunction of mitochondria through versatile activities. This study focuses on a molecular function of plant mitochondrial inner membrane-embedded i - AAA protease, FTSH4, providing its first bona fide substrate. Here, we report that the abundance of Tim17-2 protein, the essential component of the TIM17:23 translocase, is directly controlled by the proteolytic activity of FTSH4. Plants that are lacking functional FTSH4 protease are characterized by significantly enhanced capacity of preprotein import through the TIM17:23 - dependent pathway. Together with the observation that FTSH4 prevents accumulation of Tim17-2, our data points towards the role of this i - AAA protease in the regulation of mitochondrial biogenesis in plants.

  1. Nuclear export and import of human hepatitis B virus capsid protein and particles.

    Directory of Open Access Journals (Sweden)

    Hung-Cheng Li

    Full Text Available It remains unclear what determines the subcellular localization of hepatitis B virus (HBV core protein (HBc and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS, while ARD-II and ARD-IV behave like two independent nuclear export signals (NES. This conclusion is based on five independent lines of experimental evidence: i Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT. iii By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1, which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel

  2. Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

    Science.gov (United States)

    Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

    2017-03-06

    J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

  3. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

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    Zhang Heng; Wu Shengnan, E-mail: wushn@scnu.edu.cn [MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631 (China)

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  4. Yes-Associated Protein (YAP) Promotes the Nuclear Import of p73

    Science.gov (United States)

    Zhang, Heng; Wu, Shengnan

    2011-01-01

    p73 has been identified as a structural and functional homolog of the tumor suppressor p53. However, mechanisms that regulate the localization of p73 have not been fully clarified. The Yes-associated protein (YAP) is a transcriptional coactivator. As a transcriptional coactivator, YAP needs to bind transcription factors to stimulate gene expression. p73 is a reported YAP target transcription factors and YAP has been shown to positively regulate p73 in promoting apoptosis. Previous studies show that p73 interacts with YAP through its PPPY motif, and increases p73 transactivation of apoptotic genes. In this study, we focused on YAP's regulation of the localization of p73. After transient transfection into Rat pheochromocytoma (PC12) cells and Human embryonic kidney 293T cells with GFP-YAP and/or YFP-p73, and incubated for 24 hours expression. p73 was fused to YFP to allow the examination of its subcellular localization. When expressed alone, YFP-p73 was distributed throughout the cell. When coexpressed with YAP, nuclear accumulation of YFP-p73 became evident. We quantitated the effect of YAP on the redistribution of YFP-p73 by counting cells with nuclear-only YFP signal. We found that YAP can influence the subcellular distribution of p73. Altogether, coexpression with YAP affected the subcellular distribution of the p73 protein. Our studies attribute a central role to YAP in regulating p73 accumulation and YAP, at least in part, might promote the nuclear import of p73.

  5. The similarity between N-terminal targeting signals for protein import into different organelles and its evolutionary relevance

    Directory of Open Access Journals (Sweden)

    Markus eKunze

    2015-09-01

    Full Text Available The proper distribution of proteins between the cytosol and various membrane-bound compartments is crucial for the functionality of eukaryotic cells. This requires the cooperation between protein transport machineries that translocate diverse proteins from the cytosol into these compartments and targeting signal(s encoded within the primary sequence of these proteins that define their cellular destination. The mechanisms exerting protein translocation differ remarkably between the compartments, but the predominant targeting signals for mitochondria, chloroplasts and the ER share the N-terminal position, an α-helical structural element and the removal from the core protein by intraorganellar cleavage. Interestingly, similar properties have been described for the peroxisomal targeting signal type 2 mediating the import of a fraction of soluble peroxisomal proteins, whereas other peroxisomal matrix proteins encode the type 1 targeting signal residing at the extreme C-terminus. The structural similarity of N-terminal targeting signals poses a challenge to the specificity of protein transport, but allows the generation of ambiguous targeting signals that mediate dual targeting of proteins into different compartments. Dual targeting might represent an advantage for adaptation processes that involve a redistribution of proteins, because it circumvents the hierarchy of targeting signals. Thus, the co-existence of two equally functional import pathways into peroxisomes might reflect a balance between evolutionary constant and flexible transport routes.

  6. Heat shock protein 70 of Naegleria fowleri is important factor for proliferation and in vitro cytotoxicity.

    Science.gov (United States)

    Song, Kyoung-Ju; Song, Kyung-Hui; Kim, Jong-Hyun; Sohn, Hae-Jin; Lee, Yang-Jin; Park, Chang-Eun; Shin, Ho-Joon

    2008-07-01

    To evaluate the role of heat shock 70 protein (HSP70) in free-living amoeba, a constitutive and inducible heat shock 70 gene of pathogenic Naegleria fowleri has previously been cloned, characterized, and named as Nf-cHSP70. The Nf-cHSP70 is localized in the cytoplasm, pseudopodia, and phagocytic food-cups. To investigate the role of Nf-cHSP70 in the pathogenicity of N. fowleri, the synthesis of N. fowleri HSP70 was first inhibited with benzylidene lactam compound (KNK437), and Nf-cHSP70 gene was knock-downed with antisense oligomers, which were designed with a start region-specific antisense oligonucleotides (24 oligomers) and modified with phosphorothioate. KNK437 inhibited the induction of N. fowleri HSP70 in a dose-dependent manner. In addition, 300 muM KNK437 reduced the proliferation of N. fowleri to 79.4% of untreated control (100%). Nf-cHSP70 knock-downed N. fowleri with antisense oligomers showed 68.5% reduction of proliferation in comparison with untreated control (100%). The cytotoxicity of N. fowleri against CHO target cells was reduced to 42.1% by KNK437 and 68.6% by antisense oligomers. These results suggest that the cloned Nf-cHSP70 plays an important role in the proliferation and cytotoxicity of pathogenic N. fowleri.

  7. An Autophosphorylation Site of the Protein Kinase SOS2 Is Important for Salt Tolerance in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hiroaki Fujii; Jian-Kang Zhu

    2009-01-01

    The protein kinase SOS2 (Salt Overly Sensitive 2) is essential for salt-stress signaling and tolerance in Arabidopsis. SOS2 is known to be activated by calcium-SOS3 and by phosphorylation at its activation loop. SOS2 is autophosphorylated in vitro, but the autophosphorylation site and its role in salt tolerance are not known. In this study, we identified an autophosphorylation site in SOS2 and analyzed its role in the responses of Arabidopsis to salt stress. Mass spectrometry analysis showed that Ser 228 of SOS2 is autophosphorylated. When this site was mutated to Ala, the autophosphorylation rate of SOS2 decreased. The substrate phosphorylation by the mutated SOS2 was also less than that by the wild-type SOS2. In contrast, changing Ser228 to Asp to mimic the autophosphorylation enhanced substrate phosphorylation by SOS2. Complementation tests in a sos2 mutant showed that the S228A but not the S228D mutation partially disrupted the function of SOS2 in salt tolerance. We also show that activation loop phosphorylation at Thr168 and autophosphorylation at Ser228 cannot substitute for each other, suggesting that both are required for salt tolerance. Our results indicate that Ser 228 of SOS2 is autophosphorylated and that this autophosphorylation is important for SOS2 function under salt stress.

  8. Importance of fumarate and nitrate reduction regulatory protein for intestinal proliferation of Vibrio vulnificus.

    Science.gov (United States)

    Kado, Takehiro; Kashimoto, Takashige; Yamazaki, Kohei; Ueno, Shunji

    2017-01-01

    The sepsis caused by Vibrio vulnificus is characterized by an average incubation period of 26 h and a high mortality rate exceeding 50%. The fast growth and dissemination of V. vulnificus in vivo lead to poor clinical outcomes in patients. Therefore, elucidation of the proliferation mechanisms of this organism in vivo may lead to the development of an effective therapeutic strategy. In this study, we focused on the low oxygen concentration in the intestinal milieu because of its drastic difference from that in air. Fumarate and nitrate reduction regulatory protein (FNR) is known to be a global transcriptional regulator for adaptation to anaerobic conditions in various bacteria. We generated a strain of V. vulnificus in which the fnr gene was replaced with an erythromycin resistance gene (fnr::erm mutant). When the fnr::erm mutant was tested in a growth competition assay against the wild-type (WT) in vivo, the competitive index of fnr::erm mutant to WT in the intestinal loop and liver was 0.378 ± 0.192 (mean ± SD) and 0.243 ± 0.123, respectively. These data suggested that FNR is important for the proliferation of V. vulnificus in the intestine to achieve a critical mass to be able to invade the systemic circulation.

  9. The importance of Ca2+/Zn2+ signaling S100 proteins and RAGE in translational medicine.

    Science.gov (United States)

    Leclerc, Estelle; Heizmann, Claus W

    2011-06-01

    The Receptor for Advanced Glycation Endproducts (RAGE) is a multiligand receptor involved in a large number of human disorders. Identified first as the receptor for the Advanced Glycation Endproducts (AGEs), RAGE has emerged in recent years as a major receptor for many members of the S100 calcium and zinc binding protein family. The interaction with and the signaling triggered by several S100 proteins such as S100B and S100A12 have been studied in details and have shown concentration and cell type dependent signaling cascades. The S100 protein family consists of more than 20 members which present high amino-acid sequence and structural similarities. These small EF-hand calcium binding proteins interact with a large number of protein targets and are almost all been shown to be involved in cancer. In this review we discuss the recent knowledge about the role of S100 proteins and RAGE in human disorders.

  10. Specific subunits of heterotrimeric G proteins play important roles during nodulation in soybean.

    Science.gov (United States)

    Choudhury, Swarup Roy; Pandey, Sona

    2013-05-01

    Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling.

  11. The L1TD1 Protein Interactome Reveals the Importance of Post-transcriptional Regulation in Human Pluripotency

    Directory of Open Access Journals (Sweden)

    Maheswara Reddy Emani

    2015-03-01

    Full Text Available The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  12. The L1TD1 protein interactome reveals the importance of post-transcriptional regulation in human pluripotency.

    Science.gov (United States)

    Emani, Maheswara Reddy; Närvä, Elisa; Stubb, Aki; Chakroborty, Deepankar; Viitala, Miro; Rokka, Anne; Rahkonen, Nelly; Moulder, Robert; Denessiouk, Konstantin; Trokovic, Ras; Lund, Riikka; Elo, Laura L; Lahesmaa, Riitta

    2015-03-10

    The RNA-binding protein L1TD1 is one of the most specific and abundant proteins in pluripotent stem cells and is essential for the maintenance of pluripotency in human cells. Here, we identify the protein interaction network of L1TD1 in human embryonic stem cells (hESCs) and provide insights into the interactome network constructed in human pluripotent cells. Our data reveal that L1TD1 has an important role in RNA splicing, translation, protein traffic, and degradation. L1TD1 interacts with multiple stem-cell-specific proteins, many of which are still uncharacterized in the context of development. Further, we show that L1TD1 is a part of the pluripotency interactome network of OCT4, SOX2, and NANOG, bridging nuclear and cytoplasmic regulation and highlighting the importance of RNA biology in pluripotency.

  13. The importance of sequence diversity in the aggregation and evolution of proteins.

    Science.gov (United States)

    Wright, Caroline F; Teichmann, Sarah A; Clarke, Jane; Dobson, Christopher M

    2005-12-08

    Incorrect folding of proteins, leading to aggregation and amyloid formation, is associated with a group of highly debilitating medical conditions including Alzheimer's disease and late-onset diabetes. The issue of how unwanted protein association is normally avoided in a living system is particularly significant in the context of the evolution of multidomain proteins, which account for over 70% of all eukaryotic proteins, where the effective local protein concentration in the vicinity of each domain is very high. Here we describe the aggregation kinetics of multidomain protein constructs of immunoglobulin domains and the ability of different homologous domains to aggregate together. We show that aggregation of these proteins is a specific process and that the efficiency of coaggregation between different domains decreases markedly with decreasing sequence identity. Thus, whereas immunoglobulin domains with more than about 70% identity are highly prone to coaggregation, those with less than 30-40% sequence identity do not detectably interact. A bioinformatics analysis of consecutive homologous domains in large multidomain proteins shows that such domains almost exclusively have sequence identities of less than 40%, in other words below the level at which coaggregation is likely to be efficient. We propose that such low sequence identities could have a crucial and general role in safeguarding proteins against misfolding and aggregation.

  14. Molecular importance of prawn large heat shock proteins 60, 70 and 90.

    Science.gov (United States)

    Chaurasia, Mukesh Kumar; Nizam, Faizal; Ravichandran, Gayathri; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Arshad, Aziz; Elumalai, Preetham; Arockiaraj, Jesu

    2016-01-01

    Considering the importance of heat shock proteins (HSPs) in the innate immune system of prawn, a comparative molecular approach was proposed to study the crustacean large HSPs 60, 70 and 90. Three different large HSPs were identified from freshwater prawn Macrobrachium rosenbergii (Mr) cDNA library during screening. The structural and functional characteristic features of HSPs were studied using various bioinformatics tools. Also, their gene expression and mRNA regulation upon various pathogenic infections was studied by relative quantification using 2(-ΔΔCT) method. MrHSP60 contains a long chaperonin 60 domain at 46-547 which carries a chaperonin 60 signature motif between 427 and 438, whereas MrHSP70 contains a long HSP70 domain at 21-624 and MrHSP90 carries a HSP90 domain at 188-719. The two dimensional analysis showed that MrHSP60 contains more amino acids (52%) in helices, whereas MrHSP70 (40.6%) and MrHSP90 (51.8%) carried more residues in coils. Gene expression results showed significant (P < 0.05) expression of MrHSP60, 70 and 90 in haemocyte, gill and hepatopancreas, respectively. Further, the expression level was up-regulated upon bacterial (Aeromonas hydrophilla and Vibrio harveyi) and viral [white spot syndrome virus (WSSV) and M. rosenbergii nodo virus (MrNV)] infections during various time periods. The gene expression results exhibited the potential involvement of these three HSPs in the immune system of prawn. The study indicated the potentiality of these molecules, thereby protecting cells against pathogens as well as severe cellular and environmental stresses in crustaceans.

  15. Protein kinase C epsilon is important in modulating organic-dust-induced airway inflammation.

    Science.gov (United States)

    Poole, Jill A; Romberger, Debra J; Bauer, Chris; Gleason, Angela M; Sisson, Joseph H; Oldenburg, Peter J; West, William W; Wyatt, Todd A

    2012-10-01

    Organic dust samples from swine confinement facilities elicit pro-inflammatory cytokine/chemokine release from bronchial epithelial cells and monocytes, dependent, in part, upon dust-induced activation of the protein kinase C (PKC) isoform, PKCε. PKCε is also rapidly activated in murine tracheal epithelial cells following in vivo organic dust challenges, yet the functional role of PKCε in modulating dust-induced airway inflammatory outcomes is not defined. Utilizing an established intranasal inhalation animal model, experiments investigated the biologic and physiologic responses following organic dust extract (ODE) treatments in wild-type (WT) and PKCε knock-out (KO) mice. We found that neutrophil influx increased more than twofold in PKCε KO mice following both a one-time challenge and 3 weeks of daily challenges with ODE as compared with WT mice. Lung pathology revealed increased bronchiolar and alveolar inflammation, lymphoid aggregates, and T cell influx in ODE-treated PKCε KO mice. Airway hyperresponsiveness to methacholine increased in PKCε KO + ODE to a greater magnitude than WT + ODE animals. There were no significant differences in cytokine/chemokine release elicited by ODE treatment between groups. However, ODE-induced nitric oxide (NO) production differed in that ODE exposure increased nitrate levels in WT mice but not in PKCε KO mice. Moreover, ODE failed to upregulate NO from ex vivo stimulated PKCε KO lung macrophages. Collectively, these studies demonstrate that PKCε-deficient mice were hypersensitive to organic dust exposure and suggest that PKCε is important in the normative lung inflammatory response to ODE. Dampening of ODE-induced NO may contribute to these enhanced inflammatory findings.

  16. The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling.

    Science.gov (United States)

    Staines, Katherine A; MacRae, Vicky E; Farquharson, Colin

    2012-09-01

    The small integrin-binding ligand N-linked glycoprotein (SIBLING) family consists of osteopontin, bone sialoprotein, dentin matrix protein 1, dentin sialophosphoprotein and matrix extracellular phosphoglycoprotein. These proteins share many structural characteristics and are primarily located in bone and dentin. Accumulating evidence has implicated the SIBLING proteins in matrix mineralisation. Therefore, in this review, we discuss the individual role that each of the SIBLING proteins has in this highly orchestrated process. In particular, we emphasise how the nature and extent of their proteolytic processing and post-translational modification affect their functional role. Finally, we describe the likely roles of the SIBLING proteins in clinical disorders of hypophosphataemia and their potential therapeutic use.

  17. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    Directory of Open Access Journals (Sweden)

    Stacia L. Phillips

    2016-01-01

    Full Text Available Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches.

  18. The challenges in and importance of analysing protein structure and physical stability in complex formulations

    DEFF Research Database (Denmark)

    Jorgensen, L.; Jensen, Minna Grønning; Roest, N.

    2013-01-01

    In this review several analytical challenges that may be encountered during protein formulation development of complex formulations are discussed through recent examples. These examples show how selected advanced biophysical methods can greatly increase our understanding of the system under...

  19. The importance of surfactant proteins-New aspects on macrophage phagocytosis.

    Science.gov (United States)

    Tschernig, Thomas; Veith, Nils T; Diler, Ebru; Bischoff, Markus; Meier, Carola; Schicht, Martin

    2016-11-01

    Surfactant and its components have multiple functions. The so called collectins are surfactant proteins which opsonize bacteria and improve pulmonary host defense via the phagocytosis and clearance of microorganisms and particles. In this special issue of the Annals of Anatomy a new surfactant protein, Surfactant Associated 3, is highlighted. As outlined in this mini review Surfactant Associated 3 is regarded as an enhancer of phagocytosis. In addition, the role played by SP-A is updated and open research questions raised.

  20. Importance of explicit salt ions for protein stability in molecular dynamics simulation.

    Science.gov (United States)

    Ibragimova, G T; Wade, R C

    1998-06-01

    The accurate and efficient treatment of electrostatic interactions is one of the challenging problems of molecular dynamics simulation. Truncation procedures such as switching or shifting energies or forces lead to artifacts and significantly reduced accuracy. The particle mesh Ewald (PME) method is one approach to overcome these problems by providing a computationally efficient means of calculating all long-range electrostatic interactions in a periodic simulation box by use of fast Fourier transformation techniques. For the application of the PME method to the simulation of a protein with a net charge in aqueous solution, counterions are added to neutralize the system. The usual procedure is to add charge-balancing counterions close to charged residues to neutralize the protein surface. In the present article, we show that for MD simulation of a small protein of marginal stability, the YAP-WW domain, explicit modeling of 0.2 M ionic strength (in addition to the charge-balancing counterions) is necessary to maintain a stable protein structure. Without explicit ions throughout the periodic simulation box, the charge-balancing counterions on the protein surface diffuse away from the protein, resulting in destruction of the beta-sheet secondary structure of the WW domain.

  1. Characterization of the secretome of suspension cultures of Medicago species reveals proteins important for defense and development.

    Science.gov (United States)

    Kusumawati, Lucia; Imin, Nijat; Djordjevic, Michael A

    2008-10-01

    Molecular events occurring in the plant apoplast contribute to important developmental and defense responses. To define the secretome of Medicago, we used suspension cultures to isolate and identify secreted proteins as a first step to determining their functions. Proteins in the extracellular medium of the suspension cultures were examined using SDS-PAGE, tandem mass spectrometry (MALDI-TOF/TOF) and bioinformatics tools. There were 39 proteins identified in the cultures derived from M. sativa, M. truncatula 2HA (an embryogenic line), and M. truncatula sickle (an ethylene-insensitive mutant). N-Terminal secretion signals were detected in 34 proteins and five other proteins were predicted to be secreted via a nonclassical (ER-independent) route. All samples possessed defense related proteins including pathogenesis related (PR) proteins. The glycoprotein, SIEP1L, was found only in M. sativa. Three secreted proteinases were identified in M. truncatula, including a serine carboxypeptidase detected only in 2HA. Some proteins were unique to a cell culture line. Quantitative real time RT-PCR was used to determine mRNA expression of selected genes corresponding to proteins found only in 2HA or sickle or in both. The results correlate well with the proteomic data. For instance, a GDSL-lipase gene known to be regulated by ethylene was found only in 2HA but not in the ethylene insensitive mutant. Similarly, the PR1a protein, expressed from a well recognized ethylene-regulated gene, was found in 2HA but not sickle. These experiments indicate that the suspension culture systems established here are useful to avoid contamination from cytoplasmic proteins and to identify secreted proteins in Medicago, and should have application in other plant systems.

  2. Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach.

    Science.gov (United States)

    Nissen, Asger; Andersen, Pia Haubro; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

    2017-04-01

    molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.

  3. Mapping of nuclear import signal and importin {alpha}3 binding regions of 52K protein of bovine adenovirus-3

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, Carolyn P.; Ayalew, Lisanework E. [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); Tikoo, Suresh K., E-mail: suresh.tik@usask.ca [Vaccine and Infectious Disease Organization-International Vaccine Center (VIDO-InterVac), University of Saskatchewan, Saskatoon, SK S7N 5E3 Canada (Canada); Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada (Canada); School of Public Health, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada (Canada)

    2012-10-10

    The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acid 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.

  4. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

    Science.gov (United States)

    Ray, Kausik

    2015-01-01

    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  5. The emerging importance of the SPX domain-containing proteins in phosphate homeostasis.

    Science.gov (United States)

    Secco, David; Wang, Chuang; Arpat, Bulak A; Wang, Zhiye; Poirier, Yves; Tyerman, Stephen D; Wu, Ping; Shou, Huixia; Whelan, James

    2012-03-01

    Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

  6. Mathematical modeling of the intracellular protein dynamics: the importance of active transport along microtubules.

    Science.gov (United States)

    Szymańska, Zuzanna; Parisot, Martin; Lachowicz, Mirosław

    2014-12-21

    In this paper we propose a mathematical model of protein and mRNA transport inside a cell. The spatio-temporal model takes into account the active transport along microtubules in the cytoplasm as well as diffusion and is able to reproduce the oscillatory changes in protein concentration observed in many experimental data. In the model the protein and the mRNA interact with each other that allows us to classify the model as a simple gene regulatory network. The proposed model is generic and may be adapted to specific signaling pathways. On the basis of numerical simulations, we formulate a new hypothesis that the oscillatory dynamics is allowed by the mRNA active transport along microtubules from the nucleus to distant locations.

  7. Sugar-coated proteins: the importance of degree of polymerisation of oligo-galacturonic acid on protein binding and aggregation.

    Science.gov (United States)

    Xu, Amy Y; Melton, Laurence D; Ryan, Timothy M; Mata, Jitendra P; Jameson, Geoffrey B; Rekas, Agata; Williams, Martin A K; McGillivray, Duncan J

    2017-04-05

    We have simplified the structural heterogeneity of protein-polysaccharide binding by investigating protein binding to oligosaccharides. The interactions between bovine beta-lactoglobulin A (βLgA) and oligo-galacturonic acids (OGAs) with various numbers of sugar residues have been investigated with a range of biophysical techniques. We show that the βLgA-OGA interaction is critically dependent on the length of the oligosaccharide. Isothermal titration calorimetry results suggest that a minimum length of 7 or 8 sugar residues is required in order to exhibit appreciable exothermic interactions with βLgA - shorter oligosaccharides show no enthalpic interactions at any concentration ratio. When titrating βLgA into OGAs with more than 7-8 sugar residues the sample solution also became turbid with increasing amounts of βLgA, indicating the formation of macroscopic assemblies. Circular dichroism, thioflavin T fluorescence and small angle X-ray/neutron scattering experiments revealed two structural regimes during the titration. When OGAs were in excess, βLgA formed discrete assemblies upon OGA binding, and no subsequent aggregation was observed. However, when βLgA was present in excess, multi-scale structures were formed and this eventually led to the separation of the solution into two liquid-phases.

  8. Electron tomogrophy of mitochondria after the arrest of protein import associated with Tom19 depletion

    NARCIS (Netherlands)

    Perkins, Guy A.; Renken, Christian W.; Klei, Ida J. van der; Ellisman, Mark H.; Neupert, Walter; Frey, Terrence G.

    In a mutant form of Neurospora crassa, in which sheltered RIP (repeat induced point mutation) was used to deplete Tom19, protein transport through the TOM/TIM pathway is arrested by the addition of p-fluorophenylalanine (FPA). Using intermediate-voltage electron tomography, we have generated

  9. Cross-linking proteins by laccase-catalyzed oxidation: importance relative to other modifications.

    Science.gov (United States)

    Steffensen, Charlotte L; Andersen, Mogens L; Degn, Peter E; Nielsen, Jacob H

    2008-12-24

    Laccase-catalyzed oxidation was able to induce intermolecular cross-links in beta-lactoglobulin, and ferulic acid-mediated laccase-catalyzed oxidation was able to induce intermolecular cross-links in alpha-casein, whereas transglutaminase cross-linked only alpha-casein. In addition, different patterns of laccase-induced oxidative modifications were detected, including dityrosine formation, formation of fluorescent tryptophan oxidation products, and carbonyls derived from histidine, tryptophan, and methionine. Laccase-catalyzed oxidation as well as transglutaminase induced only minor changes in surface tension of the proteins, and the changes could not be correlated to protein cross-linking. The presence of ferulic acid was found to influence the effect of laccase, allowing laccase to form irreducible intermolecular cross-links in beta-lactoglobulin and resulting in proteins exercising higher surface tensions due to cross-linking as well as other oxidative modifications. The outcome of using ferulic acid-mediated laccase-catalyzed oxidation to modify the functional properties of proteinaceous food components or other biosystems is expected to be highly dependent on the protein composition, resulting in different changes of the functional properties.

  10. Importance of extracellular proteins in maintaining structural integrity of aerobic granules.

    Science.gov (United States)

    Xiong, Yanghui; Liu, Yu

    2013-12-01

    Aerobic granules developed through self-immobilization of microorganisms are compact and structured microbial consortia embedded in a matrix of extracellular polymeric substances (EPS). This study investigated the contribution of extracellular proteins (PN) to maintaining the structural integrity of aerobic granule. It was found that hydrolysis of PN induced by Proteinase K led to significant disintegration of aerobic granules, whereas a substantial reduction of extracellular polysaccharides (PS) was also observed. It was proposed that hydrolysis of extracellular proteins present in the EPS matrix of aerobic granules led to collapse of the EPS matrix, and subsequent disintegration of aerobic granule. These suggested that extracellular proteins would be essential for maintaining structural stability of EPS matrix of aerobic granules. In addition, it was revealed that production of signaling molecules, such as autoinducer-2 (AI-2) and N-acyl homoserine lactones (AHLs) was also inhibited probably due to hydrolysis of quorum sensing receptor proteins by Proteinase K. This in turn provided an additional explanation for the observed Proteinase K-triggered dispersal of aerobic granules.

  11. Perceived Importance of Dietary Protein to Prevent Weight Gain: A National Survey among Midlife Women

    Science.gov (United States)

    Aldrich, Noel D.; Perry, Courtney; Thomas, William; Raatz, Susan K.; Reicks, Marla

    2013-01-01

    Objective: Evaluate reported use of the practice of "eating more protein" to prevent weight gain among midlife women. Design: Cross-sectional national survey. Participants: One thousand eight hundred twenty-four midlife women (40-60 y) from the 9 United States geographic regions, primarily married (71%), white (76%), and well educated; half were…

  12. Abdominal obesity and smoking are important determinants of C-reactive protein in renal transplant recipients

    NARCIS (Netherlands)

    van Ree, RM; de Vries, APJ; Oterdoom, LH; The, TH; Gansevoort, RT; van der Heide, JJH; van Son, WJ; Ploeg, RJ; de Jong, PE; Gans, ROB; Bakker, SJL

    2005-01-01

    Background. C-reactive protein (CRP) is a predictor of coronary heart disease, total mortality and chronic allograft nephropathy in renal transplant recipients. The determinants of CRP have been investigated in the general population, but not in renal transplant recipients. CRP might reflect metabol

  13. Identification and characterization of functionally important elements in the multidrug resistance protein 1 COOH-terminal region.

    Science.gov (United States)

    Westlake, Christopher J; Payen, Lea; Gao, Mian; Cole, Susan P C; Deeley, Roger G

    2004-12-17

    The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

  14. Differential proteomic and oxidative profiles unveil dysfunctional protein import to adipocyte mitochondria in obesity-associated aging and diabetes

    Directory of Open Access Journals (Sweden)

    María Gómez-Serrano

    2017-04-01

    Full Text Available Human age-related diseases, including obesity and type 2 diabetes (T2DM, have long been associated to mitochondrial dysfunction; however, the role for adipose tissue mitochondria in these conditions remains unknown. We have tackled the impact of aging and T2DM on adipocyte mitochondria from obese patients by quantitating not only the corresponding abundance changes of proteins, but also the redox alterations undergone by Cys residues thereof. For that, we have resorted to a high-throughput proteomic approach based on isobaric labeling, liquid chromatography and mass spectrometry. The alterations undergone by the mitochondrial proteome revealed aging- and T2DM-specific hallmarks. Thus, while a global decrease of oxidative phosphorylation (OXPHOS subunits was found in aging, the diabetic patients exhibited a reduction of specific OXPHOS complexes as well as an up-regulation of the anti-oxidant response. Under both conditions, evidence is shown for the first time of a link between increased thiol protein oxidation and decreased protein abundance in adipose tissue mitochondria. This association was stronger in T2DM, where OXPHOS mitochondrial- vs. nuclear-encoded protein modules were found altered, suggesting impaired mitochondrial protein translocation and complex assembly. The marked down-regulation of OXPHOS oxidized proteins and the alteration of oxidized Cys residues related to protein import through the redox-active MIA (Mitochondrial Intermembrane space Assembly pathway support that defects in protein translocation to the mitochondria may be an important underlying mechanism for mitochondrial dysfunction in T2DM and physiological aging. The present draft of redox targets together with the quantification of protein and oxidative changes may help to better understand the role of oxidative stress in both a physiological process like aging and a pathological condition like T2DM.

  15. Topological analysis of protein co-abundance networks identifies novel host targets important for HCV infection and pathogenesis

    Directory of Open Access Journals (Sweden)

    McDermott Jason E

    2012-04-01

    Full Text Available Abstract Background High-throughput methods for obtaining global measurements of transcript and protein levels in biological samples has provided a large amount of data for identification of 'target' genes and proteins of interest. These targets may be mediators of functional processes involved in disease and therefore represent key points of control for viruses and bacterial pathogens. Genes and proteins that are the most highly differentially regulated are generally considered to be the most important. We present topological analysis of co-abundance networks as an alternative to differential regulation for confident identification of target proteins from two related global proteomics studies of hepatitis C virus (HCV infection. Results We analyzed global proteomics data sets from a cell culture study of HCV infection and from a clinical study of liver biopsies from HCV-positive patients. Using lists of proteins known to be interaction partners with pathogen proteins we show that the most differentially regulated proteins in both data sets are indeed enriched in pathogen interactors. We then use these data sets to generate co-abundance networks that link proteins based on similar abundance patterns in time or across patients. Analysis of these co-abundance networks using a variety of network topology measures revealed that both degree and betweenness could be used to identify pathogen interactors with better accuracy than differential regulation alone, though betweenness provides the best discrimination. We found that though overall differential regulation was not correlated between the cell culture and liver biopsy data, network topology was conserved to an extent. Finally, we identified a set of proteins that has high betweenness topology in both networks including a protein that we have recently shown to be essential for HCV replication in cell culture. Conclusions The results presented show that the network topology of protein co

  16. A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import.

    Science.gov (United States)

    Ramesh, Ajay; Peleh, Valentina; Martinez-Caballero, Sonia; Wollweber, Florian; Sommer, Frederik; van der Laan, Martin; Schroda, Michael; Alexander, R Todd; Campo, María Luisa; Herrmann, Johannes M

    2016-08-15

    Tim17 is a central, membrane-embedded subunit of the mitochondrial protein import machinery. In this study, we show that Tim17 contains a pair of highly conserved cysteine residues that form a structural disulfide bond exposed to the intermembrane space (IMS). This disulfide bond is critical for efficient protein translocation through the TIM23 complex and for dynamic gating of its preprotein-conducting channel. The disulfide bond in Tim17 is formed during insertion of the protein into the inner membrane. Whereas the import of Tim17 depends on the binding to the IMS protein Mia40, the oxidoreductase activity of Mia40 is surprisingly dispensable for Tim17 oxidation. Our observations suggest that Tim17 can be directly oxidized by the sulfhydryl oxidase Erv1. Thus, import and oxidation of Tim17 are mediated by the mitochondrial disulfide relay, though the mechanism by which the disulfide bond in Tim17 is formed differs considerably from that of soluble IMS proteins.

  17. Realizing the recognition features of model antipsychotic compounds by important protein: Photochemical and computational studies.

    Science.gov (United States)

    Ding, Fei; Peng, Wei; Chao, Ming-Wei; Peng, Yu-Kui

    2015-07-01

    Phenothiazine and its derivatives are the most effective antipsychotic drugs. They have been used in the treatment of serious mental and emotional symptoms including bipolar disorder, organic psychoses, psychotic depression and schizophrenia. However, these drugs cause serious side effects such as akathisia, hyperprolactinaemia and neuroleptic malignant syndrome. In this work we investigated the molecular recognition of two typical phenothiazine compounds, phenosafranin and safranin O by the most pivotal heme protein hemoglobin using steady state and time-resolved fluorescence, extrinsic 8-anilino-1-naphthalenesulfonic acid (ANS) fluorescent probe, circular dichroism (CD) along with computational modeling. Results show phenothiazines complex with protein via formation of adducts at 298 K with moderate strengths of 3.555×10(4) M(-1) and 2.567×10(4) M(-1) for the hemoglobin-phenosafranin and hemoglobin-safranin O, respectively. We also found phenothiazines were effectors at the protein allosteric site, which affects the allosteric equilibrium. Further, time-resolved fluorescence and hydrophobic ANS experiments showed the static mechanism is dominated for the shrinkage in the fluorescence intensity of β-37 Trp residue at the α1β2 interface. The stoichiometric proportion of the protein-drug adduct is 1:1, as derived from Job's plot. Several crucial noncovalent bonds, including hydrogen bonds, π-π stacking and hydrophobic interactions played a major role in stabilizing the noncovalent conjugates. Based on three-dimensional fluorescence, we concluded that the conformation of hemoglobin is partially destabilized after recognition with phenothiazines. These alterations were confirmed by far-UV CD spectra that showed the α-helix of protein decreased from 78.3% in free hemoglobin to 62.8% and 64.8% in hemoglobin-phenosafranin and hemoglobin-safranin O, respectively. Computer-aided molecular docking was consistent, indicating that both phenothiazines are situated

  18. DNA-protein interaction at erythroid important regulatory elements of MEL cells by in vivo footprinting

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using ligation-mediated PCR method to study the status of DNA-protein interaction at hypersensitive site 2 of locus control Region and β maj promoter of MEL cell line before and after induction, MEL cell has been cultured and induced to differentiation by Hemin and DMSO, then the live cells have been treated with dimethyl sulfate. Ligation mediated PCR has been carried out following the chemical cleavage. The results demonstrate that before and after induction, the status of DNA-protein interaction at both hypersensitive site 2 and β maj promoter change significantly, indicating that distal regulatory elements (locus control region, hypersensitive sites) as well as proximal regulatory elements (promoter, enhancer) of β -globin gene cluster participate in the regulation of developmental specificity.

  19. Proteomics reveals the importance of the dynamic redistribution of the subcellular location of proteins in breast cancer cells.

    Science.gov (United States)

    Pinto, Gabriella; Alhaiek, Abdulrab Ahmed M; Godovac-Zimmermann, Jasminka

    2015-02-01

    At the molecular level, living cells are enormously complicated complex adaptive systems in which intertwined genomic, transcriptomic, proteomic and metabolic networks all play a crucial role. At the same time, cells are spatially heterogeneous systems in which subcellular compartmentalization of different functions is ubiquitous and requires efficient cross-compartmental communication. Dynamic redistribution of multitudinous proteins to different subcellular locations in response to cellular functional state is increasingly recognized as a crucial characteristic of cellular function that seems to be at least as important as overall changes in protein abundance. Characterization of the subcellular spatial dynamics of protein distribution is a major challenge for proteomics and recent results with MCF7 breast cancer cells suggest that this may be of particular importance for cancer cells.

  20. Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution.

    Science.gov (United States)

    Jones, Justin A; Harris, Thomas I; Oliveira, Paula F; Bell, Brianne E; Alhabib, Abdulrahman; Lewis, Randolph V

    2016-11-23

    The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

  1. Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution

    Directory of Open Access Journals (Sweden)

    Justin A. Jones

    2016-11-01

    Full Text Available The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate as well as chimeric sequences (FlAS in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution.

  2. Importance of Heat and Pressure for Solubilization of Recombinant Spider Silk Proteins in Aqueous Solution

    Science.gov (United States)

    Jones, Justin A.; Harris, Thomas I.; Oliveira, Paula F.; Bell, Brianne E.; Alhabib, Abdulrahman; Lewis, Randolph V.

    2016-01-01

    The production of recombinant spider silk proteins continues to be a key area of interest for a number of research groups. Several key obstacles exist in their production as well as in their formulation into useable products. The original reported method to solubilize recombinant spider silk proteins (rSSp) in an aqueous solution involved using microwaves to quickly generate heat and pressure inside of a sealed vial containing rSSp and water. Fibers produced from this system are remarkable in their mechanical ability and demonstrate the ability to be stretched and recover 100 times. The microwave method dissolves the rSSPs with dissolution time increasing with higher molecular weight constructs, increasing concentration of rSSPs, protein type, and salt concentration. It has proven successful in solvating a number of different rSSPs including native-like sequences (MaSp1, MaSp2, piriform, and aggregate) as well as chimeric sequences (FlAS) in varied concentrations that have been spun into fibers and formed into films, foams, sponges, gels, coatings, macro and micro spheres and adhesives. The system is effective but inherently unpredictable and difficult to control. Provided that the materials that can be generated from this method of dissolution are impressive, an alternative means of applying heat and pressure that is controllable and predictable has been developed. Results indicate that there are combinations of heat and pressure (135 °C and 140 psi) that result in maximal dissolution without degrading the recombinant MaSp2 protein tested, and that heat and pressure are the key elements to the method of dissolution. PMID:27886066

  3. Cubilin is an albumin binding protein important for renal tubular albumin reabsorption

    OpenAIRE

    Birn, H.; J. C. Fyfe; Jacobsen, C.; Mounier, F.; Verroust, P. J.; Orskov, H; Willnow, T.E.; Moestrup, S K; Christensen, E I

    2000-01-01

    Using affinity chromatography and surface plasmon resonance analysis, we have identified cubilin, a 460-kDa receptor heavily expressed in kidney proximal tubule epithelial cells, as an albumin binding protein. Dogs with a functional defect in cubilin excrete large amounts of albumin in combination with virtually abolished proximal tubule reabsorption, showing the critical role for cubilin in the uptake of albumin by the proximal tubule. Also, by immunoblotting and immunocytochemistry we show ...

  4. Structural and functional importance of outer membrane proteins in Vibrio cholerae flagellum.

    Science.gov (United States)

    Bari, Wasimul; Lee, Kang-Mu; Yoon, Sang Sun

    2012-08-01

    Vibrio cholerae has a sheath-covered monotrichous flagellum that is known to contribute to virulence. Although the structural organization of the V. cholerae flagellum has been extensively studied, the involvement of outer membrane proteins as integral components in the flagellum still remains elusive. Here we show that flagella produced by V. cholerae O1 El Tor strain C6706 were two times thicker than those from two other Gram-negative bacteria. A C6706 mutant strain (SSY11) devoid of two outer membrane proteins (OMPs), OmpU and OmpT, produced thinner flagella. SSY11 showed significant defects in the flagella-mediated motility as compared to its parental strain. Moreover, increased shedding of the flagella-associated proteins was observed in the culture supernatant of SSY11. This finding was also supported by the observation that culture supernatants of the SSY11 strain induced the production of a significantly higher level of IL-8 in human colon carcinoma HT29 and alveolar epithelial A549 cells than those of the wild-type C6706 strain. These results further suggest a definite role of these two OMPs in providing the structural integrity of the V. cholerae flagellum as part of the surrounding sheath.

  5. Importance of hydrophobic cluster formation through long-range contacts in the folding transition state of two-state proteins.

    Science.gov (United States)

    Selvaraj, S; Gromiha, M Michael

    2004-06-01

    Understanding the folding pathways of proteins is a challenging task. The Phi value approach provides a detailed understanding of transition-state structures of folded proteins. In this work, we have computed the hydrophobicity associated with each residue in the folded state of 16 two-state proteins and compared the Phi values of each mutant residue. We found that most of the residues with high Phi value coincide with local maximum in surrounding hydrophobicity, or have nearby residues that show such maximum in hydrophobicity, indicating the importance of hydrophobic interactions in the transition state. We have tested our approach to different structural classes of proteins, such as alpha-helical, SH3 domains of all-beta proteins, beta-sandwich, and alpha/beta proteins, and we observed a good agreement with experimental results. Further, we have proposed a hydrophobic contact network pattern to relate the Phi values with long-range contacts, which will be helpful to understand the transition-state structures of folded proteins. The present approach could be used to identify potential hydrophobic clusters that may form through long-range contacts during the transition state.

  6. Involvement of the catalytically important Asp54 residue of Mycobacterium smegmatis DevR in protein-protein interactions between DevR and DevS.

    Science.gov (United States)

    Lee, Ha-Na; Lee, Na-On; Ko, In-Jeong; Kim, Si Wouk; Kang, Beom Sik; Oh, Jeong-Il

    2013-06-01

    The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein-protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein-protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein-protein interactions between DevR and DevS. © 2013 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  7. Distance between RBS and AUG plays an important role in overexpression of recombinant proteins.

    Science.gov (United States)

    Berwal, Sunil K; Sreejith, R K; Pal, Jayanta K

    2010-10-15

    The spacing between ribosome binding site (RBS) and AUG is crucial for efficient overexpression of genes when cloned in prokaryotic expression vectors. We undertook a brief study on the overexpression of genes cloned in Escherichia coli expression vectors, wherein the spacing between the RBS and the start codon was varied. SDS-PAGE and Western blot analysis indicated a high level of protein expression only in constructs where the spacing between RBS and AUG was approximately 40 nucleotides or more, despite the synthesis of the transcripts in the representative cases investigated.

  8. Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sung; Saunders, Adam M.; Hamaoka, Brent Y.; Beachy, Philip A.; Leahy, Daniel J. (Stanford-MED); (JHU)

    2011-09-20

    Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-{angstrom} crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, {alpha}-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

  9. Dominant negative RPW8.2 fusion proteins reveal the importance of haustorium-oriented protein trafficking for resistance against powdery mildew in Arabidopsis.

    Science.gov (United States)

    Zhang, Qiong; Berkey, Robert; Pan, Zhiyong; Wang, Wenming; Zhang, Yi; Ma, Xianfeng; King, Harlan; Xiao, Shunyuan

    2015-01-01

    Powdery mildew fungi form feeding structures called haustoria inside epidermal cells of host plants to extract photosynthates for their epiphytic growth and reproduction. The haustorium is encased by an interfacial membrane termed the extrahaustorial membrane (EHM). The atypical resistance protein RPW8.2 from Arabidopsis is specifically targeted to the EHM where RPW8.2 activates haustorium-targeted (thus broad-spectrum) resistance against powdery mildew fungi. EHM-specific localization of RPW8.2 suggests the existence of an EHM-oriented protein/membrane trafficking pathway during EHM biogenesis. However, the importance of this specific trafficking pathway for host defense has not been evaluated via a genetic approach without affecting other trafficking pathways. Here, we report that expression of EHM-oriented, nonfunctional RPW8.2 chimeric proteins exerts dominant negative effect over functional RPW8.2 and potentially over other EHM-localized defense proteins, thereby compromising both RPW8.2-mediated and basal resistance to powdery mildew. Thus, our results highlight the importance of the EHM-oriented protein/membrane trafficking pathway for host resistance against haustorium-forming pathogens such as powdery mildew fungi.

  10. Broad Distribution of Energetically Important Contacts across an Extended Protein Interface

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Lisa M.; Horne, W. Seth; Gellman, Samuel H. (UW)

    2012-02-27

    Infection of cells by HIV depends upon profound structural rearrangements within the trimeric viral protein gp41. Critical to this process is the formation of a six-helix bundle in which a set of three N-terminal heptad repeat (NHR) helices assemble to form a core displaying long grooves that provide docking sites for three C-terminal heptad repeat (CHR) helices. We report experiments designed to discriminate between two alternative hypotheses regarding the source of affinity between individual CHR helices and the complementary groove: (1) affinity is dominated by interactions of a small cluster of side chains at one end of the CHR helix; or (2) affinity depends upon interactions distributed across the long CHR helix. We have employed two complementary experimental designs, and results from both favor the latter hypothesis.

  11. Proteins Play Important Role in Intercellular Adhesion Affecting on Fruit Textural Quality

    DEFF Research Database (Denmark)

    Bahadur Adhikari, Khem; Shomer, Ilan

    2012-01-01

    strengthening was exempli ed in Medjoul date (Phoenix dactylifera L.) fruit, as a model. Fruit mesocarp sensitively responded to culture environment which was assayed in vitro at pH 3.5( pKa) in presence of organic acid molecules. The max penetration force, as a measure of ICA strength, of p......H 3.5 (incubated mesocarp (~10.5 N) was signi cantly higher than that of pH 6.5 (> pKa) incubated fruits (~2 N). The protein bands at ~29 kDa, ~75 kDa, ~32 kDa and 87 kDa were exclusively or prominently found in ICA strengthened fruits (pH 3.5

  12. Multidrug resistance proteins (MRPs, ABCCs): importance for pathophysiology and drug therapy.

    Science.gov (United States)

    Keppler, Dietrich

    2011-01-01

    The nine multidrug resistance proteins (MRPs) represent the major part of the 12 members of the MRP/CFTR subfamily belonging to the 48 human ATP-binding cassette (ABC) transporters. Cloning, functional characterization, and cellular localization of most MRP subfamily members have identified them as ATP-dependent efflux pumps with a broad substrate specificity for the transport of endogenous and xenobiotic anionic substances localized in cellular plasma membranes. Prototypic substrates include glutathione conjugates such as leukotriene C(4) for MRP1, MRP2, and MRP4, bilirubin glucuronosides for MRP2 and MRP3, and cyclic AMP and cyclic GMP for MRP4, MRP5, and MRP8. Reduced glutathione (GSH), present in living cells at millimolar concentrations, modifies the substrate specificities of several MRPs, as exemplified by the cotransport of vincristine with GSH by MRP1, or by the cotransport of GSH with bile acids or of GSH with leukotriene B(4) by MRP4.The role of MRP subfamily members in pathophysiology may be illustrated by the MRP-mediated release of proinflammatory and immunomodulatory mediators such as leukotrienes and prostanoids. Pathophysiological consequences of many genetic variants leading to a lack of functional MRP protein in the plasma membrane are observed in the hereditary MRP2 deficiency associated with conjugated hyperbilirubinemia in Dubin-Johnson syndrome, in pseudoxanthoma elasticum due to mutations in the MRP6 (ABCC6) gene, or in the type of human earwax and osmidrosis determined by single nucleotide polymorphisms in the MRP8 (ABCC8) gene. The hepatobiliary and renal elimination of many drugs and their metabolites is mediated by MRP2 in the hepatocyte canalicular membrane and by MRP4 as well as MRP2 in the luminal membrane of kidney proximal tubules. Therefore, inhibition of these efflux pumps affects pharmacokinetics, unless compensated by other ATP-dependent efflux pumps with overlapping substrate specificities.

  13. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    Science.gov (United States)

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  14. Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

    Directory of Open Access Journals (Sweden)

    Lynn G.L. Richardson

    2014-06-01

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  15. Identification of Important Process Variables for Fiber Spinning of Protein Nanotubes Generated from Waste Materials

    Science.gov (United States)

    2012-01-11

    fibers, identification of important spin dope processing and fiber spinning parameters for further opti Fibers, biopolymer , sustainable materials...engineering without disrupting their integrity 19-23, 27, 30. Due to these properties, TMV and M13 are particularly interesting scaffolds for developing...thinning along the fiber were recorded. Under polarizing light, birefringence, which is used to evaluate molecular alignment of the biopolymer chains

  16. The importance and the differences of bone morphogenetic proteins for osteoporotic hip fractures.

    Science.gov (United States)

    Dincel, V Ercan; Sepici-Dincel, Aylin

    2014-06-01

    Bone morphogenetic proteins (BMPs), major contributors to tissue repair, have become one of the most exciting fields in rheumatic and orthopaedic research. In our study we aimed to evaluate the relationship between osteoporotic hip fractures and the serum levels of BMPs to reveal their potential roles in the diagnosis of patients. The study group included 62 patients with osteoporotic hip fracture (Group 1; intertrochanteric fracture, Group 2; collum femoris fracture) and the control group. All fractures were due to low energy trauma, simple falls. For all subjects BMD measurements were in agreement for osteoporosis and no significant differences were observed between the two fracture groups. Biochemical markers; BMP-4 and BMP-7 (pg/mL) were determined by commercial Elisa kits from the serum samples. The mean and standard error values of serum samples for BMP-4 and BMP-7 in Group 1 (100.70 +/- 10.03, 74.41 +/- 6.31 respectively) and in Group 2 (112.34 +/- 11.52, 81.91 +/- 10.14 respectively) were not statistically different however for both groups only BMP-7 values increased statistically when compared to the control group. BMP-7 measurements may not only serve as potential biochemical markers for determining disease severity but also the increased levels, an osteogenic factor and bone stimulating agent in vivo, after trauma elevated levels are adaptive or protective and therefore may reduce the severity of the fracture.

  17. Effects of forest fertilization on nitrate and crude protein content in some important reindeer forage species

    Directory of Open Access Journals (Sweden)

    Gustaf Åhman

    1984-05-01

    Full Text Available When forests are fertilized with ammonia nitrate it is possible that grazing reindeer ingest ammonia nitrate by eating grains of fertilizer from the ground or by drinking contaminated water. They can also get nitrate through plants that have absorbed and disposed nitrate. This latter factor is studied in this report. In addition the effect of fertilization on crude protein content in forage plants is investigated. Fertilizing trials were done within two different areas. One was a dry scotch pine forest and the other a humid scotch pine forest. Both were situated 10 to 15 km north west of Lycksele (northern Sweden. Three different rations (75, 150 and 250 kg N/ha of ammonianitrate and one (150 kg N/ha of urea was used. Fertilization was done at two occations, in June and in July. To investigate the effect of fertilization on nitrate and crude protein content in reindeer forage plants, samples were taken of reindeer lichens (Cladina spp., heather {Calluna vulgaris, crowberry (Empetrum spp., cowberry (Vaccinium vitis ideae, blueberry (Vaccinium myrtillus and hair-grass (Deschampsia flexuosa at different times after fertilization. In this trial we could not find any higher degree of contamination of nitrate in lichens. The highest value was 0.013% nitrate-N in dry matter (table 1. Nitrate accumulation was low in shrubs and grass (table 2. The highest value (0.05% was found in heather. The concentrations were definitly below the level that could be considered as injurious to the reindeer. The effect of fertilization on crude protein content in reindeer forage plants was obvious. It was most evident in hair-grass. Four weeks after fertilization with 150 kg N/ha, crude protein content was more than doubled and reached 20% in dry matter (figure 1 and 2. In withered hair-grass in the autumn the effect was very small. One year after fertilization a small rise in crude protein was registered in both grass and shrubs (table 3. Some effect still remained

  18. The Croonian Lecture 1998. Identification of a protein kinase cascade of major importance in insulin signal transduction.

    Science.gov (United States)

    Cohen, P

    1999-02-28

    Diabetes affects 3% of the European population and 140 million people worldwide, and is largely a disease of insulin resistance in which the tissues fail to respond to this hormone. This emphasizes the importance of understanding how insulin signals to the cell's interior. We have recently dissected a protein kinase cascade that is triggered by the formation of the insulin 'second messenger' phosphatidylinositide (3,4,5) trisphosphate (PtdIns (3,4,5)P3) and which appears to mediate many of the metabolic actions of this hormone. The first enzyme in the cascade is termed 3-phosphoinositide-dependent protein kinase-1 (PDK1), because it only activates protein kinase B (PKB), the next enzyme in the pathway, in the presence of PtdIns (3,4,5)P3. PKB then inactivates glycogen synthase kinase-3 (GSK3). PDK1, PKB and GSK3 regulate many physiological events by phosphorylating a variety of intracellular proteins. In addition, PKB plays an important role in mediating protection against apoptosis by survival factors, such as insulin-like growth factor-1.

  19. Comparative binding mechanism of lupeol compounds with plasma proteins and its pharmacological importance.

    Science.gov (United States)

    Kallubai, Monika; Rachamallu, Aparna; Yeggoni, Daniel Pushparaju; Subramanyam, Rajagopal

    2015-04-01

    Lupeol, a triterpene, possesses beneficial effects like anti-inflammatory and anti-cancer properties. Binding of lupeol and its derivative (phytochemicals) to plasma proteins such as human serum albumin (HSA) and α-1-acid glycoprotein (AGP) is a major determinant in the disposition of drugs. Cytotoxic studies with mouse macrophages (RAW 246.7) and HeLa cell lines revealed anti-inflammatory and anti-cancer properties for both lupeol and lupeol derivative. Both molecules reduced the expression of pro-inflammatory cytokines in LPS induced macrophages. Further, apoptosis was observed in HeLa cell lines when they were incubated with these molecules for 24 h. The fluorescence quenching of HSA was observed upon titration with different concentrations of lupeol and lupeol derivative; their binding constants were found to be 3 ± 0.01 × 10(4) M(-1) and 6.2 ± 0.02 × 10(4) M(-1), with binding free energies of -6.59 kcal M(-1) and -7.2 kcal M(-1). With AGP, however, the lupeol and lupeol derivative showed binding constants of 0.9 ± 0.02 × 10(3) M(-1) and 2.7 ± 0.01 × 10(3) M(-1), with free energies of -4.6 kcal M(-1) and -5.1 kcal M(-1) respectively. Molecular displacement studies based on competition with site I-binding phenylbutazone (which binds site I of HSA) and ibuprofen (which binds site II) suggest that lupeol binds site II and the lupeol derivative site I. Molecular docking studies also confirmed that lupeol binds to the IIIA and the lupeol derivative to the IIA domain of HSA. Secondary structure changes were observed upon formation of HSA-lupeol/lupeol derivative complexes by circular dichroism spectroscopy. Molecular dynamics simulations support greater stability of HSA-lupeol and HSA-lupeol derivative complexes compared to that of HSA alone.

  20. RNA- binding protein Stau2 is important for spindle integrity and meiosis progression in mouse oocytes.

    Science.gov (United States)

    Cao, Yan; Du, Juan; Chen, Dandan; Wang, Qian; Zhang, Nana; Liu, Xiaoyun; Liu, Xiaoyu; Weng, Jing; Liang, Yuanjing; Ma, Wei

    2016-10-01

    Staufen2 (Stau2) is a double-stranded RNA-binding protein involved in cell fate decision by regulating mRNA transport, mRNA stability, translation, and ribonucleoprotein assembly. Little is known about Stau2 expression and function in mammalian oocytes during meiosis. Herein we report the sub-cellular distribution and function of Stau2 in mouse oocyte meiosis. Western blot analysis revealed high and stable expression of Stau2 in oocytes from germinal vesicle (GV) to metaphase II (MII). Immunofluorescence showed that Stau2 was evenly distributed in oocytes at GV stage, and assembled as filaments after germinal vesicle breakdown (GVBD), particularly, colocalized with spindle at MI and MII. Stau2 was disassembled when microtubules were disrupted with nocodazole, on the other hand, when MTs were stabilized with taxol, Stau2 was not colocalized with the stabilized microtubules, but aggregated around the chromosomes array, indicating Stau2 assembly and colocalization with microtubules require both microtubule integrity and its normal dynamics. During interphase and mitosis of BHK and MEF cells, Stau2 was not distributed on microtubules, but colocalized with cis-Golgi marker GM130, implying its association with Golgi complex but not the spindle in fully differentiated somatic cells. Specific morpholino oligo-mediated Stau2 knockdown disrupted spindle formation, chromosome alignment and microtubule-kinetochore attachment in oocytes. The majority oocytes were arrested at MI stage, with bright MAD1 at kinetochores, indicating activation of spindle assembly checkpoint (SAC). Some oocytes were stranded at telophase I (TI), implying suppressed first polar body extrution. Together these data demonstrate that Stau2 is required for spindle formation and timely meiotic progression in mouse oocytes.

  1. Intracellular localization of GBF proteins and blue light-induced import of GBF2 fusion proteins into the nucleus of cultured Arabidopsis and soybean cells.

    Science.gov (United States)

    Terzaghi, W B; Bertekap, R L; Cashmore, A R

    1997-05-01

    The G-box is an important regulatory element found in the promoters of many different genes. Four members of an Arabidopsis gene family encoding basic leucine zipper proteins (GBFs) which bind the G-box have previously been cloned. To study GBFs, a polyclonal antibody was raised against GBF1 expressed in bacteria. This antibody also recognized GBF2 and GBF3. Immunoblot analysis of nuclear and cytoplasmic fractions from Arabidopsis and soybean (SB-M) cell cultures indicated that over 90% of proteins detected with anti-GBF1 were cytoplasmic. Electrophoretic mobility shift assays indicated that over 90% of G-box binding activity was cytoplasmic. DNA affinity chromatography demonstrated that each protein detected with anti-GBF1 specifically bound the G-box. To study individual GBFs, DNA constructs fusing GBF1, GBF2 and GBF4 to GUS were made and assayed by transient expression in SB-M protoplasts. Of GUS:GBF1 proteins, 50-62% were localized in the cytoplasm under all conditions tested, while 97% of GUS:GBF4 was localized in the nucleus. By contrast, whereas about 50% of GUS:GBF2 was found in the cytoplasm of dark-grown cells, over 80% of this protein was found in the nucleus in cells cultured under blue light. Deletion analysis of GBF1 identified a region between amino acids 112 and 164 apparently required for cytoplasmic retention. These results suggest the intriguing possibility that limitation of nuclear access may be an important control on GBF activity. In particular, GBF2 is apparently specifically imported into the nucleus in response to light.

  2. Butane-1,2,3,4-tetraol-based amphiphilic stereoisomers for membrane protein study: importance of chirality in the linker region

    DEFF Research Database (Denmark)

    Das, Manabendra; Du, Yang; Mortensen, Jonas S.

    2017-01-01

    of the targeted membrane proteins depending on the chirality of the linker region. These findings indicate an important role for detergent stereochemistry in membrane protein stabilization. In addition, we generally observed enhanced detergent efficacy with increasing alkyl chain length, reinforcing...

  3. In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

    Science.gov (United States)

    Nadipuram, Santhosh M.; Kim, Elliot W.; Vashisht, Ajay A.; Lin, Andrew H.; Bell, Hannah N.; Coppens, Isabelle; Wohlschlegel, James A.

    2016-01-01

    ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. PMID:27486190

  4. Targeted axonal import (TAxI) peptide delivers functional proteins into spinal cord motor neurons after peripheral administration.

    Science.gov (United States)

    Sellers, Drew L; Bergen, Jamie M; Johnson, Russell N; Back, Heidi; Ravits, John M; Horner, Philip J; Pun, Suzie H

    2016-03-01

    A significant unmet need in treating neurodegenerative disease is effective methods for delivery of biologic drugs, such as peptides, proteins, or nucleic acids into the central nervous system (CNS). To date, there are no operative technologies for the delivery of macromolecular drugs to the CNS via peripheral administration routes. Using an in vivo phage-display screen, we identify a peptide, targeted axonal import (TAxI), that enriched recombinant bacteriophage accumulation and delivered protein cargo into spinal cord motor neurons after intramuscular injection. In animals with transected peripheral nerve roots, TAxI delivery into motor neurons after peripheral administration was inhibited, suggesting a retrograde axonal transport mechanism for delivery into the CNS. Notably, TAxI-Cre recombinase fusion proteins induced selective recombination and tdTomato-reporter expression in motor neurons after intramuscular injections. Furthermore, TAxI peptide was shown to label motor neurons in the human tissue. The demonstration of a nonviral-mediated delivery of functional proteins into the spinal cord establishes the clinical potential of this technology for minimally invasive administration of CNS-targeted therapeutics.

  5. The Molecular Chaperone HSP70 Binds to and Stabilizes NOD2, an Important Protein Involved in Crohn Disease*

    Science.gov (United States)

    Mohanan, Vishnu; Grimes, Catherine Leimkuhler

    2014-01-01

    Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand. PMID:24790089

  6. The molecular chaperone HSP70 binds to and stabilizes NOD2, an important protein involved in Crohn disease.

    Science.gov (United States)

    Mohanan, Vishnu; Grimes, Catherine Leimkuhler

    2014-07-04

    Microbes are detected by the pathogen-associated molecular patterns through specific host pattern recognition receptors. Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) is an intracellular pattern recognition receptor that recognizes fragments of the bacterial cell wall. NOD2 is important to human biology; when it is mutated it loses the ability to respond properly to bacterial cell wall fragments. To determine the mechanisms of misactivation in the NOD2 Crohn mutants, we developed a cell-based system to screen for protein-protein interactors of NOD2. We identified heat shock protein 70 (HSP70) as a protein interactor of both wild type and Crohn mutant NOD2. HSP70 has previously been linked to inflammation, especially in the regulation of anti-inflammatory molecules. Induced HSP70 expression in cells increased the response of NOD2 to bacterial cell wall fragments. In addition, an HSP70 inhibitor, KNK437, was capable of decreasing NOD2-mediated NF-κB activation in response to bacterial cell wall stimulation. We found HSP70 to regulate the half-life of NOD2, as increasing the HSP70 level in cells increased the half-life of NOD2, and down-regulating HSP70 decreased the half-life of NOD2. The expression levels of the Crohn-associated NOD2 variants were less compared with wild type. The overexpression of HSP70 significantly increased NOD2 levels as well as the signaling capacity of the mutants. Thus, our study shows that restoring the stability of the NOD2 Crohn mutants is sufficient for rescuing the ability of these mutations to signal the presence of a bacterial cell wall ligand.

  7. Protein kinase C {alpha} activity is important for contraction-induced FXYD1 phosphorylation in skeletal muscle

    DEFF Research Database (Denmark)

    Thomassen, Martin; Rose, Adam John; Jensen, Thomas Elbenhardt

    2011-01-01

    Exercise induced phosphorylation of FXYD1 is a potential important regulator of Na(+), K(+) pump activity. It was investigated if skeletal muscle contractions induce phosphorylation of FXYD1 and if Protein Kinase C a (PKCa) activity is a prerequisite for this possible mechanism. In part 1, human...... muscle biopsies were obtained at rest, after 30 s of high intensity exercise (166±31% of VO(2max)) and after a subsequent 20 min of moderate intensity exercise (79±8% of VO(2max)). In general, FXYD1 phosphorylation was increased compared to rest both after 30 s (P...

  8. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai; Nicora, Carrie D.; Aldrich, Joshua T.; Frey, Dario; Kolinko, Sebastian; Robinson, Errol W.; Jacobs, Jon M.; Adams, Paul D.; Northen, Trent R.; Simmons, Blake A.; Singer, Steven W.

    2016-08-23

    ABSTRACT

    Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacteriumThermobispora bisporathat were highly abundant in the most active consortium. Among the cellulases fromT. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite ofT. bisporahydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.

    IMPORTANCECellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose

  9. TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

    Science.gov (United States)

    Parvin, Nargis; Carrie, Chris; Pabst, Isabelle; Läßer, Antonia; Laha, Debabrata; Paul, Melanie V; Geigenberger, Peter; Heermann, Ralf; Jung, Kirsten; Vothknecht, Ute C; Chigri, Fatima

    2017-04-03

    The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  10. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18.

    Science.gov (United States)

    Smirnov, Alexandre; Entelis, Nina; Martin, Robert P; Tarassov, Ivan

    2011-06-15

    5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

  11. Adhesive ability means inhibition activities for lactobacillus against pathogens and S-layer protein plays an important role in adhesion.

    Science.gov (United States)

    Zhang, Wenming; Wang, Haifeng; Liu, Jianxin; Zhao, Yunhao; Gao, Kan; Zhang, Juan

    2013-08-01

    Eighty-five strains of lactobacillus were isolated from the pig intestine and identified by sequencing analysis based on 16S rRNA gene, from which five lactobacillus strains with high adhesive ability were selected. The inhibition ability of the five lactobacillus strains with or without S-layer proteins against adherence of Escherichia coli K88 and Salmonella enteritidis 50335 to Caco-2 was evaluated in vitro with Lactobacillus rhamnosus GG strain (LGG) as a positive control. In addition, tolerance of lactobacilli to heat, acid, bile, Zn(2+) and Cu(2+) were assessed. All five selected strains, Lactobacillus salivarius ZJ614 (JN981856), Lactobacillus reuteri ZJ616 (JN981858), L. reuteri ZJ617 (JN981859), L. reuteri ZJ621 (JN981863) and L. reuteri ZJ623 (JN981865), showed inhibition against the two pathogens, E. coli K88 and S. enteritidis 50335. L. reuteri ZJ621 showed higher inhibition ability than the others to S. enteritidis 50335 (P S-layer protein, the inhibition activities of the lactobacilli against pathogens decreased significantly (P S-layer proteins plays an important role.

  12. Crystal Structures of the uL3 Mutant Ribosome: Illustration of the Importance of Ribosomal Proteins for Translation Efficiency.

    Science.gov (United States)

    Mailliot, Justine; Garreau de Loubresse, Nicolas; Yusupova, Gulnara; Meskauskas, Arturas; Dinman, Jonathan D; Yusupov, Marat

    2016-05-22

    The ribosome has been described as a ribozyme in which ribosomal RNA is responsible for peptidyl-transferase reaction catalysis. The W255C mutation of the universally conserved ribosomal protein uL3 has diverse effects on ribosome function (e.g., increased affinities for transfer RNAs, decreased rates of peptidyl-transfer), and cells harboring this mutation are resistant to peptidyl-transferase inhibitors (e.g., anisomycin). These observations beg the question of how a single amino acid mutation may have such wide ranging consequences. Here, we report the structure of the vacant yeast uL3 W255C mutant ribosome by X-ray crystallography, showing a disruption of the A-site side of the peptidyl-transferase center (PTC). An additional X-ray crystallographic structure of the anisomycin-containing mutant ribosome shows that high concentrations of this inhibitor restore a "WT-like" configuration to this region of the PTC, providing insight into the resistance mechanism of the mutant. Globally, our data demonstrate that ribosomal protein uL3 is structurally essential to ensure an optimal and catalytically efficient organization of the PTC, highlighting the importance of proteins in the RNA-centered ribosome.

  13. The importance of connections between the cell wall integrity pathway and the unfolded protein response in filamentous fungi.

    Science.gov (United States)

    Malavazi, Iran; Goldman, Gustavo Henrique; Brown, Neil Andrew

    2014-11-01

    In the external environment, or within a host organism, filamentous fungi experience sudden changes in nutrient availability, osmolality, pH, temperature and the exposure to toxic compounds. The fungal cell wall represents the first line of defense, while also performing essential roles in morphology, development and virulence. A polarized secretion system is paramount for cell wall biosynthesis, filamentous growth, nutrient acquisition and interactions with the environment. The unique ability of filamentous fungi to secrete has resulted in their industrial adoption as fungal cell factories. Protein maturation and secretion commences in the endoplasmic reticulum (ER). The unfolded protein response (UPR) maintains ER functionality during exposure to secretion and cell wall stress. UPR, therefore, influences secretion and cell wall homeostasis, which in turn impacts upon numerous fungal traits important to pathogenesis and biotechnology. Subsequently, this review describes the relevance of the cell wall and UPR systems to filamentous fungal pathogens or industrial microbes and then highlights interconnections between the two systems. Ultimately, the possible biotechnological applications of an enhanced understanding of such regulatory systems in combating fungal disease, or the removal of natural bottlenecks in protein secretion in an industrial setting, are discussed. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  14. "Importin" signaling roles for import proteins: the function of Drosophila importin-7 (DIM-7) in muscle-tendon signaling.

    Science.gov (United States)

    Liu, Ze Cindy; Geisbrecht, Erika R

    2012-01-01

    The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.

  15. Odorant binding proteins of the red imported fire ant, Solenopsis invicta: an example of the problems facing the analysis of widely divergent proteins.

    Directory of Open Access Journals (Sweden)

    Dietrich Gotzek

    Full Text Available We describe the odorant binding proteins (OBPs of the red imported fire ant, Solenopsis invicta, obtained from analyses of an EST library and separate 454 sequencing runs of two normalized cDNA libraries. We identified a total of 18 putative functional OBPs in this ant. A third of the fire ant OBPs are orthologs to honey bee OBPs. Another third of the OBPs belong to a lineage-specific expansion, which is a common feature of insect OBP evolution. Like other OBPs, the different fire ant OBPs share little sequence similarity (∼ 20%, rendering evolutionary analyses difficult. We discuss the resulting problems with sequence alignment, phylogenetic analysis, and tests of selection. As previously suggested, our results underscore the importance for careful exploration of the sensitivity to the effects of alignment methods for data comprising widely divergent sequences.

  16. DNA2—An Important Player in DNA Damage Response or Just Another DNA Maintenance Protein?

    Directory of Open Access Journals (Sweden)

    Elzbieta Pawłowska

    2017-07-01

    Full Text Available The human DNA2 (DNA replication helicase/nuclease 2 protein is expressed in both the nucleus and mitochondria, where it displays ATPase-dependent nuclease and helicase activities. DNA2 plays an important role in the removing of long flaps in DNA replication and long-patch base excision repair (LP-BER, interacting with the replication protein A (RPA and the flap endonuclease 1 (FEN1. DNA2 can promote the restart of arrested replication fork along with Werner syndrome ATP-dependent helicase (WRN and Bloom syndrome protein (BLM. In mitochondria, DNA2 can facilitate primer removal during strand-displacement replication. DNA2 is involved in DNA double strand (DSB repair, in which it is complexed with BLM, RPA and MRN for DNA strand resection required for homologous recombination repair. DNA2 can be a major protein involved in the repair of complex DNA damage containing a DSB and a 5′ adduct resulting from a chemical group bound to DNA 5′ ends, created by ionizing radiation and several anticancer drugs, including etoposide, mitoxantrone and some anthracyclines. The role of DNA2 in telomere end maintenance and cell cycle regulation suggests its more general role in keeping genomic stability, which is impaired in cancer. Therefore DNA2 can be an attractive target in cancer therapy. This is supported by enhanced expression of DNA2 in many cancer cell lines with oncogene activation and premalignant cells. Therefore, DNA2 can be considered as a potential marker, useful in cancer therapy. DNA2, along with PARP1 inhibition, may be considered as a potential target for inducing synthetic lethality, a concept of killing tumor cells by targeting two essential genes.

  17. Salmonella typhimurium's transthyretin-like protein is a host-specific factor important in fecal survival in chickens.

    Directory of Open Access Journals (Sweden)

    Sarah C Hennebry

    Full Text Available The transthyretin-like protein (TLP from Salmonella enterica subspecies I is a periplasmic protein with high level structural similarity to a protein found in mammals and fish. In humans, the protein homologue, transthyretin, binds and carries retinol and thyroxine, and a series of other, unrelated aromatic compounds. Here we show that the amino acid sequence of the TLP from different species, subspecies and serovars of the Salmonella genus is highly conserved and demonstrate that the TLP gene is constitutively expressed in S. Typhimurium and that copper and other divalent metal ions severely inhibit enzyme activity of the TLP, a cyclic amidohydrolase that hydrolyses 5-hydroxyisourate (5-HIU. In order to determine the in vivo role of the S. Typhimurium TLP, we constructed a strain of mouse-virulent S. Typhimurium SL1344 bearing a mutation in the TLP gene (SL1344 ΔyedX. We assessed the virulence of this strain via oral inoculation of mice and chickens. Whilst SL1344 ΔyedX induced a systemic infection in both organisms, the bacterial load detected in the faeces of infected chickens was significantly reduced when compared to the load of S. Typhimurium SL1344. These data demonstrate that the TLP gene is required for survival of S. Typhimurium in a high uric acid environment such as chicken faeces, and that metabolic traits of Salmonellae in natural and contrived hosts may be fundamentally different. Our data also highlight the importance of using appropriate animal models for the study of bacterial pathogenesis especially where host-specific virulence factors or traits are the subject of the study.

  18. Functional importance of GGXG sequence motifs in putative reentrant loops of 2HCT and ESS transport proteins.

    Science.gov (United States)

    Dobrowolski, Adam; Lolkema, Juke S

    2009-08-11

    The 2HCT and ESS families are two families of secondary transporters. Members of the two families are unrelated in amino acid sequence but share similar hydropathy profiles, which suggest a similar folding of the proteins in membranes. Structural models show two homologous domains containing five transmembrane segments (TMSs) each, with a reentrant or pore loop between the fourth and fifth TMSs in each domain. Here we show that GGXG sequence motifs present in the putative reentrant loops are important for the activity of the transporters. Mutation of the conserved Gly residues to Cys in the motifs of the Na(+)-citrate transporter CitS in the 2HCT family and the Na(+)-glutamate transporter GltS in the ESS family resulted in strongly reduced transport activity. Similarly, mutation of the variable residue "X" to Cys in the N-terminal half of GltS essentially inactivated the transporter. The corresponding mutations in the N- and C-terminal halves of CitS reduced transport activity to 60 and 25% of that of the wild type, respectively. Residual activity of any of the mutants could be further reduced by treatment with the membrane permeable thiol reagent N-ethylmaleimide (NEM). The X to Cys mutation (S405C) in the cytoplasmic loop in the C-terminal half of CitS rendered the protein sensitive to the bulky, membrane impermeable thiol reagent 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AmdiS) added at the periplasmic side of the membrane, providing further evidence that this part of the loop is positioned between the transmembrane segments. The putative reentrant loop in the C-terminal half of the ESS family does not contain the GGXG motif, but a conserved stretch rich in Gly residues. Cysteine-scanning mutagenesis of a stretch of 18 residues in the GltS protein revealed two residues important for function. Mutant N356C was completely inactivated by treatment with NEM, and mutant P351C appeared to be the counterpart of mutant S405C of CitS; the mutant was

  19. Bacillus subtilis SbcC protein plays an important role in DNA inter-strand cross-link repair

    Directory of Open Access Journals (Sweden)

    Krisnamurthy Mahalakshmi

    2006-06-01

    Full Text Available Abstract Background Several distinct pathways for the repair of damaged DNA exist in all cells. DNA modifications are repaired by base excision or nucleotide excision repair, while DNA double strand breaks (DSBs can be repaired through direct joining of broken ends (non homologous end joining, NHEJ or through recombination with the non broken sister chromosome (homologous recombination, HR. Rad50 protein plays an important role in repair of DNA damage in eukaryotic cells, and forms a complex with the Mre11 nuclease. The prokaryotic ortholog of Rad50, SbcC, also forms a complex with a nuclease, SbcD, in Escherichia coli, and has been implicated in the removal of hairpin structures that can arise during DNA replication. Ku protein is a component of the NHEJ pathway in pro- and eukaryotic cells. Results A deletion of the sbcC gene rendered Bacillus subtilis cells sensitive to DNA damage caused by Mitomycin C (MMC or by gamma irradiation. The deletion of the sbcC gene in a recN mutant background increased the sensitivity of the single recN mutant strain. SbcC was also non-epistatic with AddAB (analog of Escherichia coli RecBCD, but epistatic with RecA. A deletion of the ykoV gene encoding the B. subtilis Ku protein in a sbcC mutant strain did not resulted in an increase in sensitivity towards MMC and gamma irradiation, but exacerbated the phenotype of a recN or a recA mutant strain. In exponentially growing cells, SbcC-GFP was present throughout the cells, or as a central focus in rare cases. Upon induction of DNA damage, SbcC formed 1, rarely 2, foci on the nucleoids. Different to RecN protein, which forms repair centers at any location on the nucleoids, SbcC foci mostly co-localized with the DNA polymerase complex. In contrast to this, AddA-GFP or AddB-GFP did not form detectable foci upon addition of MMC. Conclusion Our experiments show that SbcC plays an important role in the repair of DNA inter-strand cross-links (induced by MMC, most likely

  20. The diagnostic and prognostic importance of oxidative stress biomarkers and acute phase proteins in Urinary Tract Infection (UTI) in camels.

    Science.gov (United States)

    El-Deeb, Wael M; Buczinski, Sébastien

    2015-01-01

    The present study aimed to investigate the diagnostic and prognostic importance of oxidative stress biomarkers and acute phase proteins in urinary tract infection (UTI) in camels. We describe the clinical, bacteriological and biochemical findings in 89 camels. Blood and urine samples from diseased (n = 74) and control camels (n = 15) were submitted to laboratory investigations. The urine analysis revealed high number of RBCS and pus cells. The concentrations of serum and erythrocytic malondialdehyde (sMDA & eMDA), Haptoglobin (Hp), serum amyloid A (SAA), Ceruloplasmin (Cp), fibrinogen (Fb), albumin, globulin and interleukin 6 (IL-6) were higher in diseased camels when compared to healthy ones. Catalase, super oxide dismutase and glutathione levels were lower in diseased camels when compared with control group. Forty one of 74 camels with UTI were successfully treated. The levels of malondialdehyde, catalase, super oxide dismutase, glutathione, Hp, SAA, Fb, total protein, globulin and IL-6 were associated with the odds of treatment failure. The MDA showed a great sensitivity (Se) and specificity (Sp) in predicting treatment failure (Se 85%/Sp 100%) as well as the SAA (Se 92%/Sp 87%) and globulin levels (Se 85%/Sp 100%) when using the cutoffs that maximizes the sum of Se + Sp. Multivariate logistic regression analysis revealed that two models had a high accuracy to predict failure with the first model including sex, sMDA and Hp as covariates (area under the receiver operating characteristic curve (AUC) = 0.92) and a second model using sex, SAA and Hp (AUC = 0.89). Conclusively, the oxidative stress biomarkers and acute phase proteins could be used as diagnostic and prognostic biomarkers in camel UTI management. Efforts should be forced to investigate such biomarkers in other species with UTI.

  1. Drought and Recovery: Independently Regulated Processes Highlighting the Importance of Protein Turnover Dynamics and Translational Regulation in Medicago truncatula.

    Science.gov (United States)

    Lyon, David; Castillejo, Maria Angeles; Mehmeti-Tershani, Vlora; Staudinger, Christiana; Kleemaier, Christoph; Wienkoop, Stefanie

    2016-06-01

    Climate change in conjunction with population growth necessitates a systems biology approach to characterize plant drought acclimation as well as a more thorough understanding of the molecular mechanisms of stress recovery. Plants are exposed to a continuously changing environment. Extremes such as several weeks of drought are followed by rain. This requires a molecular plasticity of the plant enabling drought acclimation and the necessity of deacclimation processes for recovery and continuous growth.During drought stress and subsequent recovery, the metabolome and proteome are regulated through a sequence of molecular processes including synthesis and degradation and molecular interaction networks are part of this regulatory process. In order to study this complex regulatory network, a comprehensive analysis is presented for the first time, investigating protein turnover and regulatory classes of proteins and metabolites during a stress recovery scenario in the model legume Medicago truncatula The data give novel insights into the molecular capacity and differential processes required for acclimation and deacclimation of severe drought stressed plants.Functional cluster and network analyses unraveled independent regulatory mechanisms for stress and recovery with different dynamic phases that during the course of recovery define the plants deacclimation from stress. The combination of relative abundance levels and turnover analysis revealed an early transition phase that seems key for recovery initiation through water resupply and is independent from renutrition. Thus, a first indication for a metabolite and protein-based load capacity was observed necessary for the recovery from drought, an important but thus far ignored possible feature toward tolerance. The data indicate that apart from the plants molecular stress response mechanisms, plasticity may be related to the nutritional status of the plant prior to stress initiation. A new perspective and possible new

  2. A Cytoplasmic Protein Ssl3829 Is Important for NDH-1 Hydrophilic Arm Assembly in Synechocystis sp. Strain PCC 6803.

    Science.gov (United States)

    Wang, Xiaozhuo; Gao, Fudan; Zhang, Jingsong; Zhao, Jiaohong; Ogawa, Teruo; Ma, Weimin

    2016-06-01

    Despite significant progress in clarifying the subunit compositions and functions of the multiple NDH-1 complexes in cyanobacteria, the assembly factors and their roles in assembling these NDH-1 complexes remain elusive. Two mutants sensitive to high light for growth and impaired in NDH-1-dependent cyclic electron transport around photosystem I were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-tagged library. Both mutants were tagged in the ssl3829 gene encoding an unknown protein, which shares significant similarity with Arabidopsis (Arabidopsis thaliana) CHLORORESPIRATORY REDUCTION7. The ssl3829 product was localized in the cytoplasm and associates with an NDH-1 hydrophilic arm assembly intermediate (NAI) of about 300 kD (NAI300) and an NdhI maturation factor, Slr1097. Upon deletion of Ssl3829, the NAI300 complex was no longer visible on gels, thereby impeding the assembly of the NDH-1 hydrophilic arm. The deletion also abolished Slr1097 and consequently reduced the amount of mature NdhI in the cytoplasm, which repressed the dynamic assembly process of the NDH-1 hydrophilic arm because mature NdhI was essential to stabilize all functional NAIs. Therefore, Ssl3829 plays an important role in the assembly of the NDH-1 hydrophilic arm by accumulating the NAI300 complex and Slr1097 protein in the cytoplasm.

  3. Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication.

    Science.gov (United States)

    Khalil, Mohamed I; Che, Xibing; Sung, Phillip; Sommer, Marvin H; Hay, John; Arvin, Ann M

    2016-05-01

    VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.

  4. Importance of two Enterococcus faecium loci encoding Gls-like proteins for in vitro bile salts stress response and virulence.

    Science.gov (United States)

    Choudhury, Tina; Singh, Kavindra V; Sillanpää, Jouko; Nallapareddy, Sreedhar R; Murray, Barbara E

    2011-04-15

    General stress proteins, Gls24 and GlsB, were previously shown to be involved in bile salts resistance of Enterococcus faecalis and in virulence. Here, we identified 2 gene clusters in Enterococcus faecium each encoding a homolog of Gls24 (Gls33 and Gls20; designated on the basis of their predicted sizes) and of GlsB (GlsB and GlsB1). The sequences of the gls33 and gls20 gene clusters from available genomes indicate distinct lineages, with those of hospital-associated CC17 isolates differing from non-CC17 by ∼7% and ∼3.5%, respectively. Deletion of an individual locus did not have a significant effect on virulence in a mouse peritonitis model, whereas a double-deletion mutant was highly attenuated (Pimportant for adaptation to the intestinal environment, in addition to being important for virulence functions.

  5. Identification of an Allosteric Pocket on Human Hsp70 Reveals a Mode of Inhibition of This Therapeutically Important Protein

    Science.gov (United States)

    Rodina, Anna; Patel, Pallav D.; Kang, Yanlong; Patel, Yogita; Baaklini, Imad; Wong, Michael J.H.; Taldone, Tony; Yan, Pengrong; Yang, Chenghua; Maharaj, Ronnie; Gozman, Alexander; Patel, Maulik R.; Patel, Hardik J.; Chirico, William; Erdjument-Bromage, Hediye; Talele, Tanaji T.; Young, Jason C.; Chiosis, Gabriela

    2014-01-01

    SUMMARY Hsp70s are important cancer chaperones that act upstream of Hsp90 and exhibit independent anti-apoptotic activities. To develop chemical tools for the study of human Hsp70, we developed a homology model that unveils a previously unknown allosteric site located in the nucleotide binding domain of Hsp70. Combining structure-based design and phenotypic testing, we discovered a previously unknown inhibitor of this site, YK5. In cancer cells, this compound is a potent and selective binder of the cytosolic but not the organellar human Hsp70s and has biological activity partly by interfering with the formation of active oncogenic Hsp70/Hsp90/client protein complexes. YK5 is a small molecule inhibitor rationally designed to interact with an allosteric pocket of Hsp70 and represents a previously unknown chemical tool to investigate cellular mechanisms associated with Hsp70. PMID:24239008

  6. The importance of protein binding for the in vitro-in vivo extrapolation (IVIVE)-example of ibuprofen, a highly protein-bound substance.

    Science.gov (United States)

    Mielke, H; Di Consiglio, E; Kreutz, R; Partosch, F; Testai, E; Gundert-Remy, U

    2017-04-01

    A physiologically based human kinetic model (PBHKM) was used to predict the in vivo ibuprofen dose leading to the same concentration-time profile as measured in cultured human hepatic cells (Truisi et al. in Toxicol Lett 233(2):172-186, 2015). We parameterized the PBHKM with data from an in vivo study. Tissue partition coefficients were calculated by an algorithm and also derived from the experimental in vitro data for the liver. The predicted concentration-time profile in plasma was in excellent agreement with human experimental data when the liver partition coefficient was calculated by the algorithm (3.01) demonstrating values in line with findings obtained from human postmortem tissues. The results were less adequate when the liver partition coefficient was based on the experimental in vitro data (11.1). The in vivo doses necessary to reach the in vitro concentrations in the liver cells were 3610 mg using the best fitting model with a liver partition coefficient of 3.01 compared to 2840 mg with the in vitro liver partition coefficient of 11.1. We found that this difference is possibly attributable to the difference between protein binding in vivo (99.9 %) and in vitro (nearly zero) as the partition coefficient is highly dependent on protein binding. Hence, the fraction freely diffusible in the liver tissue is several times higher in vitro than in vivo. In consequence, when extrapolating from in vitro to in vivo liver toxicity, it is important to consider non-intended in vitro/in vivo differences in the tissue concentration which may occur due to a low protein content of the medium.

  7. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Xiaoying Zhou

    2012-09-01

    Full Text Available Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1(ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1(ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant

  8. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    Science.gov (United States)

    Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

    2012-09-01

    Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1(ΔAB) only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1(ΔAB) transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

  9. Nucleus-encoded light-harvesting chlorophyll a/b proteins are imported normally into chlorophyll b-free chloroplasts of Arabidopsis.

    Science.gov (United States)

    Nick, Sabine; Meurer, Jörg; Soll, Jürgen; Ankele, Elisabeth

    2013-05-01

    Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light-harvesting chlorophyll a/b-binding proteins (LHCPs). After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO (chlorophyll a oxigenase) which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant (cao-1). We imported in vitro translated and (35)S-labeled precursor proteins of light-harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex (LHC) proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.

  10. Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

    Energy Technology Data Exchange (ETDEWEB)

    Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo (Egypt); Che, Xibing; Sung, Phillip; Sommer, Marvin H. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States); Hay, John [Department of Microbiology and Immunology, School of Medicine and Biomedical Science, University at Buffalo, Buffalo, NY (United States); Arvin, Ann M. [Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA (United States)

    2016-05-15

    VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.

  11. The mitochondrial protein Mcu1 plays important roles in carbon source utilization, filamentation, and virulence in Candida albicans.

    Science.gov (United States)

    Guan, Guobo; Wang, Haitao; Liang, Weihong; Cao, Chengjun; Tao, Li; Naseem, Shamoon; Konopka, James B; Wang, Yue; Huang, Guanghua

    2015-08-01

    The fungus Candida albicans is both a pathogen and a commensal in humans. The ability to utilize different carbon sources available in diverse host niches is vital for both commensalism and pathogenicity. N-acetylglucosamine (GlcNAc) is an important signaling molecule as well as a carbon source in C. albicans. Here, we report the discovery of a novel gene MCU1 essential for GlcNAc utilization. Mcu1 is located in mitochondria and associated with multiple energy- and metabolism-related proteins including Por1, Atp1, Pet9, and Mdh1. Consistently, inactivating Por1 impaired GlcNAc utilization as well. Deletion of MCU1 also caused defects in utilizing non-fermentable carbon sources and amino acids. Furthermore, MCU1 is required for filamentation in several inducing conditions and virulence in a mouse systemic infection model. We also deleted TGL99 and GUP1, two genes adjacent to MCU1, and found that the gup1/gup1 mutant exhibited mild defects in the utilization of several carbon sources including GlcNAc, maltose, galactose, amino acids, and ethanol. Our results indicate that MCU1 exists in a cluster of genes involved in the metabolism of carbon sources. Given its importance in metabolism and lack of a homolog in humans, Mcu1 could be a potential target for developing antifungal agents.

  12. The Chloroplast Import Receptor Toc90 Partially Restores the Accumulation of Toc159 Client Proteins in the Arabidopsis thaliana ppi2 Mutant

    Institute of Scientific and Technical Information of China (English)

    Sibylle Infanger; Sylvain Bischof; Andreas Hiltbrunner; Birgit Agne; Sacha Baginsky; Felix Kessler

    2011-01-01

    Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- (translocon at the outer chloroplast membrane) and Tic- (translocon at the inner chloroplast membrane) complexes. In Arabidopsis thaliana,precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families: Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied,but the function of Toc90 still remains unclear. Here,we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.

  13. Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

    Science.gov (United States)

    Weinheimer-Haus, Eileen M.; Mirza, Rita E.; Koh, Timothy J.

    2015-01-01

    The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing. PMID:25793779

  14. Nod-like receptor protein-3 inflammasome plays an important role during early stages of wound healing.

    Science.gov (United States)

    Weinheimer-Haus, Eileen M; Mirza, Rita E; Koh, Timothy J

    2015-01-01

    The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.

  15. Could structural similarity of specific domains between animal globins and plant antenna proteins provide hints important for the photoprotection mechanism?

    Science.gov (United States)

    Ioannidis, Nikolaos E; Kotzabasis, Kiriakos

    2015-01-07

    Non photochemical quenching is a fundamental mechanism in photosynthesis, which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. In the last decades hundreds of papers have appeared that describe the role of antenna regulation in protection or the so called qE response. However, the exact quenching site is still obscure. Previously overlooked features of the antenna may provide hints towards the elucidation of its functionality and of the quenching mechanism. Recently it was demonstrated that the catalytic domain of human myoglobin that binds the pigment (i.e. heme) is similar in structure to the domain of the light harvesting complex II of pea that binds Chl a 614 (former known as b3). In addition, it is well accepted that conformational changes of the chlorophyll macrocycle result in reversible changes of fluorescence (the lowest fluorescence corresponds to non planar macrocycle). Here we put forward a hypothesis regarding the molecular mechanism that leads to the formation of a quenching center inside the antenna proteins. Our main suggestion is that a conformational change of helix H5 (known also as helix D) forces conformational changes in the macrocycle of Chl a 614 is implicated in the ΔA535 absorbance change and quenching during photoprotective qE. The specific features (some of them similar to those of heme domain of globins) of the b3 domain account for these traits. The model predicts that antenna proteins having b3 pigments (i.e. LHCII, CP29, CP26) can act as potential quenchers.

  16. Osteopontin (OPN is an important protein to mediate improvements in the biocompatibility of C ion-implanted silicone rubber.

    Directory of Open Access Journals (Sweden)

    Shao-liang Wang

    Full Text Available Medical device implants are drawing increasing amounts of interest from modern medical practitioners. However, this attention is not evenly spread across all such devices; most of these implantable devices can cause adverse reactions such as inflammation, fibrosis, thrombosis, and infection. In this work, the biocompatibility of silicone rubber (SR was improved through carbon (C ion implantation. Scanning electron microscopy (SEM, atomic force microscopy (AFM, X-ray photoelectron spectroscopy (XPS, and X-ray diffraction (XRD results confirmed that these newly generated carbon-implanted silicone rubbers (C-SRs had large, irregular peaks and deep valleys on their surfaces. The water contact angle of the SR surface decreased significantly after C ion implantation. C ion implantation also changed the surface charge distribution, silicone oxygen rate, and chemical-element distribution of SR to favor cell attachment. The dermal fibroblasts cultured on the surface C-SR grew faster and showed more typical fibroblastic shapes. The expression levels of major adhesion proteins, including talin-1, zyxin, and vinculin, were significantly higher in dermal fibroblasts cultured on C-SR coated plates than in dermal fibroblasts cultured on SR. Those same dermal fibroblasts on C-SRs showed more pronounced adhesion and migration abilities. Osteopontin (OPN, a critical extracellular matrix (ECM protein, was up-regulated and secreted from dermal fibroblasts cultured on C-SR. Matrix metalloproteinase-9 (MMP-9 activity was also increased. These cells were highly mobile and were able to adhere to surfaces, but these abilities were inhibited by the monoclonal antibody against OPN, or by shRNA-mediated MMP-9 knockdown. Together, these results suggest that C ion implantation significantly improves SR biocompatibility, and that OPN is important to promote cell adhesion to the C-SR surface.

  17. Nuclear Import Analysis of Two Different Fluorescent Marker Proteins into Hepatocyte Cell Lines (HuH-7 Cell

    Directory of Open Access Journals (Sweden)

    Aris Haryanto

    2015-10-01

    Full Text Available The application of fluorescent proteins as expression markers and protein fusion partners has provedimmensely valuable for resolving the organization of biological events in living cells. EGFP and DsRed2 arecommonly fluorescent marker protein which is used for biotechnology and cell biology research. The presentstudy was designed to identify the expression vector that suitable to ligate with DNA encoding HBV coreprotein for intracellular localization study in hepatocyte cell, which were expressed as fusion proteins. We alsocompared and quantified the expressed fluorescent protein which predominantly localized in the cellcompartment. The results indicated that DsRed2 shown as less than ideal for intracellular localization study ofthan EGFP, because of its tetrameric structure of the fluorescent protein and when fused to a protein of interest,the fusion protein often forms aggregates in the living cells. In contrast, EGFP fluorescent protein shown a muchhigher proportion of cytoplasmic localization, thus being more suitable for analysis of intracellular localizationthan DsRed2 fluorescent protein. EGFP fluorescent protein is also capable to produce a strong green fluorescencewhen excited by blue light, without any exogenously added substrate or cofactor, events inside living cell canthus be visualized in a non-invasive way. Based on our present quantitative data and some reasons above shownthat EGFP is more suitable than DsRed2 as a fluorescent marker protein for intracellular localization study intoHuH-7 cell.Keywords: EGFP, DsRed2 fluorescent protein , HuH-7 cell, HBV, intracellular localization

  18. The importance of making ends meet: mutations in genes and altered expression of proteins of the MRN complex and cancer.

    Science.gov (United States)

    Dzikiewicz-Krawczyk, Agnieszka

    2008-01-01

    The MRN protein complex, consisting of MRE1, RAD50 and NBS1, plays a crucial role in sensing DNA double-strand breaks (DSBs), and it is involved in cell cycle control. This makes the MRN complex an important guard of genome stability. Hypomorphic mutations in NBS1 result in the Nijmegen breakage syndrome (NBS), which is characterized by, among other things, an increased predisposition to malignancies, especially leukemia/lymphoma. Relatives of NBS patients carrying heterozygous mutations are also more prone to cancer development. This review summarizes several studies searching for associations between heterozygous mutations in NBS1, MRE11, and RAD50 and cancer and examining the levels of expression of proteins coded by these genes in tumor tissues. The results indicate that both decreased and increased expression of NBS1 may contribute to tumorigenesis, whereas overexpressed RAD50 has an anti-tumoric effect. MRE11 and RAD50 are also affected in tumors with microsatellite instability. However, the outcomes of association studies, which concerned primarily lymphomas/leukemias and breast cancer, were inconclusive. Heterozygous NBS1 mutations and molecular variants 657del5, I171V, R215W and E185Q were most commonly analyzed. Among these, an association with cancer was found most frequently for 657del5 (in leukemia/lymphoma and breast cancer) and I171V (in leukemia, breast, head and neck and colorectal cancers); however, other studies gave contradictory results. For other NBS1 as well as MRE11 and RAD50 variants, too little data were available to assess their role in cancer risk. Overall, the results suggest that heterozygous MRN complex mutations and molecular variants may contribute only to a limited fraction of tumors. This may be caused by several factors: various frequencies of the variants in specific populations, different criteria used for selection of control groups, possible effects of environmental factors, and potential interactions with variants of other

  19. Functional constraints on SoxE proteins in neural crest development: The importance of differential expression for evolution of protein activity.

    Science.gov (United States)

    Lee, Eric M; Yuan, Tian; Ballim, Reyna D; Nguyen, Kristy; Kelsh, Robert N; Medeiros, Daniel M; McCauley, David W

    2016-10-01

    Vertebrate SoxE genes (Sox8, 9, and 10) are key regulators of neural crest cell (NCC) development. These genes arose by duplication from a single SoxE gene in the vertebrate ancestor. Although SoxE paralogs are coexpressed early in NCC development, later, Sox9 is restricted to skeletogenic lineages in the head, and Sox10 to non-skeletogenic NCC in the trunk and head. When this subfunctionalization evolved and its possible role in the evolution of the neural crest are unknown. Sea lampreys are basal vertebrates that also possess three SoxE genes, while only a single SoxE is present in the cephalochordate amphioxus. In order to address the functional divergence of SoxE genes, and to determine if differences in their biochemical functions may be linked to changes in neural crest developmental potential, we examined the ability of lamprey and amphioxus SoxE genes to regulate differentiation of NCC derivatives in zebrafish colourless (cls) mutants lacking expression of sox10. Our findings suggest that the proto-vertebrate SoxE gene possessed both melanogenic and neurogenic capabilities prior to SoxE gene duplication. Following the agnathan-gnathostome split, lamprey SoxE1 and SoxE3 largely lost their melanogenic and/or enteric neurogenic properties, while gnathostome SoxE paralogs have retained functional conservation. We posit that this difference in protein subfunctionalization is a direct consequence of the independent regulation of SoxE paralog expression between the two lineages. Specifically, we propose that the overlapping expression of gnathostome SoxE paralogs in early neural crest largely constrained the function of gnathostome SoxE proteins. In contrast, the largely non-overlapping expression of lamprey SoxE paralogs allowed them to specialize with regard to their DNA-binding and/or protein interaction properties. Restriction of developmental potential among cranial and trunk neural crest in lampreys may be related to constraints on SoxE activity among

  20. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Directory of Open Access Journals (Sweden)

    Jihong Li

    2008-05-01

    Full Text Available Clostridium perfringens is a major cause of food poisoning (FP in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  1. Multidrug Resistance-Associated Protein 3 Plays an Important Role in Protection against Acute Toxicity of Diclofenac.

    Science.gov (United States)

    Scialis, Renato J; Csanaky, Iván L; Goedken, Michael J; Manautou, José E

    2015-07-01

    Diclofenac (DCF) is a nonsteroidal anti-inflammatory drug commonly prescribed to reduce pain in acute and chronic inflammatory diseases. One of the main DCF metabolites is a reactive diclofenac acyl glucuronide (DCF-AG) that covalently binds to biologic targets and may contribute to adverse drug reactions arising from DCF use. Cellular efflux of DCF-AG is partially mediated by multidrug resistance-associated proteins (Mrp). The importance of Mrp2 during DCF-induced toxicity has been established, yet the role of Mrp3 remains largely unexplored. In the present work, Mrp3-null (KO) mice were used to study the toxicokinetics and toxicodynamics of DCF and its metabolites. DCF-AG plasma concentrations were 90% lower in KO mice than in wild-type (WT) mice, indicating that Mrp3 mediates DCF-AG basolateral efflux. In contrast, there were no differences in DCF-AG biliary excretion between WT and KO, suggesting that only DCF-AG basolateral efflux is compromised by Mrp3 deletion. Susceptibility to toxicity was also evaluated after a single high DCF dose. No signs of injury were detected in livers and kidneys; however, ulcers were found in the small intestines. Furthermore, the observed intestinal injuries were consistently more severe in KO compared with WT. DCF covalent adducts were observed in liver and small intestines; however, staining intensity did not correlate with the severity of injuries, implying that tissues respond differently to covalent modification. Overall, the data provide strong evidence that (1) in vivo Mrp3 plays an important role in DCF-AG disposition and (2) compromised Mrp3 function can enhance injury in the gastrointestinal tract after DCF treatment.

  2. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Science.gov (United States)

    Li, Jihong; McClane, Bruce A

    2008-05-02

    Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  3. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    Science.gov (United States)

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity.

  4. Polymorphisms at activated protein C cleavage sites of factor V: Are they important in the absence of factor V Leiden?

    Directory of Open Access Journals (Sweden)

    Ehsan Kheradmand

    2017-01-01

    Full Text Available Background: Activated protein C (APC inactivates factor V (FV by cleavage of its heavy chain at Arg306, Arg506, Arg679, and Lys994. Mutational changes, which abolish APC cleavage sites, may predispose thrombosis by altering the inactivation process of FV. FV Leiden (FVL (Arg506Glu has been demonstrated as a strong risk factor for thrombosis. In the current study, we have studied whether mutations in the cleavage sites of FV for APC, not due to FVL, would have a role in presenting APC resistance (APCR and initiation of a cerebral thrombotic event.Methods: A group of 22 patients with a history of cerebral venous thrombosis (CVT, who were not carriers of FVL enrolled in the study. The patients who had conditions associated with acquired APCR were excluded from the study. APCR test was performed on the remaining 16 patients, which showed APCR in 4 plasma samples. DNA sequencing was performed on four exons of FV of APCR patients, encoding Arg306, Arg506, Arg679, and Lys994.Results: Mutations were not found within nucleotides encoding the cleavage sites; neither was found within their close upstream and downstream sequences.Conclusion: Our results show that polymorphisms affecting cleavage sites of FV other than Arg506Glu it would be less likely to be the basis for APCR and its increased thrombosis susceptibility. In addition, it emphasizes on the importance of screening for APCR in the patients diagnosed with CVT.

  5. Dual interaction of scaffold protein Tim44 of mitochondrial import motor with channel-forming translocase subunit Tim23.

    Science.gov (United States)

    Ting, See-Yeun; Yan, Nicholas L; Schilke, Brenda A; Craig, Elizabeth A

    2017-04-25

    Proteins destined for the mitochondrial matrix are targeted to the inner membrane Tim17/23 translocon by their presequences. Inward movement is driven by the matrix-localized, Hsp70-based motor. The scaffold Tim44, interacting with the matrix face of the translocon, recruits other motor subunits and binds incoming presequence. The basis of these interactions and their functional relationships remains unclear. Using site-specific in vivo crosslinking and genetic approaches in Saccharomyces cerevisiae, we found that both domains of Tim44 interact with the major matrix-exposed loop of Tim23, with the C-terminal domain (CTD) binding Tim17 as well. Results of in vitro experiments showed that the N-terminal domain (NTD) is intrinsically disordered and binds presequence near a region important for interaction with Hsp70 and Tim23. Our data suggest a model in which the CTD serves primarily to anchor Tim44 to the translocon, whereas the NTD is a dynamic arm, interacting with multiple components to drive efficient translocation.

  6. Controls on the production, incorporation and decomposition of glomalin - a novel fungal soil protein important to soil carbon

    Energy Technology Data Exchange (ETDEWEB)

    Matthias C. Rillig

    2003-11-20

    OAK B263 Glomalin is an operationally defined soil protein, produced by arbuscular mycorrhizal fungi (AMF), with importance in soil carbon sequestration through its relationship with soil aggregation. The goal of the project was to further explore the natural history of glomalin and to address several questions regarding basic behavior of this compound in soil (production, incorporation, decomposition). We have obtained a significant amount of novel information on the arbuscular mycorrhizal fungal soil protein, concerning factors controlling its production to mechanisms of incorporation and decomposition. These findings have resulted in 10 publications in peer-reviewed journals, with several more submitted or in preparation, and 16 contributed presentations at meetings. I have sought collaborative opportunities whenever they fit within the research proposed to enhance our productivity. Additionally, although not part of the original proposed work, we have made a significant effort to elucidate the molecular biology of glomalin (in response to Program Officer suggestions). In addition to peer-reviewed publications there have also been a number of invited presentations, including a keynote address delivered by the PI at the International Conference on Mycorrhizae (ICOM4) in Montreal, summer 2003. Two Master's students have been trained (and graduated), and a postdoctoral associate has been mentored, as well as numerous undergraduate researchers at UM. In this report I summarize the major findings of the project in the areas of glomalin production control (host factors, elevated CO2), incorporation, and decomposition. Section D is newly added and describes recent progress in molecular biology. Briefly, we found that glomalin production is influenced by the host, as shown by host species effects and responses to elevated CO2. We have recently made a significant breakthrough in understanding how glomalin may become deposited into soil; apparently the dominant

  7. The importance of CELF control: molecular and biological roles of the CUG-BP, Elav-like family of RNA-binding proteins.

    Science.gov (United States)

    Dasgupta, Twishasri; Ladd, Andrea N

    2012-01-01

    RNA processing is important for generating protein diversity and modulating levels of protein expression. The CUG-BP, Elav-like family (CELF) of RNA-binding proteins regulate several steps of RNA processing in the nucleus and cytoplasm, including pre-mRNA alternative splicing, C to U RNA editing, deadenylation, mRNA decay, and translation. In vivo, CELF proteins have been shown to play roles in gametogenesis and early embryonic development, heart and skeletal muscle function, and neurosynaptic transmission. Dysregulation of CELF-mediated programs has been implicated in the pathogenesis of human diseases affecting the heart, skeletal muscles, and nervous system.

  8. Specific Subunits of Heterotrimeric G Proteins Play Important Roles during Nodulation in Soybean1[W][OA

    Science.gov (United States)

    Choudhury, Swarup Roy; Pandey, Sona

    2013-01-01

    Heterotrimeric G proteins comprising Gα, Gβ, and Gγ subunits regulate many fundamental growth and development processes in all eukaryotes. Plants possess a relatively limited number of G-protein components compared with mammalian systems, and their detailed functional characterization has been performed mostly in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). However, the presence of single Gα and Gβ proteins in both these species has significantly undermined the complexity and specificity of response regulation in plant G-protein signaling. There is ample pharmacological evidence for the role of G proteins in regulation of legume-specific processes such as nodulation, but the lack of genetic data from a leguminous species has restricted its direct assessment. Our recent identification and characterization of an elaborate G-protein family in soybean (Glycine max) and the availability of appropriate molecular-genetic resources have allowed us to directly evaluate the role of G-protein subunits during nodulation. We demonstrate that all G-protein genes are expressed in nodules and exhibit significant changes in their expression in response to Bradyrhizobium japonicum infection and in representative supernodulating and nonnodulating soybean mutants. RNA interference suppression and overexpression of specific G-protein components results in lower and higher nodule numbers, respectively, validating their roles as positive regulators of nodule formation. Our data further show preferential usage of distinct G-protein subunits in the presence of an additional signal during nodulation. Interestingly, the Gα proteins directly interact with the soybean nodulation factor receptors NFR1α and NFR1β, suggesting that the plant G proteins may couple with receptors other than the canonical heptahelical receptors common in metazoans to modulate signaling. PMID:23569109

  9. Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

    Science.gov (United States)

    Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

    2012-06-01

    Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

  10. PfEMP1 – A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis

    DEFF Research Database (Denmark)

    Hviid, Lars; Jensen, Anja T R

    2015-01-01

    to be a central element in the pathogenesis of the disease. It is mediated by the interaction of parasite ligands on the erythrocyte surface and a range of host receptor molecules in many organs and tissues. Among several proteins and protein families implicated in this process, the P. falciparum erythrocyte...... membrane protein 1 (PfEMP1) family of high-molecular weight and highly variable antigens appears to be the most prominent. In this chapter, we aim to provide a systematic overview of the current knowledge about these proteins, their structure, their function, how they are presented on the erythrocyte...

  11. Immunoproteomic Analysis of Antibody Responses to Extracellular Proteins of Candida albicans Revealing the Importance of Glycosylation for Antigen Recognition.

    Science.gov (United States)

    Luo, Ting; Krüger, Thomas; Knüpfer, Uwe; Kasper, Lydia; Wielsch, Natalie; Hube, Bernhard; Kortgen, Andreas; Bauer, Michael; Giamarellos-Bourboulis, Evangelos J; Dimopoulos, George; Brakhage, Axel A; Kniemeyer, Olaf

    2016-08-05

    During infection, the human pathogenic fungus Candida albicans undergoes a yeast-to-hypha transition, secretes numerous proteins for invasion of host tissues, and modulates the host's immune response. Little is known about the interplay of C. albicans secreted proteins and the host adaptive immune system. Here, we applied a combined 2D gel- and LC-MS/MS-based approach for the characterization of C. albicans extracellular proteins during the yeast-to-hypha transition, which led to a comprehensive C. albicans secretome map. The serological responses to C. albicans extracellular proteins were investigated by a 2D-immunoblotting approach combined with MS for protein identification. On the basis of the screening of sera from candidemia and three groups of noncandidemia patients, a core set of 19 immunodominant antibodies against secreted proteins of C. albicans was identified, seven of which represent potential diagnostic markers for candidemia (Xog1, Lip4, Asc1, Met6, Tsa1, Tpi1, and Prx1). Intriguingly, some secreted, strongly glycosylated protein antigens showed high cross-reactivity with sera from noncandidemia control groups. Enzymatic deglycosylation of proteins secreted from hyphae significantly impaired sera antibody recognition. Furthermore, deglycosylation of the recombinantly produced, secreted aspartyl protease Sap6 confirmed a significant contribution of glycan epitopes to the recognition of Sap6 by antibodies in patient's sera.

  12. Bottom–up protein identifications from microliter quantities of individual human tear samples. Important steps towards clinical relevance.

    Directory of Open Access Journals (Sweden)

    Peter Raus

    2015-12-01

    With 375 confidently identified proteins in the healthy adult tear, the obtained results are comprehensive and in large agreement with previously published observations on pooled samples of multiple patients. We conclude that, to a limited extent, bottom–up tear protein identifications from individual patients may have clinical relevance.

  13. Increasing the catalytic activity of Bilirubin oxidase from Bacillus pumilus: Importance of host strain and chaperones proteins.

    Science.gov (United States)

    Gounel, Sébastien; Rouhana, Jad; Stines-Chaumeil, Claire; Cadet, Marine; Mano, Nicolas

    2016-07-20

    Aggregation of recombinant proteins into inclusion bodies (IBs) is the main problem of the expression of multicopper oxidase in Escherichia coli. It is usually attributed to inefficient folding of proteins due to the lack of copper and/or unavailability of chaperone proteins. The general strategies reported to overcome this issue have been focused on increasing the intracellular copper concentration. Here we report a complementary method to optimize the expression in E. coli of a promising Bilirubin oxidase (BOD) isolated from Bacillus pumilus. First, as this BOD has a disulfide bridge, we switched E.coli strain from BL21 (DE3) to Origami B (DE3), known to promote the formation of disulfide bridges in the bacterial cytoplasm. In a second step, we investigate the effect of co-expression of chaperone proteins on the protein production and specific activity. Our strategy allowed increasing the final amount of enzyme by 858% and its catalytic rate constant by 83%.

  14. The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum.

    Science.gov (United States)

    Mirshafiee, Vahid; Kim, Raehyun; Mahmoudi, Morteza; Kraft, Mary L

    2016-06-01

    Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the "protein corona". To simplify studies of protein-NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography-mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.

  15. Identification, expression, and immuno-reactivity of Sol i 2 & Sol i 4 venom proteins of queen red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae).

    Science.gov (United States)

    Lockwood, Stephanie A; Haghipour-Peasley, Jilla; Hoffman, Donald R; Deslippe, Richard J

    2012-10-01

    We report on two low-molecular weight proteins that are stored in the venom of queen red imported fire ants (Solenopsis invicta). Translated amino acid sequences identified one protein to have 74.8% identity with the Sol i 2w worker allergen, and the other protein was found to have 96/97% identity with Sol i 4.01w/4.02w worker allergens. Both Sol i 2 and Sol i 4 queen and worker proteins were expressed using pEXP1-DEST vector in SHuffle™ T7 Express lysY Escherichia coli. Proteins were expressed at significant concentrations, as opposed to the μg/ml amounts by our previous expression methods, enabling further study of these proteins. Sol i 2q protein bound weakly to human IgE, sera pooled from allergic patients, whereas Sol i 2w, Sol i 4.01w, and Sol i 4q proteins bound strongly. Despite Sol i 2w and Sol i 2q proteins having 74.8% identity, the queen protein is less immuno-reactive than the worker allergen. This finding is consistent with allergic individuals being less sensitive to queen than worker venom. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration.

    Science.gov (United States)

    Park, Hee Ho; Sohn, Youngsoo; Yeo, Ji Woo; Park, Ju Hyun; Lee, Hong Jai; Ryu, Jina; Rhee, Won Jong; Park, Tai Hyun

    2014-12-01

    Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.

  17. IDENTIFICATION AND EXPRESSION ANALYSIS OF TWO 14-3-3 PROTEINS IN THE MOSQUITO Aedes aegypti, AN IMPORTANT ARBOVIRUSES VECTOR.

    Science.gov (United States)

    Trujillo-Ocampo, Abel; Cázares-Raga, Febe Elena; Celestino-Montes, Antonio; Cortés-Martínez, Leticia; Rodríguez, Mario H; Hernández-Hernández, Fidel de la Cruz

    2016-11-01

    The 14-3-3 proteins are evolutionarily conserved acidic proteins that form a family with several isoforms in many cell types of plants and animals. In invertebrates, including dipteran and lepidopteran insects, only two isoforms have been reported. 14-3-3 proteins are scaffold molecules that form homo- or heterodimeric complexes, acting as molecular adaptors mediating phosphorylation-dependent interactions with signaling molecules involved in immunity, cell differentiation, cell cycle, proliferation, apoptosis, and cancer. Here, we describe the presence of two isoforms of 14-3-3 in the mosquito Aedes aegypti, the main vector of dengue, yellow fever, chikungunya, and zika viruses. Both isoforms have the conserved characteristics of the family: two protein signatures (PS1 and PS2), an annexin domain, three serine residues, targets for phosphorylation (positions 58, 184, and 233), necessary for their function, and nine alpha helix-forming segments. By sequence alignment and phylogenetic analysis, we found that the molecules correspond to Ɛ and ζ isoforms (Aeae14-3-3ε and Aeae14-3-3ζ). The messengers and protein products were present in all stages of the mosquito life cycle and all the tissues analyzed, with a small predominance of Aeae14-3-3ζ except in the midgut and ovaries of adult females. The 14-3-3 proteins in female midgut epithelial cells were located in the cytoplasm. Our results may provide insights to further investigate the functions of these proteins in mosquitoes.

  18. A cis-regulatory mutation in troponin-I of Drosophila reveals the importance of proper stoichiometry of structural proteins during muscle assembly.

    Science.gov (United States)

    Firdaus, Hena; Mohan, Jayaram; Naz, Sarwat; Arathi, Prabhashankar; Ramesh, Saraf R; Nongthomba, Upendra

    2015-05-01

    Rapid and high wing-beat frequencies achieved during insect flight are powered by the indirect flight muscles, the largest group of muscles present in the thorax. Any anomaly during the assembly and/or structural impairment of the indirect flight muscles gives rise to a flightless phenotype. Multiple mutagenesis screens in Drosophila melanogaster for defective flight behavior have led to the isolation and characterization of mutations that have been instrumental in the identification of many proteins and residues that are important for muscle assembly, function, and disease. In this article, we present a molecular-genetic characterization of a flightless mutation, flightless-H (fliH), originally designated as heldup-a (hdp-a). We show that fliH is a cis-regulatory mutation of the wings up A (wupA) gene, which codes for the troponin-I protein, one of the troponin complex proteins, involved in regulation of muscle contraction. The mutation leads to reduced levels of troponin-I transcript and protein. In addition to this, there is also coordinated reduction in transcript and protein levels of other structural protein isoforms that are part of the troponin complex. The altered transcript and protein stoichiometry ultimately culminates in unregulated acto-myosin interactions and a hypercontraction muscle phenotype. Our results shed new insights into the importance of maintaining the stoichiometry of structural proteins during muscle assembly for proper function with implications for the identification of mutations and disease phenotypes in other species, including humans.

  19. Emerging Evidence for the Importance of Dietary Protein Source on Glucoregulatory Markers and Type 2 Diabetes: Different Effects of Dairy, Meat, Fish, Egg, and Plant Protein Foods

    Directory of Open Access Journals (Sweden)

    Kevin B. Comerford

    2016-07-01

    Full Text Available Observational studies provide evidence that a higher intake of protein from plant-based foods and certain animal-based foods is associated with a lower risk for type 2 diabetes. However, there are few distinguishable differences between the glucoregulatory qualities of the proteins in plant-based foods, and it is likely their numerous non-protein components (e.g., fibers and phytochemicals that drive the relationship with type 2 diabetes risk reduction. Conversely, the glucoregulatory qualities of the proteins in animal-based foods are extremely divergent, with a higher intake of certain animal-based protein foods showing negative effects, and others showing neutral or positive effects on type 2 diabetes risk. Among the various types of animal-based protein foods, a higher intake of dairy products (such as milk, yogurt, cheese and whey protein consistently shows a beneficial relationship with glucose regulation and/or type 2 diabetes risk reduction. Intervention studies provide evidence that dairy proteins have more potent effects on insulin and incretin secretion compared to other commonly consumed animal proteins. In addition to their protein components, such as insulinogenic amino acids and bioactive peptides, dairy products also contain a food matrix rich in calcium, magnesium, potassium, trans-palmitoleic fatty acids, and low-glycemic index sugars—all of which have been shown to have beneficial effects on aspects of glucose control, insulin secretion, insulin sensitivity and/or type 2 diabetes risk. Furthermore, fermentation and fortification of dairy products with probiotics and vitamin D may improve a dairy product’s glucoregulatory effects.

  20. Emerging Evidence for the Importance of Dietary Protein Source on Glucoregulatory Markers and Type 2 Diabetes: Different Effects of Dairy, Meat, Fish, Egg, and Plant Protein Foods.

    Science.gov (United States)

    Comerford, Kevin B; Pasin, Gonca

    2016-07-23

    Observational studies provide evidence that a higher intake of protein from plant-based foods and certain animal-based foods is associated with a lower risk for type 2 diabetes. However, there are few distinguishable differences between the glucoregulatory qualities of the proteins in plant-based foods, and it is likely their numerous non-protein components (e.g., fibers and phytochemicals) that drive the relationship with type 2 diabetes risk reduction. Conversely, the glucoregulatory qualities of the proteins in animal-based foods are extremely divergent, with a higher intake of certain animal-based protein foods showing negative effects, and others showing neutral or positive effects on type 2 diabetes risk. Among the various types of animal-based protein foods, a higher intake of dairy products (such as milk, yogurt, cheese and whey protein) consistently shows a beneficial relationship with glucose regulation and/or type 2 diabetes risk reduction. Intervention studies provide evidence that dairy proteins have more potent effects on insulin and incretin secretion compared to other commonly consumed animal proteins. In addition to their protein components, such as insulinogenic amino acids and bioactive peptides, dairy products also contain a food matrix rich in calcium, magnesium, potassium, trans-palmitoleic fatty acids, and low-glycemic index sugars-all of which have been shown to have beneficial effects on aspects of glucose control, insulin secretion, insulin sensitivity and/or type 2 diabetes risk. Furthermore, fermentation and fortification of dairy products with probiotics and vitamin D may improve a dairy product's glucoregulatory effects.

  1. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang

    2016-01-01

    are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene-cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously...... developed xylene-linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein-coupled receptor (GPCR), some agents such as MGA-C13 and MGA-C14 resulted in markedly enhanced stability of membrane...

  2. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-01-01

    The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised.

  3. Leachability of protein and metals incorporated into aquatic invertebrates: are species and metals-exposure history important?

    Science.gov (United States)

    Meyer, J.S.; Suedkamp, M.J.; Morris, J.M.; Farag, A.M.

    2005-01-01

    To partially simulate conditions in fish intestinal tracts, we leached six groups of metals-contaminated invertebrates at pH 2 and pH 7, and analyzed the concentrations of four metals (Cd, Cu, Pb, and Zn) and total protein in the leachates. Four of the groups of invertebrates were benthic macroinvertebrates collected from metals-contaminated rivers (the Clark Fork River in Montana and the Coeur d’Alene River in Idaho, USA); the other two groups of invertebrates (one of which was exposed to metals in the laboratory) were laboratory-reared brine shrimp (Artemia sp.). Additionally, we fractionated the pH 2 leachates using size-exclusion chromatography (SEC). Protein content was 1.3 to 1.4× higher in Artemia than in the benthic macroinvertebrates, and leachability of metals and protein differed considerably among several of the groups of invertebrates. In SEC fractions of the pH 2 leachates from both groups of Artemia, Cu and protein co-eluted; however, Cu and protein did not co-elute in SEC fractions of the leachates from any of the benthic macroinvertebrate groups. Although none of the other three metals co-eluted with protein in any of the pH 2 leachates, one or more of the metals co-eluted with lower-molecular-weight molecules in the leachates from all of the groups of invertebrates. These results suggest fundamental differences in metal-binding properties and protein leachability among some invertebrates. Thus, different invertebrates and different histories of metals exposure might lead to different availability of metals and protein to predators.

  4. Degradation of proteins by enzymes exuded by Allium porrum roots - a potentially important strategy for acquiring organic nitrogen by plants.

    Science.gov (United States)

    Adamczyk, Bartosz; Godlewski, Mirosław; Smolander, Aino; Kitunen, Veikko

    2009-10-01

    Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography-mass spectrometry (LC-MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.

  5. Topology of eukaryotic type II membrane proteins: importance of N-terminal positively charged residues flanking the hydrophobic domain.

    Science.gov (United States)

    Parks, G D; Lamb, R A

    1991-02-22

    We have tested the role of different charged residues flanking the sides of the signal/anchor (S/A) domain of a eukaryotic type II (N(cyt)C(exo)) integral membrane protein in determining its topology. The removal of positively charged residues on the N-terminal side of the S/A yields proteins with an inverted topology, while the addition of positively charged residues to only the C-terminal side has very little effect on orientation. Expression of chimeric proteins composed of domains from a type II protein (HN) and the oppositely oriented membrane protein M2 indicates that the HN N-terminal domain is sufficient to confer a type II topology and that the M2 N-terminal ectodomain can direct a type II topology when modified by adding positively charged residues. These data suggest that eukaryotic membrane protein topology is governed by the presence or absence of an N-terminal signal for retention in the cytoplasm that is composed in part of positive charges.

  6. The presence of the iron-sulfur motif is important for the conformational stability of the antiviral protein, Viperin.

    Directory of Open Access Journals (Sweden)

    Shubhasis Haldar

    Full Text Available Viperin, an antiviral protein, has been shown to contain a CX(3CX(2C motif, which is conserved in the radical S-adenosyl-methionine (SAM enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α-β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity.

  7. Is Asp-His-Ser/Thr-Trp tetrad hydrogen-bond network important to WD40-repeat proteins: a statistical and theoretical study.

    Science.gov (United States)

    Wu, Xian-Hui; Zhang, Hui; Wu, Yun-Dong

    2010-04-01

    WD40-repeat proteins are abundant and play important roles in forming protein complexes. The domain usually has seven WD40 repeats, which folds into a seven beta-sheet propeller with each beta-sheet in a four-strand structure. An analysis of 20 available WD40-repeat proteins in Protein Data Bank reveals that each protein has at least one Asp-His-Ser/Thr-Trp (D-H-S/T-W) hydrogen-bonded tetrad, and some proteins have up to six or seven such tetrads. The relative positions of the four residues in the tetrads are also found to be conserved. A sequence alignment analysis of 560 WD40-repeat protein sequences in human reveals very similar features, indicating that such tetrad may be a general feature of WD40-repeat proteins. We carried out density functional theory and found that these tetrads can lead to significant stabilization including hydrogen-bonding cooperativity. The hydrogen bond involving Trp is significant. These results lead us to propose that the tetrads may be critical to the stability and the mechanism of folding of these proteins.

  8. Comparative genomic studies suggest that the cyanobacterial endosymbionts of the amoeba Paulinella chromatophora possess an import apparatus for nuclear-encoded proteins.

    Science.gov (United States)

    Bodył, A; Mackiewicz, P; Stiller, J W

    2010-07-01

    Plastids evolved from free-living cyanobacteria through a process of primary endosymbiosis. The most widely accepted hypothesis derives three ancient lineages of primary plastids, i.e. those of glaucophytes, red algae and green plants, from a single cyanobacterial endosymbiosis. This hypothesis was originally predicated on the assumption that transformations of endosymbionts into organelles must be exceptionally rare because of the difficulty in establishing efficient protein trafficking between a host cell and incipient organelle. It turns out, however, that highly integrated endosymbiotic associations are more common than once thought. Among them is the amoeba Paulinella chromatophora, which harbours independently acquired cyanobacterial endosymbionts functioning as plastids. Sequencing of the Paulinella endosymbiont genome revealed an absence of essential genes for protein trafficking, suggesting their residence in the host nucleus and import of protein products back into the endosymbiont. To investigate this hypothesis, we searched the Paulinella endosymbiont genome for homologues of higher plant translocon proteins that form the import apparatus in two-membrane envelopes of primary plastids. We found homologues of Toc12, Tic21 and Tic32, but genes for other key translocon proteins (e.g. Omp85/Toc75 and Tic20) were missing. We propose that these missing genes were transferred to the Paulinella nucleus and their products are imported and integrated into the endosymbiont envelope membranes, thereby creating an effective protein import apparatus. We further suggest that other bacterial/cyanobacterial endosymbionts found in protists, plants and animals could have evolved efficient protein import systems independently and, therefore, reached the status of true cellular organelles.

  9. Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts.

    Science.gov (United States)

    Chen, Kuan-Yu; Li, Hsou-min

    2007-01-01

    The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.

  10. EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency.

    Science.gov (United States)

    Lee, E F; Chen, L; Yang, H; Colman, P M; Huang, D C S; Fairlie, W D

    2008-10-01

    Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x(L) by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim(S), which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity.

  11. Fission yeast ATF/CREB family protein Atf21 plays important roles in production of normal spores.

    Science.gov (United States)

    Morita, Tomohiko; Yamada, Takatomi; Yamada, Shintaro; Matsumoto, Kouji; Ohta, Kunihiro

    2011-02-01

    Activating transcription factor/cAMP response element binding protein (ATF/CREB) family transcription factors play central roles in maintaining cellular homeostasis. They are activated in response to environmental stimuli, bind to CRE sequences in the promoters of stress-response genes and regulate transcription. Although ATF/CREB proteins are widely conserved among most eukaryotes, their characteristics are highly diverse. Here, we investigated the functions of a fission yeast ATF/CREB protein Atf21 to find out its unique properties. We show that Atf21 is dispensable for the adaptive response to several stresses such as nitrogen starvation and for meiotic events including nuclear divisions. However, spores derived from atf21Δ mutants are not as mature as wild-type ones and are unable to form colonies under nutrition-rich conditions. Furthermore, we demonstrate that the Atf21 protein, which is scarce in early meiosis, gradually accumulates as meiosis proceeds; it reaches maximum levels approximately 8 h after nitrogen starvation and is present during germination. These results suggest that Atf21 is expressed and functions long after nitrogen starvation. Given that other well-characterized fission yeast ATF/CREB proteins Atf1 and Pcr1 accumulate and function promptly upon exposure to environmental stresses, we propose that Atf21 is a distinct member of the ATF/CREB family in fission yeast. © 2010 The Authors. Journal compilation © 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  12. In vitro and in vivo studies identify important features of dengue virus pr-E protein interactions.

    Directory of Open Access Journals (Sweden)

    Aihua Zheng

    2010-10-01

    Full Text Available Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors.

  13. In Vitro and In Vivo Studies Identify Important Features of Dengue Virus pr-E Protein Interactions

    Science.gov (United States)

    Zheng, Aihua; Umashankar, Mahadevaiah; Kielian, Margaret

    2010-01-01

    Flaviviruses bud into the endoplasmic reticulum and are transported through the secretory pathway, where the mildly acidic environment triggers particle rearrangement and allows furin processing of the prM protein to pr and M. The peripheral pr peptide remains bound to virus at low pH and inhibits virus-membrane interaction. Upon exocytosis, the release of pr at neutral pH completes virus maturation to an infectious particle. Together this evidence suggests that pr may shield the flavivirus fusion protein E from the low pH environment of the exocytic pathway. Here we developed an in vitro system to reconstitute the interaction of dengue virus (DENV) pr with soluble truncated E proteins. At low pH recombinant pr bound to both monomeric and dimeric forms of E and blocked their membrane insertion. Exogenous pr interacted with mature infectious DENV and specifically inhibited virus fusion and infection. Alanine substitution of E H244, a highly conserved histidine residue in the pr-E interface, blocked pr-E interaction and reduced release of DENV virus-like particles. Folding, membrane insertion and trimerization of the H244A mutant E protein were preserved, and particle release could be partially rescued by neutralization of the low pH of the secretory pathway. Thus, pr acts to silence flavivirus fusion activity during virus secretion, and this function can be separated from the chaperone activity of prM. The sequence conservation of key residues involved in the flavivirus pr-E interaction suggests that this protein-protein interface may be a useful target for broad-spectrum inhibitors. PMID:20975939

  14. Importance of interferon inducible trans-membrane proteins and retinoic acid inducible gene I for influenza virus replication: A review.

    Science.gov (United States)

    Suo, Siqingaowa; Ren, Xiaofeng

    2016-01-01

    Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.

  15. MS(E) based multiplex protein analysis quantified important allergenic proteins and detected relevant peptides carrying known epitopes in wheat grain extracts.

    Science.gov (United States)

    Uvackova, Lubica; Skultety, Ludovit; Bekesova, Slavka; McClain, Scott; Hajduch, Martin

    2013-11-01

    The amount of clinically relevant, allergy-related proteins in wheat grain is still largely unknown. The application of proteomics may create a platform not only for identification and characterization, but also for quantitation of these proteins. The aim of this study was to evaluate the data-independent quantitative mass spectrometry (MS(E)) approach in combination with 76 wheat allergenic sequences downloaded from the AllergenOnline database ( www.allergenonline.org ) as a starting point. Alcohol soluble extracts of gliadin and glutenin proteins were analyzed. This approach has resulted in identification and quantification of 15 allergenic protein isoforms that belong to amylase/trypsin inhibitors, γ-gliadins, and high or low molecular weight glutenins. Additionally, several peptides carrying four previously discovered epitopes of γ-gliadin B precursor have been detected. These data were validated against the UniProt database, which contained 11764 Triticeae protein sequences. The identified allergens are discussed in relation to Baker's asthma, food allergy, wheat dependent exercise induced anaphylaxis, atopic dermatitis, and celiac disease (i.e., gluten-sensitive enteropathy). In summary, the results showed that the MS(E) approach is suitable for quantitative analysis and allergens profiling in wheat varieties and/or other food matrices.

  16. Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

    Science.gov (United States)

    Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

  17. PfEMP1 - A Parasite Protein Family of Key Importance in Plasmodium falciparum Malaria Immunity and Pathogenesis.

    Science.gov (United States)

    Hviid, Lars; Jensen, Anja T R

    2015-04-01

    Plasmodium falciparum causes the most severe form of malaria and is responsible for essentially all malaria-related deaths. The accumulation in various tissues of erythrocytes infected by mature P. falciparum parasites can lead to circulatory disturbances and inflammation, and is thought to be a central element in the pathogenesis of the disease. It is mediated by the interaction of parasite ligands on the erythrocyte surface and a range of host receptor molecules in many organs and tissues. Among several proteins and protein families implicated in this process, the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family of high-molecular weight and highly variable antigens appears to be the most prominent. In this chapter, we aim to provide a systematic overview of the current knowledge about these proteins, their structure, their function, how they are presented on the erythrocyte surface, and how the var genes encoding them are regulated. The role of PfEMP1 in the pathogenesis of malaria, PfEMP1-specific immune responses, and the prospect of PfEMP1-specific vaccination against malaria are also covered briefly. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. The peptide antibiotic microcin 25 is imported through the TonB pathway and the SbmA protein.

    OpenAIRE

    Salomón, R A; Farías, R N

    1995-01-01

    Selection of spontaneous mutants for insensitivity to the peptide antibiotic microcin 25 led to the isolation of five categories of mutants. Phenotypic and mapping studies showed the mutations to be located in the fhuA, exb, tonB, and sbmA genes. The latter encodes a cytoplasmic membrane protein which is also required for the penetration of microcin B17.

  19. Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis.

    Science.gov (United States)

    Hiras, Jennifer; Wu, Yu-Wei; Deng, Kai; Nicora, Carrie D; Aldrich, Joshua T; Frey, Dario; Kolinko, Sebastian; Robinson, Errol W; Jacobs, Jon M; Adams, Paul D; Northen, Trent R; Simmons, Blake A; Singer, Steven W

    2016-08-23

    Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora, the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered. Cellulose is the most abundant organic polymer on earth, and its enzymatic hydrolysis is a key reaction in the global carbon cycle and the conversion of plant biomass to biofuels. The glycoside hydrolases that depolymerize crystalline cellulose have been primarily characterized from isolates. In this study, we demonstrate that adapting microbial consortia from compost to grow on crystalline cellulose generated communities whose soluble enzymes exhibit differential abilities to hydrolyze crystalline

  20. Cold-inducible RNA-binding protein is an important mediator of alcohol-induced brain inflammation.

    Directory of Open Access Journals (Sweden)

    Salil R Rajayer

    Full Text Available Binge drinking has been associated with cerebral dysfunction. Ethanol induced microglial activation initiates an inflammatory process that causes upregulation of proinflammatory cytokines which in turn creates neuronal inflammation and damage. However, the molecular mechanism is not fully understood. We postulate that cold-inducible RNA-binding protein (CIRP, a novel proinflammatory molecule, can contribute to alcohol-induced neuroinflammation. To test this theory male wild-type (WT mice were exposed to alcohol at concentrations consistent to binge drinking and blood and brain tissues were collected. At 5 h after alcohol, a significant increase of 53% in the brain of CIRP mRNA was observed and its expression remained elevated at 10 h and 15 h. Brain CIRP protein levels were increased by 184% at 10 h and remained high at 15 h. We then exposed male WT and CIRP knockout (CIRP(-/- mice to alcohol, and blood and brain tissues were collected at 15 h post-alcohol infusion. Serum levels of tissue injury markers (AST, ALT and LDH were significantly elevated in alcohol-exposed WT mice while they were less increased in the CIRP(-/- mice. Brain TNF-α mRNA and protein expressions along with IL-1β protein levels were significantly increased in WT mice, which was not seen in the CIRP(-/- mice. In cultured BV2 cells (mouse microglia, ethanol at 100 mM showed an increase of CIRP mRNA by 274% and 408% at 24 h and 48 h respectively. Corresponding increases in TNF-α and IL-1β were also observed. CIRP protein levels were markedly increased in the medium, suggesting that CIRP was secreted by the BV2 cells. From this we conclude that alcohol exposure activates microglia to produce and secrete CIRP and possibly induce pro-inflammatory response and thereby causing neuroinflammation. CIRP could be a novel mediator of alcohol-induced brain inflammation.

  1. ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.

    Science.gov (United States)

    Hou, Jie M; D'Lima, Nadia G; Rigel, Nathan W; Gibbons, Henry S; McCann, Jessica R; Braunstein, Miriam; Teschke, Carolyn M

    2008-07-01

    The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

  2. Histidine and Aspartic Acid Residues Important for Immunoglobulin G Endopeptidase Activity of the Group A Streptococcus Opsonophagocytosis-Inhibiting Mac Protein

    Science.gov (United States)

    Lei, Benfang; Liu, Mengyao; Meyers, Elishia G.; Manning, Heather M.; Nagiec, Michael J.; Musser, James M.

    2003-01-01

    The secreted Mac protein made by serotype M1 group A Streptococcus (GAS) (designated Mac5005) inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear neutrophils. This protein also has cysteine endopeptidase activity against human immunoglobulin G (IgG). Site-directed mutagenesis was used to identify histidine and aspartic acid residues important for Mac IgG endopeptidase activity. Replacement of His262 with Ala abolished Mac5005 IgG endopeptidase activity. Asp284Ala and Asp286Ala mutant proteins had compromised enzymatic activity, whereas 21 other Asp-to-Ala mutant proteins cleaved human IgG at the apparent wild-type level. The results suggest that His262 is an active-site residue and that Asp284 and Asp286 are important for the enzymatic activity or structure of Mac protein. These Mac mutants provide new information about structure-activity relationships in this protein and will assist study of the mechanism of inhibition of opsonophagocytosis and killing of GAS by Mac. PMID:12704162

  3. Large-scale evaluation of dynamically important residues in proteins predicted by the perturbation analysis of a coarse-grained elastic model

    Directory of Open Access Journals (Sweden)

    Tekpinar Mustafa

    2009-07-01

    Full Text Available Abstract Backgrounds It is increasingly recognized that protein functions often require intricate conformational dynamics, which involves a network of key amino acid residues that couple spatially separated functional sites. Tremendous efforts have been made to identify these key residues by experimental and computational means. Results We have performed a large-scale evaluation of the predictions of dynamically important residues by a variety of computational protocols including three based on the perturbation and correlation analysis of a coarse-grained elastic model. This study is performed for two lists of test cases with >500 pairs of protein structures. The dynamically important residues predicted by the perturbation and correlation analysis are found to be strongly or moderately conserved in >67% of test cases. They form a sparse network of residues which are clustered both in 3D space and along protein sequence. Their overall conservation is attributed to their dynamic role rather than ligand binding or high network connectivity. Conclusion By modeling how the protein structural fluctuations respond to residue-position-specific perturbations, our highly efficient perturbation and correlation analysis can be used to dissect the functional conformational changes in various proteins with a residue level of detail. The predictions of dynamically important residues serve as promising targets for mutational and functional studies.

  4. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    Science.gov (United States)

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process.

  5. Electrochemical determination of heme-linked pKa values and the importance of using fluoride binding in heme proteins.

    Science.gov (United States)

    Cerda, Jose F; Roeder, Margaret H; Houchins, Danielle N; Guzman, Carmen X; Amendola, Emily J; Castorino, Jacquelyn D; Fritz, Andrea L

    2013-12-01

    The ultraviolet-visible (UV-vis) spectroelectrochemical measurements of heme proteins in the presence of a heme-bound fluoride ion can be used as a probe for heme-linked ionizations of acid-base groups in the heme pocket. A detailed study of the pH dependence of the midpoint potential of skeletal horse myoglobin (Mb) with a heme-bound fluoride ion (Mb-F) reveals how protonation of the distal histidine (H64) changes the redox properties of the protein with a determined pKa of 5.3. In addition, fluoride binding in myoglobin provides a stabilization of -1.9 kcal/mol of the ferric Mb-F relative to ferric Mb without fluoride.

  6. LDRD Final Report (08-ERD-037): Important Modes to Drive Protein MD Simulations to the Next Conformational Level

    Energy Technology Data Exchange (ETDEWEB)

    Sadigh, B

    2011-04-07

    Every action in biology is performed by dynamic proteins that convert between multiple states in order to engage their functions. Often binding to various ligands is essential for the rates of desired transitions to be enhanced. The goal of computational biology is to study these transitions and discover the different states to fully understand the protein's normal and diseased function, design drugs to target/bias specific states, and understand all of the interactions in between. We have developed a new methodology that is capable of calculating the absolute free energy of proteins while taking into account all the interactions with the solvent molecules. The efficiency of the new scheme is an order of magnitude greater than any existing technique. This method is now implemented in the massively parallel popular MD program package NAMD. This now makes it possible to calculate the relative stability of different conformational states of biological macromolecules as well as their binding free energies to various ligands.

  7. Nuclear Import and Dimerization of Tomato ASR1, a Water Stress-Inducible Protein Exclusive to Plants

    Science.gov (United States)

    Ricardi, Martiniano M.; Guaimas, Francisco F.; González, Rodrigo M.; Burrieza, Hernán P.; López-Fernández, María P.; Estévez, José M.; Iusem, Norberto D.

    2012-01-01

    The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an “NLS” of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE. PMID:22899993

  8. Nuclear import and dimerization of tomato ASR1, a water stress-inducible protein exclusive to plants.

    Directory of Open Access Journals (Sweden)

    Martiniano M Ricardi

    Full Text Available The ASR (for ABA/water stress/ripening protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.

  9. Nuclear import and dimerization of tomato ASR1, a water stress-inducible protein exclusive to plants.

    Science.gov (United States)

    Ricardi, Martiniano M; Guaimas, Francisco F; González, Rodrigo M; Burrieza, Hernán P; López-Fernández, María P; Jares-Erijman, Elizabeth A; Estévez, José M; Iusem, Norberto D

    2012-01-01

    The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE.

  10. Role of the C-terminal extension peptide of plastid located glutamine synthetase from Medicago truncatula: Crucial for enzyme activity and needless for protein import into the plastids.

    Science.gov (United States)

    Ferreira, Maria João; Vale, Diogo; Cunha, Luis; Melo, Paula

    2017-02-01

    Glutamine synthetase (GS), a key enzyme in plant nitrogen metabolism, is encoded by a small family of highly homologous nuclear genes that produce cytosolic (GS1) and plastidic (GS2) isoforms. Compared to GS1, GS2 proteins have two extension peptides, one at the N- and the other at the C-terminus, which show a high degree of conservation among plant species. It has long been known that the N-terminal peptide acts as a transit peptide, targeting the protein to the plastids however, the function of the C-terminal extension is still unknown. To investigate whether the C-terminal extension influences the activity of the enzyme, we produced a C-terminal truncated version of Medicago truncatula GS2a in Escherechia coli and studied its catalytic properties. The activity of the truncated protein was found to be lower than that of MtGS2a and with less affinity for glutamate. The importance of the C-terminal extension for the protein import into the chloroplast was also assessed by transient expression of fluorescently-tagged MtGS2a truncated at the C-terminus, which was correctly detected in the chloroplast. The results obtained in this work demonstrate that the C-terminal extension of M. truncatula GS2a is important for the activity of the enzyme and does not contain crucial information for the import process.

  11. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import.

    LENUS (Irish Health Repository)

    Gu, Lili

    2011-03-14

    Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS) by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s) predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein) as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  12. Intermolecular masking of the HIV-1 Rev NLS by the cellular protein HIC: Novel insights into the regulation of Rev nuclear import

    Directory of Open Access Journals (Sweden)

    Sheehy Noreen

    2011-03-01

    Full Text Available Abstract Background The HIV-1 regulatory protein Rev, which is essential for viral replication, mediates the nuclear export of unspliced viral transcripts. Rev nuclear function requires active nucleocytoplasmic shuttling, and Rev nuclear import is mediated by the recognition of its Nuclear Localisation Signal (NLS by multiple import factors, which include transportin and importin β. However, it remains unclear which nuclear import pathway(s predominate in vivo, and the cellular environment that modulates Rev nucleocytoplasmic shuttling remains to be characterised. Results In our study, we have identified the cellular protein HIC (Human I-mfa domain-Containing protein as a novel interactor of HIV-1 Rev. We demonstrate that HIC selectively interferes with Rev NLS interaction with importin β and impedes its nuclear import and function, but does not affect Rev nuclear import mediated by transportin. Hence, the molecular determinants mediating Rev-NLS recognition by importin β and transportin appear to be distinct. Furthermore, we have employed HIC and M9 M, a peptide specifically designed to inhibit the transportin-mediated nuclear import pathway, to characterise Rev nuclear import pathways within different cellular environments. Remarkably, we could show that in 293T, HeLa, COS7, Jurkat, U937, THP-1 and CEM cells, Rev nuclear import is cell type specific and alternatively mediated by transportin or importin β, in a mutually exclusive fashion. Conclusions Rev cytoplasmic sequestration by HIC may represent a novel mechanism for the control of Rev function. These studies highlight that the multivalent nature of the Rev NLS for different import receptors enables Rev to adapt its nuclear trafficking strategy.

  13. A "coiled-coil" motif is important for oligomerization and DNA binding properties of human cytomegalovirus protein UL77.

    Directory of Open Access Journals (Sweden)

    Christina Sylvia Meissner

    Full Text Available Human cytomegalovirus (HCMV UL77 gene encodes the essential protein UL77, its function is characterized in the present study. Immunoprecipitation identified monomeric and oligomeric pUL77 in HCMV infected cells. Immunostaining of purified virions and subviral fractions showed that pUL77 is a structural protein associated with capsids. In silico analysis revealed the presence of a coiled-coil motif (CCM at the N-terminus of pUL77. Chemical cross-linking of either wild-type pUL77 or CCM deletion mutant (pUL77ΔCCM implicated that CCM is critical for oligomerization of pUL77. Furthermore, co-immunoprecipitations of infected and transfected cells demonstrated that pUL77 interacts with the capsid-associated DNA packaging motor components, pUL56 and pUL104, as well as the major capsid protein. The ability of pUL77 to bind dsDNA was shown by an in vitro assay. Binding to certain DNA was further confirmed by an assay using biotinylated 36-, 250-, 500-, 1000-meric dsDNA and 966-meric HCMV-specific dsDNA designed for this study. The binding efficiency (BE was determined by image processing program defining values above 1.0 as positive. While the BE of the pUL56 binding to the 36-mer bio-pac1 containing a packaging signal was 10.0 ± 0.63, the one for pUL77 was only 0.2±0.03. In contrast to this observation the BE of pUL77 binding to bio-500 bp or bio-1000 bp was 2.2 ± 0.41 and 4.9 ± 0.71, respectively. By using pUL77ΔCCM it was demonstrated that this protein could not bind to dsDNA. These data indicated that pUL77 (i could form homodimers, (ii CCM of pUL77 is crucial for oligomerization and (iii could bind to dsDNA in a sequence independent manner.

  14. Proteomics Profiling Reveals Carbohydrate Metabolic Enzymes and 14-3-3 Proteins Play Important Roles for Starch Accumulation during Cassava Root Tuberization.

    Science.gov (United States)

    Wang, Xuchu; Chang, Lili; Tong, Zheng; Wang, Dongyang; Yin, Qi; Wang, Dan; Jin, Xiang; Yang, Qian; Wang, Liming; Sun, Yong; Huang, Qixing; Guo, Anping; Peng, Ming

    2016-01-01

    Cassava is one of the most important root crops as a reliable source of food and carbohydrates. Carbohydrate metabolism and starch accumulation in cassava storage root is a cascade process that includes large amounts of proteins and cofactors. Here, comparative proteomics were conducted in cassava root at nine developmental stages. A total of 154 identified proteins were found to be differentially expressed during starch accumulation and root tuberization. Many enzymes involved in starch and sucrose metabolism were significantly up-regulated, and functional classification of the differentially expressed proteins demonstrated that the majority were binding-related enzymes. Many proteins were took part in carbohydrate metabolism to produce energy. Among them, three 14-3-3 isoforms were induced to be clearly phosphorylated during storage root enlargement. Overexpression of a cassava 14-3-3 gene in Arabidopsis thaliana confirmed that the older leaves of these transgenic plants contained higher sugar and starch contents than the wild-type leaves. The 14-3-3 proteins and their binding enzymes may play important roles in carbohydrate metabolism and starch accumulation during cassava root tuberization. These results not only deepened our understanding of the tuberous root proteome, but also uncovered new insights into carbohydrate metabolism and starch accumulation during cassava root enlargement.

  15. Conserved structural motifs at the C-terminus of baculovirus protein IE0 are important for its functions in transactivation and supporting hr5-mediated DNA replication.

    Science.gov (United States)

    Luria, Neta; Lu, Liqun; Chejanovsky, Nor

    2012-05-01

    IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  16. Conserved Structural Motifs at the C-Terminus of Baculovirus Protein IE0 are Important for its Functions in Transactivation and Supporting hr5-mediated DNA Replication

    Directory of Open Access Journals (Sweden)

    Neta Luria

    2012-05-01

    Full Text Available IE0 and IE1 are transactivator proteins of the most studied baculovirus, the Autographa californica multiple nucleopolyhedrovirus (AcMNPV. IE0 is a 72.6 kDa protein identical to IE1 with the exception of its 54 N-terminal amino acid residues. To gain some insight about important structural motifs of IE0, we expressed the protein and C‑terminal mutants of it under the control of the Drosophila heat shock promoter and studied the transactivation and replication functions of the transiently expressed proteins. IE0 was able to promote replication of a plasmid bearing the hr5 origin of replication of AcMNPV in transient transfections with a battery of eight plasmids expressing the AcMNPV genes dnapol, helicase, lef-1, lef-2, lef-3, p35, ie-2 and lef-7. IE0 transactivated expression of the baculovirus 39K promoter. Both functions of replication and transactivation were lost after introduction of selected mutations at the basic domain II and helix-loop-helix conserved structural motifs in the C-terminus of the protein. These IE0 mutants were unable to translocate to the cell nucleus. Our results point out the important role of some structural conserved motifs to the proper functioning of IE0.

  17. On the importance of the distance measures used to train and test knowledge-based potentials for proteins.

    Science.gov (United States)

    Carlsen, Martin; Koehl, Patrice; Røgen, Peter

    2014-01-01

    Knowledge-based potentials are energy functions derived from the analysis of databases of protein structures and sequences. They can be divided into two classes. Potentials from the first class are based on a direct conversion of the distributions of some geometric properties observed in native protein structures into energy values, while potentials from the second class are trained to mimic quantitatively the geometric differences between incorrectly folded models and native structures. In this paper, we focus on the relationship between energy and geometry when training the second class of knowledge-based potentials. We assume that the difference in energy between a decoy structure and the corresponding native structure is linearly related to the distance between the two structures. We trained two distance-based knowledge-based potentials accordingly, one based on all inter-residue distances (PPD), while the other had the set of all distances filtered to reflect consistency in an ensemble of decoys (PPE). We tested four types of metric to characterize the distance between the decoy and the native structure, two based on extrinsic geometry (RMSD and GTD-TS*), and two based on intrinsic geometry (Q* and MT). The corresponding eight potentials were tested on a large collection of decoy sets. We found that it is usually better to train a potential using an intrinsic distance measure. We also found that PPE outperforms PPD, emphasizing the benefits of capturing consistent information in an ensemble. The relevance of these results for the design of knowledge-based potentials is discussed.

  18. Hematopoietic lineage cell specific protein 1 (HS1) is a functionally important signaling molecule in platelet activation.

    Science.gov (United States)

    Kahner, Bryan N; Dorsam, Robert T; Mada, Sripal R; Kim, Soochong; Stalker, Timothy J; Brass, Lawrence F; Daniel, James L; Kitamura, Daisuke; Kunapuli, Satya P

    2007-10-01

    Collagen activates platelets through an intracellular signaling cascade downstream of glycoprotein VI (GPVI). We have investigated the contribution of hematopoietic lineage cell-specific protein 1 (HS1) downstream of GPVI in platelet activation. Stimulation of GPVI leads to tyrosine phosphorylation of HS1, which is blocked by Src-family kinase inhibitors. Coimmunoprecipitation experiments revealed that HS1 associates with Syk and phosphatidylinositol 3-kinases. HS1-null mice displayed increased bleeding times and increased time to occlusion in the FeCl(3) in vivo thrombosis model compared with their wild-type littermates. In addition, aggregation and secretion responses were diminished in HS1-null mouse platelets after stimulation of GPVI and protease-activated receptor 4 (PAR-4) agonists compared with wild-type littermate mouse platelets. Finally, Akt phosphorylation was diminished after GPVI or PAR-4 stimulation in platelets from HS1-null mice compared with their wild-type littermates. These results demonstrate that phosphorylation of the HS1 protein occurs downstream of GPVI stimulation and that HS1 plays a significant functional role in platelet activation downstream of GPVI and PARs.

  19. The Popeye domain containing protein family – A novel class of cAMP effectors with important functions in multiple tissues

    Science.gov (United States)

    Schindler, Roland F.R.; Brand, Thomas

    2016-01-01

    Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle. PMID:26772438

  20. The Popeye domain containing protein family--A novel class of cAMP effectors with important functions in multiple tissues.

    Science.gov (United States)

    Schindler, Roland F R; Brand, Thomas

    2016-01-01

    Popeye domain containing (Popdc) proteins are a unique family, which combine several different properties and functions in a surprisingly complex fashion. They are expressed in multiple tissues and cell types, present in several subcellular compartments, interact with different classes of proteins, and are associated with a variety of physiological and pathophysiological processes. Moreover, Popdc proteins bind the second messenger cAMP with high affinity and it is thought that they act as a novel class of cAMP effector proteins. Here, we will review the most important findings about the Popdc family, which accumulated since its discovery about 15 years ago. We will be focussing on Popdc protein interaction and function in striated muscle tissue. However, as a full picture only emerges if all aspects are taken into account, we will also describe what is currently known about the role of Popdc proteins in epithelial cells and in various types of cancer, and discuss these findings with regard to their relevance for cardiac and skeletal muscle.

  1. Protein

    Science.gov (United States)

    ... Food Service Resources Additional Resources About FAQ Contact Protein Protein is found throughout the body—in muscle, ... the heart and respiratory system, and death. All Protein Isn’t Alike Protein is built from building ...

  2. THE IMPORTANCE OF MONITORING CHANGES IN MILK FAT TO MILK PROTEIN RATIO IN HOLSTEIN COWS DURING LACTATION

    Directory of Open Access Journals (Sweden)

    Vladimír ČEJNA

    2006-07-01

    Full Text Available Milk samples of 24 pure-bred Holstein cows were taken during lactation and changes in milk fat to milk protein ratio and quality of rennet gel were evaluated. The cows were either fi rst (n=12 or second (n=12 calvers, in the same phase of lactation. The individual milk samples were taken on average on the day 25, 45, 73, 101, 133, 166, 199, 224, 253 and 280 of lactation and the following mean values of the F/P ratio were found: 1.91; 1.45; 1.38; 1.28; 1.22; 1.14; 1.26; 1.21; 1.09; 1.18. High values in the fi rst phase suggest a great energy defi ciency. The quality of rennet gel was also worst in the fi rst phase of lactation.

  3. On the importance of the distance measures used to train and test knowledge-based potentials for proteins

    DEFF Research Database (Denmark)

    Carlsen, Martin; Koehl, Patrice; Røgen, Peter

    2014-01-01

    protein structures into energy values, while potentials from the second class are trained to mimic quantitatively the geometric differences between incorrectly folded models and native structures. In this paper, we focus on the relationship between energy and geometry when training the second class...... of knowledge-based potentials. We assume that the difference in energy between a decoy structure and the corresponding native structure is linearly related to the distance between the two structures. We trained two distance-based knowledge-based potentials accordingly, one based on all inter-residue distances...... geometry (Q* and MT). The corresponding eight potentials were tested on a large collection of decoy sets. We found that it is usually better to train a potential using an intrinsic distance measure. We also found that PPE outperforms PPD, emphasizing the benefits of capturing consistent information...

  4. A nuclear fraction of turnip crinkle virus capsid protein is important for elicitation of the host resistance response.

    Science.gov (United States)

    Kang, Sung-Hwan; Qu, Feng; Morris, T Jack

    2015-12-01

    The N-terminal 25 amino acids (AAs) of turnip crinkle virus (TCV) capsid protein (CP) are recognized by the resistance protein HRT to trigger a hypersensitive response (HR) and systemic resistance to TCV infection. This same region of TCV CP also contains a motif that interacts with the transcription factor TIP, as well as a nuclear localization signal (NLS). However, it is not yet known whether nuclear localization of TCV CP is needed for the induction of HRT-mediated HR and resistance. Here we present new evidence suggesting a tight correlation between nuclear inclusions formed by CP and the manifestation of HR. We show that a fraction of TCV CP localized to cell nuclei to form discrete inclusion-like structures, and a mutated CP (R6A) known to abolish HR failed to form nuclear inclusions. Notably, TIP-CP interaction augments the inclusion-forming activity of CP by tethering inclusions to the nuclear membrane. This TIP-mediated augmentation is also critical for HR resistance, as another CP mutant (R8A) known to elicit a less restrictive HR, though still self-associated into nuclear inclusions, failed to direct inclusions to the nuclear membrane due to its inability to interact with TIP. Finally, exclusion of CP from cell nuclei abolished induction of HR. Together, these results uncovered a strong correlation between nuclear localization and nuclear inclusion formation by TCV CP and induction of HR, and suggest that CP nuclear inclusions could be the key trigger of the HRT-dependent, yet TIP-reinforced, resistance to TCV.

  5. On the importance of the distance measures used to train and test knowledge-based potentials for proteins.

    Directory of Open Access Journals (Sweden)

    Martin Carlsen

    Full Text Available Knowledge-based potentials are energy functions derived from the analysis of databases of protein structures and sequences. They can be divided into two classes. Potentials from the first class are based on a direct conversion of the distributions of some geometric properties observed in native protein structures into energy values, while potentials from the second class are trained to mimic quantitatively the geometric differences between incorrectly folded models and native structures. In this paper, we focus on the relationship between energy and geometry when training the second class of knowledge-based potentials. We assume that the difference in energy between a decoy structure and the corresponding native structure is linearly related to the distance between the two structures. We trained two distance-based knowledge-based potentials accordingly, one based on all inter-residue distances (PPD, while the other had the set of all distances filtered to reflect consistency in an ensemble of decoys (PPE. We tested four types of metric to characterize the distance between the decoy and the native structure, two based on extrinsic geometry (RMSD and GTD-TS*, and two based on intrinsic geometry (Q* and MT. The corresponding eight potentials were tested on a large collection of decoy sets. We found that it is usually better to train a potential using an intrinsic distance measure. We also found that PPE outperforms PPD, emphasizing the benefits of capturing consistent information in an ensemble. The relevance of these results for the design of knowledge-based potentials is discussed.

  6. 70-kDa Heat Shock Cognate Protein hsc70 Mediates Calmodulin-dependent Nuclear Import of the Sex-determining Factor SRY*

    Science.gov (United States)

    Kaur, Gurpreet; Lieu, Kim G.; Jans, David A.

    2013-01-01

    We recently showed that the developmentally important family of SOX (SRY (sex determining region on the Y chromosome)-related high mobility group (HMG) box) proteins require the calcium-binding protein calmodulin (CaM) for optimal nuclear accumulation, with clinical mutations in SRY that specifically impair nuclear accumulation via this pathway resulting in XY sex reversal. However, the mechanism by which CaM facilitates nuclear accumulation is unknown. Here, we show, for the first time, that the 70-kDa heat shock cognate protein hsc70 plays a key role in CaM-dependent nuclear import of SRY. Using a reconstituted nuclear import assay, we show that antibodies to hsc70 significantly reduce nuclear accumulation of wild type SRY and mutant derivatives thereof that retain CaM-dependent nuclear import, with an increased rate of nuclear accumulation upon addition of both CaM and hsc70, in contrast to an SRY mutant derivative with impaired CaM binding. siRNA knockdown of hsc70 in intact cells showed similar results, indicating clear dependence upon hsc70 for CaM-dependent nuclear import. Analysis using the technique of fluorescence recovery after photobleaching indicated that hsc70 is required for the maximal rate of SRY nuclear import in living cells but has no impact upon SRY nuclear retention/nuclear dynamics. Finally, we demonstrate direct binding of hsc70 to the SRY·CaM complex, with immunoprecipitation experiments from cell extracts showing association of hsc70 with wild type SRY, but not with a mutant derivative with impaired CaM binding, dependent on Ca2+. Our novel findings strongly implicate hsc70 in CaM-dependent nuclear import of SRY. PMID:23235156

  7. Importance of polarization effect in the study of metalloproteins: application of polarized protein specific charge scheme in predicting the reduction potential of azurin.

    Science.gov (United States)

    Wei, Caiyi; Lazim, Raudah; Zhang, Dawei

    2014-09-01

    Molecular dynamics (MD) simulation is commonly used in the study of protein dynamics, and in recent years, the extension of MD simulation to the study of metalloproteins is gaining much interest. Choice of force field is crucial in MD studies, and the inclusion of metal centers complicates the process of accurately describing the electrostatic environment that surrounds the redox centre. Herein, we would like to explore the importance of including electrostatic contribution from both protein and solvent in the study of metalloproteins. MD simulations with the implementation of thermodynamic integration will be conducted to model the reduction process of azurin from Pseudomonas aeruginosa. Three charge schemes will be used to derive the partial charges of azurin. These charge schemes differ in terms of the amount of immediate environment, respective to copper, considered during charge fitting, which ranges from the inclusion of copper and residues in the first coordination sphere during density functional theory charge fitting to the comprehensive inclusion of protein and solvent effect surrounding the metal centre using polarized protein-specific charge scheme. From the simulations conducted, the relative reduction potential of the mutated azurins respective to that of wild-type azurin (ΔEcal) were calculated and compared with experimental values. The ΔEcal approached experimental value with increasing consideration of environmental effect hence substantiating the importance of polarization effect in the study of metalloproteins. This study also attests the practicality of polarized protein-specific charge as a computational tool capable of incorporating both protein environment and solvent effect into MD simulations.

  8. Importance of anisotropy in detachment rates for force production and cargo transport by a team of motor proteins.

    Science.gov (United States)

    Takshak, Anjneya; Kunwar, Ambarish

    2016-05-01

    Many cellular processes are driven by collective forces generated by a team consisting of multiple molecular motor proteins. One aspect that has received less attention is the detachment rate of molecular motors under mechanical force/load. While detachment rate of kinesin motors measured under backward force increases rapidly for forces beyond stall-force; this scenario is just reversed for non-yeast dynein motors where detachment rate from microtubule decreases, exhibiting a catch-bond type behavior. It has been shown recently that yeast dynein responds anisotropically to applied load, i.e. detachment rates are different under forward and backward pulling. Here, we use computational modeling to show that these anisotropic detachment rates might help yeast dynein motors to improve their collective force generation in the absence of catch-bond behavior. We further show that the travel distance of cargos would be longer if detachment rates are anisotropic. Our results suggest that anisotropic detachment rates could be an alternative strategy for motors to improve the transport properties and force production by the team.

  9. Pseudomonas fluorescens filamentous hemagglutinin, an iron-regulated protein, is an important virulence factor that modulates bacterial pathogenicity

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    Yuan-yuan Sun

    2016-08-01

    Full Text Available Pseudomonas fluorescens is a common bacterial pathogen to a wide range of aquaculture animals including various species of fish. In this study, we employed proteomic analysis and identified filamentous hemagglutinin (FHA as an iron-responsive protein secreted by TSS, a pathogenic P. fluorescens isolate. In vitro study showed that compared to the wild type, the fha mutant TSSfha (i exhibited a largely similar vegetative growth profile but significantly retarded in the ability of biofilm growth and producing extracellular matrix, (ii displayed no apparent flagella and motility, (iii was defective in the attachment to host cells and unable to form self-aggregation, (iv displayed markedly reduced capacity of hemagglutination and surviving in host serum. In vivo infection analysis revealed that TSSfha was significantly attenuated in the ability of dissemination in fish tissues and inducing host mortality, and that antibody blocking of the natural FHA produced by the wild type TSS impaired the infectivity of the pathogen. Furthermore, when introduced into turbot as a subunit vaccine, recombinant FHA elicited a significant protection against lethal TSS challenge. Taken together, these results indicate for the first time that P. fluorescens FHA is a key virulence factor essential to multiple biological processes associated with pathogenicity.

  10. Prion protein is expressed on long-term repopulating hematopoietic stem cells and is important for their self-renewal.

    Science.gov (United States)

    Zhang, Cheng Cheng; Steele, Andrew D; Lindquist, Susan; Lodish, Harvey F

    2006-02-14

    Although the wild-type prion protein (PrP) is abundant and widely expressed in various types of tissues and cells, its physiological function(s) remain unknown, and PrP knockout mice do not exhibit overt and undisputed phenotypes. Here we showed that PrP is expressed on the surface of several bone marrow cell populations successively enriched in long-term (LT) hematopoietic stem cells (HSCs) using flow cytometry analysis. Affinity purification of the PrP-positive and -negative fractions from these populations, followed by competitive bone marrow reconstitution assays, shows that all LT HSCs express PrP. HSCs from PrP-null bone marrow exhibited impaired self-renewal in serial transplantation of lethally irradiated mouse recipients both in the presence and absence of competitors. When treated with a cell cycle-specific myelotoxic agent, the animals reconstituted with PrP-null HSCs exhibit increased sensitivity to hematopoietic cell depletion. Ectopic expression of PrP in PrP-null bone marrow cells by retroviral infection rescued the defective hematopoietic engraftment during serial transplantation. Therefore, PrP is a marker for HSCs and supports their self-renewal.

  11. Quantum coupled mutation finder: predicting functionally or structurally important sites in proteins using quantum Jensen-Shannon divergence and CUDA programming.

    Science.gov (United States)

    Gültas, Mehmet; Düzgün, Güncel; Herzog, Sebastian; Jäger, Sven Joachim; Meckbach, Cornelia; Wingender, Edgar; Waack, Stephan

    2014-04-03

    The identification of functionally or structurally important non-conserved residue sites in protein MSAs is an important challenge for understanding the structural basis and molecular mechanism of protein functions. Despite the rich literature on compensatory mutations as well as sequence conservation analysis for the detection of those important residues, previous methods often rely on classical information-theoretic measures. However, these measures usually do not take into account dis/similarities of amino acids which are likely to be crucial for those residues. In this study, we present a new method, the Quantum Coupled Mutation Finder (QCMF) that incorporates significant dis/similar amino acid pair signals in the prediction of functionally or structurally important sites. The result of this study is twofold. First, using the essential sites of two human proteins, namely epidermal growth factor receptor (EGFR) and glucokinase (GCK), we tested the QCMF-method. The QCMF includes two metrics based on quantum Jensen-Shannon divergence to measure both sequence conservation and compensatory mutations. We found that the QCMF reaches an improved performance in identifying essential sites from MSAs of both proteins with a significantly higher Matthews correlation coefficient (MCC) value in comparison to previous methods. Second, using a data set of 153 proteins, we made a pairwise comparison between QCMF and three conventional methods. This comparison study strongly suggests that QCMF complements the conventional methods for the identification of correlated mutations in MSAs. QCMF utilizes the notion of entanglement, which is a major resource of quantum information, to model significant dissimilar and similar amino acid pair signals in the detection of functionally or structurally important sites. Our results suggest that on the one hand QCMF significantly outperforms the previous method, which mainly focuses on dissimilar amino acid signals, to detect essential sites

  12. The role and importance of glycosylation of acute phase proteins with focus on alpha-1 antitrypsin in acute and chronic inflammatory conditions.

    Science.gov (United States)

    McCarthy, Cormac; Saldova, Radka; Wormald, Mark R; Rudd, Pauline M; McElvaney, Noel G; Reeves, Emer P

    2014-07-03

    Acute phase proteins (APPs) are a group of circulating plasma proteins which undergo changes quantitatively or qualitatively at the time of inflammation. Many of these APPs are glycosylated, and it has been shown that alterations in glycosylation may occur in inflammatory and malignant conditions. Changes in glycosylation have been studied as potential biomarkers in cancer and also in chronic inflammatory conditions and have been shown to correlate with disease severity in certain conditions. Serine protease inhibitors (serpins), many of which are also APPs, are proteins involved in the control of proteases in numerous pathways. Alpha-1 Antitrypsin (AAT) is the most abundant serpin within the circulation and is an APP which has been shown to increase in response to inflammation. The primary role of AAT is maintaining the protease/antiprotease balance in the lung, but it also possesses important anti-inflammatory and immune-modulating properties. Several glycoforms of AAT exist, and they possess differing properties in regard to plasma half-life and stability. Glycosylation may also be important in determining the immune modulatory properties of AAT. The review will focus on the role and importance of glycosylation in acute phase proteins with particular attention to AAT and its use as a biomarker of disease. The review describes the processes involved in glycosylation, how glycosylation changes in differing disease states, and the alterations that occur to glycans of APPs with disease and inflammation. Finally, the review explores the importance of changes in glycosylation of AAT at times of inflammation and in malignant conditions and how this may impact upon the functions of AAT.

  13. Rice TUTOU1 Encodes a Suppressor of cAMP Receptor-Like Protein That Is Important for Actin Organization and Panicle Development.

    Science.gov (United States)

    Bai, Jiaoteng; Zhu, Xudong; Wang, Qing; Zhang, Jian; Chen, Hongqi; Dong, Guojun; Zhu, Lei; Zheng, Huakun; Xie, Qingjun; Nian, Jinqiang; Chen, Fan; Fu, Ying; Qian, Qian; Zuo, Jianru

    2015-10-01

    Panicle development, a key event in rice (Oryza sativa) reproduction and a critical determinant of grain yield, forms a branched structure containing multiple spikelets. Genetic and environmental factors can perturb panicle development, causing panicles to degenerate and producing characteristic whitish, small spikelets with severely reduced fertility and yield; however, little is known about the molecular basis of the formation of degenerating panicles in rice. Here, we report the identification and characterization of the rice panicle degenerative mutant tutou1 (tut1), which shows severe defects in panicle development. The tut1 also shows a pleiotropic phenotype, characterized by short roots, reduced plant height, and abnormal development of anthers and pollen grains. Molecular genetic studies revealed that TUT1 encodes a suppressor of cAMP receptor/Wiskott-Aldrich syndrome protein family verprolin-homologous (SCAR/WAVE)-like protein. We found that TUT1 contains conserved functional domains found in eukaryotic SCAR/WAVE proteins, and was able to activate Actin-related protein2/3 to promote actin nucleation and polymerization in vitro. Consistently, tut1 mutants show defects in the arrangement of actin filaments in trichome. These results indicate that TUT1 is a functional SCAR/WAVE protein and plays an important role in panicle development. © 2015 American Society of Plant Biologists. All Rights Reserved.

  14. GP-9s are ubiquitous proteins unlikely involved in olfactory mediation of social organization in the red imported fire ant, Solenopsis invicta.

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    Walter S Leal

    Full Text Available The red imported fire ant (RIFA, Solenopsis invicta, is an invasive species, accidentally introduced in the United States that can cause painful (sometimes life-threatening stings to human, pets, and livestock. Their colonies have two social forms: monogyne and polygyne that have a single and multiple functional queens, respectively. A major gene (Gp-9, identified as a putative pheromone-binding protein on the basis of a modest amino acid sequence identity, has been suggested to influence the expression of colony social organization. Monogyne queens are reported to possess only the GP-9B alleles, whereas polygyne queens possess both GP-9B and GP-9b. Thus, both social forms are reported to express GP-9B, with GP-9b being a marker expressed in polygynes but it is absent in monogynes. Here, we report two types of polygyne colonies, one that does not express GP-9b (monogyne-like and the other expressing both proteins, GP-9B and GP-9b. Given their expression pattern, GP-9s are hemolymph proteins, which are more likely to be involved in the transport of lipids and small ligands within the homocoel. GP-9B existed in two forms, one of them is phosphorylated. The helical-rich content of the protein resembles the secondary structures of a beetle hemolymph protein and moth pheromone-binding proteins. An olfactory role is unlikely given the lack of specific expression in the sensillar lymph. In marked contrast to GP-9s, a chemosensory protein, SinvCSP, is demonstrated to be specifically expressed in the antennae. Within the antennae, expression of SinvCSP is restricted to the last two segments, which are known to house olfactory sensilla.

  15. Phosphorylation of Serine 148 in Giardia lamblia End-binding 1 Protein is Important for Cell Division.

    Science.gov (United States)

    Kim, Juri; Lee, Hye-Yeon; Lee, Kyu-Ho; Park, Soon-Jung

    2017-07-01

    Giardia lamblia is a unicellular organism, showing a polarity with two nuclei and cytoskeletal structures. Accurate positioning of these organelles is essential for division of G. lamblia, which is poorly understood. Giardia lamblia end-binding 1 (GlEB1) protein and G. lamblia aurora kinase (GlAK) have been shown to modulate microtubule (MT) distribution during cytokinesis. A direct association between GlEB1 and GlAK was demonstrated. Like GlEB1, GlAK was also found at nuclear envelopes and median bodies of G. lamblia. In vitro kinase assays using Giardia lysates immunoprecipitated with anti-GlAK antibodies or recombinant GlAK suggested that GlEB1 is a substrate of GlAK. Site-directed mutagenesis indicated that threonine-205 in GlAK was auto-phosphorylated and that GlAK phosphorylated serine (Ser)-148 in GlEB1. Ectopic expression of a mutant GlEB1 (with conversion of Ser-148 into alanine of GlEB1) resulted in an increased number of Giardia cells with division defects. Treatment of G. lamblia with an AK inhibitor triggered cytokinesis defects, and ectopic expression of a phospho-mimetic mutant GlEB1 (with conversion of Ser-148 into aspartate) rescued the defects in Giardia cell division caused by the AK inhibitor. These results suggested that phosphorylation of GlEB1 played a role in cytokinesis in G. lamblia. © 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.

  16. A Petunia homeodomain-leucine zipper protein, PhHD-Zip, plays an important role in flower senescence.

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    Xiaoxiao Chang

    Full Text Available Flower senescence is initiated by developmental and environmental signals, and regulated by gene transcription. A homeodomain-leucine zipper transcription factor, PhHD-Zip, is up-regulated during petunia flower senescence. Virus-induced gene silencing of PhHD-Zip extended flower life by 20% both in unpollinated and pollinated flowers. Silencing PhHD-Zip also dramatically reduced ethylene production and the abundance of transcripts of genes involved in ethylene (ACS, ACO, and ABA (NCED biosynthesis. Abundance of transcripts of senescence-related genes (SAG12, SAG29 was also dramatically reduced in the silenced flowers. Over-expression of PhHD-Zip accelerated petunia flower senescence. Furthermore, PhHD-Zip transcript abundance in petunia flowers was increased by application of hormones (ethylene, ABA and abiotic stresses (dehydration, NaCl and cold. Our results suggest that PhHD-Zip plays an important role in regulating petunia flower senescence.

  17. The Proapoptotic Bcl-2 Protein Bax Plays an Important Role in the Pathogenesis of Reovirus Encephalitis ▿

    Science.gov (United States)

    Berens, Heather M.; Tyler, Kenneth L.

    2011-01-01

    Encephalitis induced by reovirus serotype 3 (T3) strains results from the apoptotic death of infected neurons. Extrinsic apoptotic signaling is activated in reovirus-infected neurons in vitro and in vivo, but the role of intrinsic apoptosis signaling during encephalitis is largely unknown. Bax plays a key role in intrinsic apoptotic signaling in neurons by allowing the release of mitochondrial cytochrome c. We found Bax activation and cytochrome c release in neurons following infection of neonatal mice with T3 reoviruses. Bax−/− mice infected with T3 Abney (T3A) have reduced central nervous system (CNS) tissue injury and decreased apoptosis, despite viral replication that is similar to that in wild-type (WT) Bax+/+ mice. In contrast, in the heart, T3A-infected Bax−/− mice have viral growth, caspase activation, and injury comparable to those in WT mice, indicating that the role of Bax in pathogenesis is organ specific. Nonmyocarditic T3 Dearing (T3D)-infected Bax−/− mice had delayed disease and enhanced survival compared to WT mice. T3D-infected Bax−/− mice had significantly lower viral titers and levels of activated caspase 3 in the brain despite unaffected transneuronal spread of virus. Cytochrome c and Smac release occurred in some reovirus-infected neurons in the absence of Bax; however, this was clearly reduced compared to levels seen in Bax+/+ wild-type mice, indicating that Bax is necessary for efficient activation of proapoptotic mitochondrial signaling in infected neurons. Our studies suggest that Bax is important for reovirus growth and pathogenesis in neurons and that the intrinsic pathway of apoptosis, mediated by Bax, is important for full expression of disease, CNS tissue injury, apoptosis, and viral growth in the CNS of reovirus-infected mice. PMID:21307199

  18. Mitogen-activated protein kinase signal pathways play an important role in right ventricular hypertrophy of tetralogy of Fallot

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hong-shan; WU Qing-yu; XU Ming; ZHOU Yu-xiang; SHUI Chao-xiang

    2012-01-01

    Background Tetralogy of Fallot (TOF) is the most common malformation of children with an incidence of approximately 10% of congenital heart disease patients.There can be a wide spectrum to the severity of the anatomic defects,which include ventricular septal defect,aortic override,right ventricular outflow tract obstruction,and right ventricular hypertrophy.We examined the relationship between right ventricular hypertrophy in patients with TOF and the gene expression of factors in the mitogen-activated protein kinase (MAPK) signal pathway.Methods To gain insight into the characteristic gene(s) involved in molecular mechanisms of right ventricular hypertrophy in TOF,differential mRNA and micro RNA expression profiles were assessed using expression-based micro array technology on right ventricular biopsies from young TOF patients who underwent primary correction and on normal heart tissue.We then analyzed the gene expression of the MAPK signal pathway using reverse transcription-polymerase chain reaction (RT-PCR) in normals and TOF patients.Results Using the micro RNA chip V3.0 and human whole genome oligonucleotide microarray V1.0 to detect the gene expression,we found 1068 genes showing altered expression of at least two-fold in TOF patients compared to the normal hearts,and 47 micro RNAs that showed a significant difference of at least two-fold in TOF patients.We then analyzed these mRNAs and micro RNAs by target gene predicting software Microcosm Targets version 5.0,and determined those mRNA highly relevant to the right ventricular hypertrophy by RT-PCR method.There were obvious differences in the gene expression of factors in the MAPK signal pathway when using RT-PCR,which was consistent to the results of the cDNA microarray.Conclusion The upregulation of genes in the MAPK signal pathway may be the key events that contribute to right ventricular hypertroohv and stunted angiogenesis in patients with TOF.

  19. Toward accurate prediction of pKa values for internal protein residues: the importance of conformational relaxation and desolvation energy.

    Science.gov (United States)

    Wallace, Jason A; Wang, Yuhang; Shi, Chuanyin; Pastoor, Kevin J; Nguyen, Bao-Linh; Xia, Kai; Shen, Jana K

    2011-12-01

    Proton uptake or release controls many important biological processes, such as energy transduction, virus replication, and catalysis. Accurate pK(a) prediction informs about proton pathways, thereby revealing detailed acid-base mechanisms. Physics-based methods in the framework of molecular dynamics simulations not only offer pK(a) predictions but also inform about the physical origins of pK(a) shifts and provide details of ionization-induced conformational relaxation and large-scale transitions. One such method is the recently developed continuous constant pH molecular dynamics (CPHMD) method, which has been shown to be an accurate and robust pK(a) prediction tool for naturally occurring titratable residues. To further examine the accuracy and limitations of CPHMD, we blindly predicted the pK(a) values for 87 titratable residues introduced in various hydrophobic regions of staphylococcal nuclease and variants. The predictions gave a root-mean-square deviation of 1.69 pK units from experiment, and there were only two pK(a)'s with errors greater than 3.5 pK units. Analysis of the conformational fluctuation of titrating side-chains in the context of the errors of calculated pK(a) values indicate that explicit treatment of conformational flexibility and the associated dielectric relaxation gives CPHMD a distinct advantage. Analysis of the sources of errors suggests that more accurate pK(a) predictions can be obtained for the most deeply buried residues by improving the accuracy in calculating desolvation energies. Furthermore, it is found that the generalized Born implicit-solvent model underlying the current CPHMD implementation slightly distorts the local conformational environment such that the inclusion of an explicit-solvent representation may offer improvement of accuracy.

  20. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

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    Anna Garbelli

    Full Text Available DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  1. A motif unique to the human DEAD-box protein DDX3 is important for nucleic acid binding, ATP hydrolysis, RNA/DNA unwinding and HIV-1 replication.

    Science.gov (United States)

    Garbelli, Anna; Beermann, Sandra; Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-05-12

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

  2. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    Science.gov (United States)

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  3. WD40 domain divergence is important for functional differences between the fission yeast Tup11 and Tup12 co-repressor proteins.

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    Monica E Ferreira

    Full Text Available We have previously demonstrated that subsets of Ssn6/Tup target genes have distinct requirements for the Schizosaccharomyces pombe homologs of the Tup1/Groucho/TLE co-repressor proteins, Tup11 and Tup12. The very high level of divergence in the histone interacting repression domains of the two proteins suggested that determinants distinguishing Tup11 and Tup12 might be located in this domain. Here we have combined phylogenetic and structural analysis as well as phenotypic characterization, under stress conditions that specifically require Tup12, to identify and characterize the domains involved in Tup12-specific action. The results indicate that divergence in the repression domain is not generally relevant for Tup12-specific function. Instead, we show that the more highly conserved C-terminal WD40 repeat domain of Tup12 is important for Tup12-specific function. Surface amino acid residues specific for the WD40 repeat domain of Tup12 proteins in different fission yeasts are clustered in blade 3 of the propeller-like structure that is characteristic of WD40 repeat domains. The Tup11 and Tup12 proteins in fission yeasts thus provide an excellent model system for studying the functional divergence of WD40 repeat domains.

  4. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    Science.gov (United States)

    Nuss, Jonathan E; Kehn-Hall, Kylene; Benedict, Ashwini; Costantino, Julie; Ward, Michael; Peyser, Brian D; Retterer, Cary J; Tressler, Lyal E; Wanner, Laura M; McGovern, Hugh F; Zaidi, Anum; Anthony, Scott M; Kota, Krishna P; Bavari, Sina; Hakami, Ramin M

    2014-01-01

    Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV). Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry) with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90), as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

  5. Multi-faceted proteomic characterization of host protein complement of Rift Valley fever virus virions and identification of specific heat shock proteins, including HSP90, as important viral host factors.

    Directory of Open Access Journals (Sweden)

    Jonathan E Nuss

    Full Text Available Rift Valley fever is a potentially fatal disease of humans and domestic animals caused by Rift Valley fever virus (RVFV. Infection with RVFV in ruminants can cause near 100% abortion rates and recent outbreaks in naïve human populations have suggested case fatality rates of greater than thirty percent. To elucidate the roles that host proteins play during RVFV infection, proteomic analysis of RVFV virions was conducted using complementary analytical approaches, followed by functional validation studies of select identified host factors. Coupling the more traditional Gel LC/MS/MS approach (SDS PAGE followed by liquid chromatography tandem mass spectrometry with an alternative technique that preserves protein complexes allowed the protein complement of these viral particles to be thoroughly examined. In addition to viral proteins present within the virions and virion-associated host proteins, multiple macromolecular complexes were identified. Bioinformatic analysis showed that host chaperones were among over-represented protein families associated with virions, and functional experiments using siRNA gene silencing and small molecule inhibitors identified several of these heat shock proteins, including heat shock protein 90 (HSP90, as important viral host factors. Further analysis indicated that HSP inhibition effects occur during the replication/transcription phase of the virus life cycle, leading to significant lowering of viral titers without compromising the functional capacity of released virions. Overall, these studies provide much needed further insight into interactions between RVFV and host cells, increasing our understanding of the infection process and suggesting novel strategies for anti-viral development. In particular, considering that several HSP90 inhibitors have been advancing through clinical trials for cancer treatment, these results also highlight the exciting potential of repurposing HSP90 inhibitors to treat RVF.

  6. Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

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    Aris Haryanto

    2015-10-01

    Full Text Available Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC and typically requires a specific nuclear localization signal (NLS. In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc, phosphorylated recombinant HBV core (rHBc and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

  7. SCR96, a small cysteine-rich secretory protein of Phytophthora cactorum, can trigger cell death in the Solanaceae and is important for pathogenicity and oxidative stress tolerance.

    Science.gov (United States)

    Chen, Xiao-Ren; Li, Yan-Peng; Li, Qi-Yuan; Xing, Yu-Ping; Liu, Bei-Bei; Tong, Yun-Hui; Xu, Jing-You

    2016-05-01

    Peptides and small molecules produced by both the plant pathogen Phytophthora and host plants in the apoplastic space mediate the relationship between the interplaying organisms. Various Phytophthora apoplastic effectors, including small cysteine-rich (SCR) secretory proteins, have been identified, but their roles during interaction remain to be determined. Here, we identified an SCR effector encoded by scr96, one of three novel genes encoding SCR proteins in P. cactorum with similarity to the P. cactorum phytotoxic protein PcF. Together with the other two genes, scr96 was transcriptionally induced throughout the developmental and infection stages of the pathogen. These genes triggered plant cell death (PCD) in the Solanaceae, including Nicotiana benthamiana and tomato. The scr96 gene did not show single nucleotide polymorphisms in a collection of P. cactorum isolates from different countries and host plants, suggesting that its role is essential and non-redundant during infection. Homologues of SCR96 were identified only in oomycetes, but not in fungi and other organisms. A stable protoplast transformation protocol was adapted for P. cactorum using green fluorescent protein as a marker. The silencing of scr96 in P. cactorum caused gene-silenced transformants to lose their pathogenicity on host plants and these transformants were significantly more sensitive to oxidative stress. Transient expression of scr96 partially recovered the virulence of gene-silenced transformants on plants. Overall, our results indicate that the P. cactorum scr96 gene encodes an important virulence factor that not only causes PCD in host plants, but is also important for pathogenicity and oxidative stress tolerance.

  8. Mapping of a Region of the PA-X Protein of Influenza A Virus That Is Important for Its Shutoff Activity.

    Science.gov (United States)

    Oishi, Kohei; Yamayoshi, Seiya; Kawaoka, Yoshihiro

    2015-08-01

    Influenza A virus PA-X comprises an N-terminal PA endonuclease domain and a C-terminal PA-X-specific domain. PA-X reduces host and viral mRNA accumulation via its endonuclease function. Here, we found that the N-terminal 15 amino acids, particularly six basic amino acids, in the C-terminal PA-X-specific region are important for PA-X shutoff activity. These six basic amino acids enabled a PA deletion mutant to suppress protein expression at a level comparable to that of wild-type PA-X.

  9. Protein kinase a dependent phosphorylation of apical membrane antigen 1 plays an important role in erythrocyte invasion by the malaria parasite.

    Directory of Open Access Journals (Sweden)

    Kerstin Leykauf

    2010-06-01

    Full Text Available Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels itself into the host-cell, the junction moves down the length of the parasite. This process must be tightly regulated and signalling is likely to play a role in this event. One crucial protein for tight-junction formation is the apical membrane antigen 1 (AMA1. Here we have investigated the phosphorylation status of this key player in the invasion process in the human malaria parasite Plasmodium falciparum. We show that the cytoplasmic tail of P. falciparum AMA1 is phosphorylated at serine 610. We provide evidence that the enzyme responsible for serine 610 phosphorylation is the cAMP regulated protein kinase A (PfPKA. Importantly, mutation of AMA1 serine 610 to alanine abrogates phosphorylation of AMA1 in vivo and dramatically impedes invasion. In addition to shedding unexpected new light on AMA1 function, this work represents the first time PKA has been implicated in merozoite invasion.

  10. The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression

    Directory of Open Access Journals (Sweden)

    Jennina Taylor-Wells

    2014-01-01

    Full Text Available The organic anion transporting polypeptides (OATPs encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1 mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL, spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

  11. The Signature Sequence Region of the Human Drug Transporter Organic Anion Transporting Polypeptide 1B1 Is Important for Protein Surface Expression.

    Science.gov (United States)

    Taylor-Wells, Jennina; Meredith, David

    2014-01-01

    The organic anion transporting polypeptides (OATPs) encompass a family of membrane transport proteins responsible for the uptake of xenobiotic compounds. Human organic anion transporting polypeptide 1B1 (OATP1B1) mediates the uptake of clinically relevant compounds such as statins and chemotherapeutic agents into hepatocytes, playing an important role in drug delivery and detoxification. The OATPs have a putative 12-transmembrane domain topology and a highly conserved signature sequence (human OATP1B1: DSRWVGAWWLNFL), spanning the extracellular loop 3/TM6 boundary. The presence of three conserved tryptophan residues at the TM interface suggests a structural role for the sequence. This was investigated by site-directed mutagenesis of selected amino acids within the sequence D251E, W254F, W258/259F, and N261A. Transport was measured using the substrate estrone-3-sulfate and surface expression detected by luminometry and confocal microscopy, facilitated by an extracellular FLAG epitope. Uptake of estrone-3-sulfate and the surface expression of D251E, W254F, and W258/259F were both significantly reduced from the wild type OATP1B1-FLAG in transfected HEK293T cells. Confocal microscopy revealed that protein was produced but was retained intracellularly. The uptake and expression of N261A were not significantly different. The reduction in surface expression and intracellular protein retention indicates a structural and/or membrane localization role for these signature sequence residues in the human drug transporter OATP1B1.

  12. HER2 and β-catenin protein location: importance in the prognosis of breast cancer patients and their correlation when breast cancer cells suffer stressful situations.

    Science.gov (United States)

    Cuello-Carrión, F Darío; Shortrede, Jorge E; Alvarez-Olmedo, Daiana; Cayado-Gutiérrez, Niubys; Castro, Gisela N; Zoppino, Felipe C M; Guerrero, Martín; Martinis, Estefania; Wuilloud, Rodolfo; Gómez, Nidia N; Biaggio, Verónica; Orozco, Javier; Gago, Francisco E; Ciocca, Leonardo A; Fanelli, Mariel A; Ciocca, Daniel R

    2015-02-01

    In human breast cancer, β-catenin localization has been related with disease prognosis. Since HER2-positive patients are an important subgroup, and that in breast cancer cells a direct interaction of β-catenin/HER2 has been reported, in the present study we have explored whether β-catenin location is related with the disease survival. The study was performed in a tumor bank from patients (n = 140) that did not receive specific anti-HER2 therapy. The proteins were detected by immunohistochemistry in serial sections, 47 (33.5%) patients were HER2-positive with a long follow-up. HER2-positive patients that displayed β-catenin at the plasma membrane (completely surrounding the tumour cells) showed a significant better disease-free survival and overall survival than the patients showing the protein on other locations. Then we explored the dynamics of the co-expression of β-catenin and HER2 in human MCF-7 and SKBR3 cells exposed to different stressful situations. In untreated conditions MCF-7 and SKBR3 cells showed very different β-catenin localization. In MCF-7 cells, cadmium administration caused a striking change in β-catenin localization driving it from plasma membrane to cytoplasmic and perinuclear areas and HER2 showed a similar localization patterns. The changes induced by cadmium were compared with heat shock, H2O2 and tamoxifen treatments. In conclusion, this study shows the dynamical associations of HER2 and β-catenin and their changes in subcellular localizations driven by stressful situations. In addition, we report for the first time the correlation between plasma membrane associated β-catenin in HER2-positive breast cancer and survival outcome, and the importance of the protein localization in breast cancer samples.

  13. The importance of a critical protonation state and the fate of the catalytic steps in class A beta-lactamases and penicillin-binding proteins.

    Science.gov (United States)

    Golemi-Kotra, Dasantila; Meroueh, Samy O; Kim, Choonkeun; Vakulenko, Sergei B; Bulychev, Alexey; Stemmler, Ann J; Stemmler, Timothy L; Mobashery, Shahriar

    2004-08-13

    Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.

  14. Universal Stress Proteins Are Important for Oxidative and Acid Stress Resistance and Growth of Listeria monocytogenes EGD-e In Vitro and In Vivo

    Science.gov (United States)

    Mohamed, Walid; Mraheil, Mobarak Abu; Mukherjee, Krishnendu; Billion, André; Aharonowitz, Yair; Chakraborty, Trinad; Hain, Torsten

    2011-01-01

    Background Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA) and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species. Methods and Findings We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673) were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models. Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection. Conclusion This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions. PMID:21980369

  15. Universal stress proteins are important for oxidative and acid stress resistance and growth of Listeria monocytogenes EGD-e in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Christa Seifart Gomes

    Full Text Available BACKGROUND: Pathogenic bacteria maintain a multifaceted apparatus to resist damage caused by external stimuli. As part of this, the universal stress protein A (UspA and its homologues, initially discovered in Escherichia coli K-12 were shown to possess an important role in stress resistance and growth in several bacterial species. METHODS AND FINDINGS: We conducted a study to assess the role of three homologous proteins containing the UspA domain in the facultative intracellular human pathogen Listeria monocytogenes under different stress conditions. The growth properties of three UspA deletion mutants (Δlmo0515, Δlmo1580 and Δlmo2673 were examined either following challenge with a sublethal concentration of hydrogen peroxide or under acidic conditions. We also examined their ability for intracellular survival within murine macrophages. Virulence and growth of usp mutants were further characterized in invertebrate and vertebrate infection models. Tolerance to acidic stress was clearly reduced in Δlmo1580 and Δlmo0515, while oxidative stress dramatically diminished growth in all mutants. Survival within macrophages was significantly decreased in Δlmo1580 and Δlmo2673 as compared to the wild-type strain. Viability of infected Galleria mellonella larvae was markedly higher when injected with Δlmo1580 or Δlmo2673 as compared to wild-type strain inoculation, indicating impaired virulence of bacteria lacking these usp genes. Finally, we observed severely restricted growth of all chromosomal deletion mutants in mice livers and spleens as compared to the load of wild-type bacteria following infection. CONCLUSION: This work provides distinct evidence that universal stress proteins are strongly involved in listerial stress response and survival under both in vitro and in vivo growth conditions.

  16. Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes.

    Science.gov (United States)

    Khrustalev, Vladislav Victorovich; Barkovsky, Eugene Victorovich

    2012-12-01

    Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.

  17. Putative recombination events and evolutionary history of five economically important viruses of fruit trees based on coat protein-encoding gene sequence analysis.

    Science.gov (United States)

    Boulila, Moncef

    2010-06-01

    To enhance the knowledge of recombination as an evolutionary process, 267 accessions retrieved from GenBank were investigated, all belonging to five economically important viruses infecting fruit crops (Plum pox, Apple chlorotic leaf spot, Apple mosaic, Prune dwarf, and Prunus necrotic ringspot viruses). Putative recombinational events were detected in the coat protein (CP)-encoding gene using RECCO and RDP version 3.31beta algorithms. Based on RECCO results, all five viruses were shown to contain potential recombination signals in the CP gene. Reconstructed trees with modified topologies were proposed. Furthermore, RECCO performed better than the RDP package in detecting recombination events and exhibiting their evolution rate along the sequences of the five viruses. RDP, however, provided the possible major and minor parents of the recombinants. Thus, the two methods should be considered complementary.

  18. A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of N-Terminal β-Barrel in Microsomal Triglyceride Transfer Protein Function

    Science.gov (United States)

    Walsh, Meghan T.; Iqbal, Jahangir; Josekutty, Joby; Soh, James; Di Leo, Enza; Özaydin, Eda; Gündüz, Mehmet; Tarugi, Patrizia; Hussain, M. Mahmood

    2015-01-01

    Background The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias due to associated hepatosteatosis and gastrointestinal adverse effects. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia have revealed that the central α-helical and C-terminal β-sheet domains are important for protein disulfide isomerase (PDI) binding and lipid transfer activity. Our aim was to identify and characterize mutations in the N-terminal domain to understand its function. Methods and Results We identified a novel missense mutation (D169V) in a 4-month old Turkish male with severe signs of ABL. To study the effect of this mutation on MTP function, we created mutants via site-directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apoB17, it was unable to bind PDI, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion, without affecting apoB17 binding. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities. Conclusions D169V is detrimental because it disrupts an internal salt bridge leading to loss of PDI binding and lipid transfer activities; however, it does not affect apoB-binding. Thus, the N-terminal domain of MTP is also important for its lipid transfer activity. PMID:26224785

  19. The atypical IκB protein IκB(NS) is important for Toll-like receptor-induced interleukin-10 production in B cells.

    Science.gov (United States)

    Miura, Minami; Hasegawa, Naoki; Noguchi, Mitsuo; Sugimoto, Kenkichi; Touma, Maki

    2016-04-01

    Although a major function of B cells is to mediate humoral immunity by producing antigen-specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). However, the mechanism by which IL-10 is induced in B cells has not been fully elucidated. Here, we report that IκBNS , an inducible nuclear IκB protein, is important for Toll-like receptor (TLR)-mediated IL-10 production in B cells. Studies using IκB(NS) knockout mice revealed that the number of IL-10-producing B cells is reduced in IκB(NS)(-/-) spleens and that the TLR-mediated induction of cytoplasmic IL-10-positive cells and IL-10 secretion in B cells are impaired in the absence of IκB(NS). The impairment of IL-10 production by a lack of IκB(NS) was not observed in TLR-triggered macrophages or T-cell-receptor-stimulated CD4(+) CD25(+) T cells. In addition, IκB(NS)-deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL-10(+) CD138(+) plasmablasts. These results suggest that IκB(NS) is selectively required for IL-10 production in B cells responding to TLR signals, so defining an additional role for IκB(NS) in the control of the B-cell-mediated immune responses.

  20. Factor H-binding protein is important for meningococcal survival in human whole blood and serum and in the presence of the antimicrobial peptide LL-37.

    Science.gov (United States)

    Seib, K L; Serruto, D; Oriente, F; Delany, I; Adu-Bobie, J; Veggi, D; Aricò, B; Rappuoli, R; Pizza, M

    2009-01-01

    Factor H-binding protein (fHBP; GNA1870) is one of the antigens of the recombinant vaccine against serogroup B Neisseria meningitidis, which has been developed using reverse vaccinology and is the basis of a meningococcal B vaccine entering phase III clinical trials. Binding of factor H (fH), an inhibitor of the complement alternative pathway, to fHBP enables N. meningitidis to evade killing by the innate immune system. All fHBP null mutant strains analyzed were sensitive to killing in ex vivo human whole blood and serum models of meningococcal bacteremia with respect to the isogenic wild-type strains. The fHBP mutant strains of MC58 and BZ83 (high fHBP expressors) survived in human blood and serum for less than 60 min (decrease of >2 log(10) CFU), while NZ98/254 (intermediate fHBP expressor) and 67/00 (low fHBP expressor) showed decreases of >1 log(10) CFU after 60 to 120 min of incubation. In addition, fHBP is important for survival in the presence of the antimicrobial peptide LL-37 (decrease of >3 log(10) CFU after 2 h of incubation), most likely due to electrostatic interactions between fHBP and the cationic LL-37 molecule. Hence, the expression of fHBP by N. meningitidis strains is important for survival in human blood and human serum and in the presence of LL-37, even at low levels. The functional significance of fHBP in mediating resistance to the human immune response, in addition to its widespread distribution and its ability to induce bactericidal antibodies, indicates that it is an important component of the serogroup B meningococcal vaccine.

  1. Importance of light in temporal organization of photoreceptor proteins and melatonin-producing system in the pineal of carp Catla catla.

    Science.gov (United States)

    Seth, Mohua; Maitra, Saumen Kumar

    2010-05-01

    The importance of light in the temporal organization of photoreceptor proteins and melatonin-producing system has been investigated for the first time in the pineal of a tropical fish. In this study, an identical experimental paradigm was followed during the four distinct phases of an annual cycle in adult carps (Catla catla) maintained either under natural photoperiod (NP) or continuous illumination (LL) or darkness (DD) for 30 days. At the end of each experiment, the pineal from fish in each experimental group was collected either at 06:00, 12:00, 18:00, or 24:00 in a daily cycle and assessed by Western blot analysis for pineal rod-like opsin, alpha-transducin, and AANAT. The same animals were also used for measurement of serum melatonin levels, and the serum as well as intra-pineal Ca(++) levels at each timepoint. The study revealed a daily rhythmicity with a peak at 12:00 h and nadir at 24:00 h in the band intensity of pineal rod-like opsin and alpha-transducin in NP fish, while the band intensities of these photo-pigment proteins remained high under LL and low under DD, irrespective of clock hour during the 24 h cycle. The band intensity of pineal AANAT, levels of serum melatonin, and both serum Ca(++) and intra-pineal Ca(++) were maximum at 24:00 h and minimum at 12:00h in NP fish, and they were significantly lower under LL and higher under DD at each point of study. The results showed loss of daily rhythm in each studied variable in both LL and DD carps, suggesting that their circadian organization is dependent on the external light-dark conditions, rather than an endogenous circadian oscillator in the pineal.

  2. The Importance of G Protein-Coupled Receptor Kinase 4 (GRK4 in Pathogenesis of Salt Sensitivity, Salt Sensitive Hypertension and Response to Antihypertensive Treatment

    Directory of Open Access Journals (Sweden)

    Brian Rayner

    2015-03-01

    Full Text Available Salt sensitivity is probably caused by either a hereditary or acquired defect of salt excretion by the kidney, and it is reasonable to consider that this is the basis for differences in hypertension between black and white people. Dopamine acts in an autocrine/paracrine fashion to promote natriuresis in the proximal tubule and thick ascending loop of Henle. G-protein receptor kinases (or GRKs are serine and threonine kinases that phosphorylate G protein-coupled receptors in response to agonist stimulation and uncouple the dopamine receptor from its G protein. This results in a desensitisation process that protects the cell from repeated agonist exposure. GRK4 activity is increased in spontaneously hypertensive rats, and infusion of GRK4 antisense oligonucleotides attenuates the increase in blood pressure (BP. This functional defect is replicated in the proximal tubule by expression of GRK4 variants namely p.Arg65Leu, p.Ala142Val and p.Val486Ala, in cell lines, with the p.Ala142Val showing the most activity. In humans, GRK4 polymorphisms were shown to be associated with essential hypertension in Australia, BP regulation in young adults, low renin hypertension in Japan and impaired stress-induced Na excretion in normotensive black men. In South Africa, GRK4 polymorphisms are more common in people of African descent, associated with impaired Na excretion in normotensive African people, and predict blood pressure response to Na restriction in African patients with mild to moderate essential hypertension. The therapeutic importance of the GRK4 single nucleotide polymorphisms (SNPs was emphasised in the African American Study of Kidney Disease (AASK where African-Americans with hypertensive nephrosclerosis were randomised to receive amlodipine, ramipril or metoprolol. Men with the p.Ala142Val genotype were less likely to respond to metoprolol, especially if they also had the p.Arg65Leu variant. Furthermore, in the analysis of response to treatment in

  3. Conserved leucines in N-terminal heptad repeat HR1 of envelope fusion protein F of group II nucleopolyhedroviruses are important for correct processing and essential for fusogenicity

    NARCIS (Netherlands)

    Long, G.; Pan, X.; Vlak, J.M.

    2008-01-01

    The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common fe

  4. Mitochondrial Ribosomal Protein L12 Is Required for POLRMT Stability and Exists as Two Forms Generated by Alternative Proteolysis during Import.

    Science.gov (United States)

    Nouws, Jessica; Goswami, Arvind V; Bestwick, Megan; McCann, Beverly Jo; Surovtseva, Yulia V; Shadel, Gerald S

    2016-01-08

    To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein.

    Science.gov (United States)

    Schuijt, Tim J; Bakhtiari, Kamran; Daffre, Sirlei; Deponte, Kathleen; Wielders, Simone J H; Marquart, J Arnoud; Hovius, Joppe W; van der Poll, Tom; Fikrig, Erol; Bunce, Matthew W; Camire, Rodney M; Nicolaes, Gerry A F; Meijers, Joost C M; van 't Veer, Cornelis

    2013-07-16

    Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

  6. Tissue, developmental, and caste-specific expression of odorant binding proteins in a eusocial insect, the red imported fire ant, Solenopsis invicta

    Science.gov (United States)

    Zhang, Wei; Wanchoo, Arun; Ortiz-Urquiza, Almudena; Xia, Yuxian; Keyhani, Nemat O.

    2016-01-01

    Insects interact with the surrounding environment via chemoreception, and in social insects such as ants, chemoreception functions to mediate diverse behaviors including food acquisition, self/non-self recognition, and intraspecific communication. The invasive red imported fire ant, Solenopsis invicta, has spread worldwide, displaying a remarkable environmental adaptability. Odorant binding proteins (OBPs) are chemical compound carriers, involved in diverse physiological processes including odor detection and chemical transport. S. invicta contains a highly divergent 17-member OBP gene family, that includes an ant-specific expansion and the social organization implicated Gp-9 (OBP3) gene. A systematic gene expression analysis of the SiOBP repertoire was performed across social caste (workers, male and female alates), tissues (antennae, head, thorax, and abdomen), and developmental stages (egg, larvae, and pupae), revealing that although SiOBPs were expressed in the antennae, the major regions of expression were in the head and thorax across all castes, and the abdomen in male and female alates. SiOBPs were very highly expressed in female alates and at somewhat lower levels in male alates and workers. SiOBPs were differentially expressed, with unique signatures in various castes and tissues, suggesting functionality of SiOBPs beyond olfaction Expression patterns of SiOBP subgroups also showed relationships with their evolutionary relatedness. PMID:27765943

  7. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    Directory of Open Access Journals (Sweden)

    Olgica Nedić

    2015-08-01

    Full Text Available Post-translational modifications (PTM of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM, which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues.

  8. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    Science.gov (United States)

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh I S

    2015-08-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically, depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of nePTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues.

  9. A disulfide-bonded dimer of the core protein of hepatitis C virus is important for virus-like particle production.

    Science.gov (United States)

    Kushima, Yukihiro; Wakita, Takaji; Hijikata, Makoto

    2010-09-01

    Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

  10. The importance of RSV F protein conformation in VLPs in stimulation of neutralizing antibody titers in mice previously infected with RSV.

    Science.gov (United States)

    Cullen, Lori M; Schmidt, Madelyn R; Morrison, Trudy G

    2017-06-12

    Respiratory syncytial virus (RSV) is a significant respiratory pathogen but no vaccine is available. RSV infections present 2 major, unique problems. First, humans can experience repeated infections caused by the same virus sero-group indicating that protective memory responses to RSV infection are defective. Second, most people have been infected with RSV by age 5. Immune responses to these infections, while poorly protective, could impact the effectiveness of a vaccine. The goal of this study was to assess the generation of protective immune responses in mice previously infected with RSV by virus-like particle (VLP) vaccine candidates containing a stabilized pre-fusion form of the RSV F protein or a stabilized post-fusion F protein. We report that a single immunization of RSV-experienced animals with a stabilized pre-fusion F protein VLP stimulated high titers of neutralizing antibody while a single injection of a post-fusion F protein VLP or a second RSV infection only weakly stimulated neutralizing antibody titers. These results suggest that prior RSV infection can induce neutralizing antibody memory responses, which can be activated by pre-F protein VLPs but not by post-F protein VLPs or a subsequent infection. Thus the F protein conformation has a major impact on enhancing production of neutralizing antibodies in RSV-experienced animals. Furthermore, although both VLPs contained the same RSV G protein, the pre-F VLP stimulated significantly higher titers of total anti-G protein IgG than the post-F VLP in both naïve and RSV-experienced animals. Thus the F protein conformation also influences anti-G protein responses.

  11. Mucopolysaccharidosis type I in 21 Czech and Slovak patients: Mutation analysis suggests a functional importance of C-terminus of the IDUA protein

    Science.gov (United States)

    Vazna, Alzbeta; Beesley, Clare; Berna, Linda; Stolnaja, Larisa; Myskova, Helena; Bouckova, Michaela; Vlaskova, Hana; Poupetova, Helena; Zeman, Jiri; Magner, Martin; Hlavata, Anna; Winchester, Bryan; Hrebicek, Martin; Dvorakova, Lenka

    2009-01-01

    Abstract Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α-l-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein. © 2009 Wiley-Liss, Inc. PMID:19396826

  12. Mass spectrometric peptide analysis of 2DE-separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation.

    Science.gov (United States)

    Mikkat, Stefan; Kischstein, Timo; Kreutzer, Michael; Glocker, Michael O

    2013-06-01

    Asparagine deamidation is a common nonenzymatic post-translational modification comprising the conversion of asparaginyl residues to aspartyl and isoaspartyl residues, respectively. As a result an additional negative charge is introduced that can affect the tertiary structure as well as the biological activity of a protein. Since deamidation reduces the protein's pI value, differentially deamidated forms of a protein can be separated in 2D gels. We have analyzed a dataset of 430 protein spots from 2D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a Coomassie-stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin-2, V-type proton ATPase subunit B, and protein disulfide-isomerase A3 were also found in 2D gels of proteins extracted from rat hippocampus. All identified deamidation sites contained a glycine residue on the carboxyl side of the asparaginyl residue. Strikingly, a second glycine residue at the +3 position was found in the majority of the deamidated peptides. We propose that the NGxG motif confers exceptional susceptibility to in vivo asparagine deamidation.

  13. Sequence and expression analyses of KIX domain proteins suggest their importance in seed development and determination of seed size in rice, and genome stability in Arabidopsis.

    Science.gov (United States)

    Thakur, Jitendra Kumar; Agarwal, Pinky; Parida, Swarup; Bajaj, Deepak; Pasrija, Richa

    2013-08-01

    The KIX domain, which mediates protein-protein interactions, was first discovered as a motif in the large multidomain transcriptional activator histone acetyltransferase p300/CBP. Later, the domain was also found in Mediator subunit MED15, where it interacts with many transcription factors. In both proteins, the KIX domain is a target of activation domains of diverse transcription activators. It was found to be an essential component of several specific gene-activation pathways in fungi and metazoans. Not much is known about KIX domain proteins in plants. This study aims to characterize all the KIX domain proteins encoded by the genomes of Arabidopsis and rice. All identified KIX domain proteins are presented, together with their chromosomal locations, phylogenetic analysis, expression and SNP analyses. KIX domains were found not only in p300/CBP- and MED15-like plant proteins, but also in F-box proteins in rice and DNA helicase in Arabidopsis, suggesting roles of KIX domains in ubiquitin-mediated proteasomal degradation and genome stability. Expression analysis revealed overlapping expression of OsKIX_3, OsKIX_5 and OsKIX_7 in different stages of rice seeds development. Moreover, an association analysis of 136 in silico mined SNP loci in 23 different rice genotypes with grain-length information identified three non-synonymous SNP loci in these three rice genes showing strong association with long- and short-grain differentiation. Interestingly, these SNPs were located within KIX domain encoding sequences. Overall, this study lays a foundation for functional analysis of KIX domain proteins in plants.

  14. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    DEFF Research Database (Denmark)

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh

    2015-01-01

    , depend on the ternary structure of proteins and can be damaging. It is often observed that inactive and abnormal proteins accumulate in old cells and tissues. The nature, site and extent of nePTM give rise to a population of that specific protein with alterations in structure and function ranging from...... being fully active to totally inactive molecules. Determination of the type and the amount (abundance) of nePTM is essential for establishing connection between specific protein structure and specific biological role. This article summarizes analytical demands for reliable quantification of ne......PTM, including requirements for the assay performance, standardization and quality control, and points to the difficulties, uncertainties and un-resolved issues....

  15. The amino-terminal region of the neuraminidase protein from avian H5N1 influenza virus is important for its biosynthetic transport to the host cell surface.

    Science.gov (United States)

    Qian, Guomin; Wang, Song; Chi, Xiaojuan; Li, Hua; Wei, Haitao; Zhu, Xiaomei; Chen, Yuhai; Chen, Ji-Long

    2014-12-01

    Influenza virus neuraminidase (NA) is a major viral envelope glycoprotein, which plays a critical role in viral infection. Although NA functional domains have been determined previously, the precise role of the amino acids located at the N-terminus of avian H5N1 NA for protein expression and intracellular transport to the host plasma membrane is not fully understood. In the present study, a series of N-terminal truncation or deletion mutants of H5N1 NA were generated and their expression and intracellular trafficking were investigated. Protein expression from mutants NAΔ20, NAΔ35, NAΔ40, NAΔ7-20 and NAΔ7-35 was undetectable by immunoblotting and by performing NA activity assays. Mutants NAΔ6, NAΔ11 and NAΔ15-20 showed a marked decreased in protein expression, whereas mutants NAΔ7-15 and NAΔ15 displayed a slight increase in protein expression, compared with that of the native NA protein. These data suggest that amino acid residues 16-20 are vital for NA protein expression, while amino acids 7-15 might suppress NA protein expression. In deletion mutants NAΔ7-15 and NAΔ15 there was an accumulation of NA protein at the juxta-nuclear region, with reduced expression of NA at the cell surface. Although active Cdc42 could promote transport of wild-type NA to the host cell surface, this member of the Rho family of GTPases failed to regulate transport of mutants NAΔ7-15 and NAΔ15. The results of the study reveal that amino acid residues 7-15 of H5N1 NA are critical for its biosynthetic transport to the host cell surface.

  16. Proteome analysis identifies the Dpr protein of Streptococcus mutans as an important factor in the presence of early streptococcal colonizers of tooth surfaces.

    Directory of Open Access Journals (Sweden)

    Akihiro Yoshida

    Full Text Available Oral streptococci are primary colonizers of tooth surfaces and Streptococcus mutans is the principal causative agent of dental caries in humans. A number of proteins are involved in the formation of monospecies biofilms by S. mutans. This study analyzed the protein expression profiles of S. mutans biofilms formed in the presence or absence of S. gordonii, a pioneer colonizer of the tooth surface, by two-dimensional gel electrophoresis (2-DE. After identifying S. mutans proteins by Mass spectrometric analysis, their expression in the presence of S. gordonii was analyzed. S. mutans was inoculated with or without S. gordonii DL1. The two species were compartmentalized using 0.2-μl Anopore membranes. The biofilms on polystyrene plates were harvested, and the solubilized proteins were separated by 2-DE. When S. mutans biofilms were formed in the presence of S. gordonii, the peroxide resistance protein Dpr of the former showed 4.3-fold increased expression compared to biofilms that developed in the absence of the pioneer colonizer. In addition, we performed a competition assay using S. mutans antioxidant protein mutants together with S. gordonii and other initial colonizers. Growth of the dpr-knockout S. mutans mutant was significantly inhibited by S. gordonii, as well as by S. sanguinis. Furthermore, a cell viability assay revealed that the viability of the dpr-defective mutant was significantly attenuated compared to the wild-type strain when co-cultured with S. gordonii. Therefore, these results suggest that Dpr might be one of the essential proteins for S. mutans survival on teeth in the presence of early colonizing oral streptococci.

  17. Gene Network Analysis of Metallo Beta Lactamase Family Proteins Indicates the Role of Gene Partners in Antibiotic Resistance and Reveals Important Drug Targets.

    Science.gov (United States)

    Parimelzaghan, Anitha; Anbarasu, Anand; Ramaiah, Sudha

    2016-06-01

    Metallo Beta (β) Lactamases (MBL) are metal dependent bacterial enzymes that hydrolyze the β-lactam antibiotics. In recent years, MBL have received considerable attention because it inactivates most of the β-lactam antibiotics. Increase in dissemination of MBL encoding antibiotic resistance genes in pathogenic bacteria often results in unsuccessful treatments. Gene interaction network of MBL provides a complete understanding on the molecular basis of MBL mediated antibiotic resistance. In our present study, we have constructed the MBL network of 37 proteins with 751 functional partners from pathogenic bacterial spp. We found 12 highly interconnecting clusters. Among the 37 MBL proteins considered in the present study, 22 MBL proteins are from B3 subclass, 14 are from B1 subclass and only one is from B2 subclass. Global topological parameters are used to calculate and compare the probability of interactions in MBL proteins. Our results indicate that the proteins associated within the network have a strong influence in antibiotic resistance mechanism. Interestingly, several drug targets are identified from the constructed network. We believe that our results would be helpful for researchers exploring MBL-mediated antibiotic resistant mechanisms.

  18. IMPORTANT NOTIFICATION

    CERN Multimedia

    HR Department

    2009-01-01

    Green plates, removals and importation of personal effects Please note that, as from 1 April 2009, formalities relating to K and CD special series French vehicle plates (green plates), removals and importation of personal effects into France and Switzerland will be dealt with by GS Department (Building 73/3-014, tel. 73683/74407). Importation and purchase of tax-free vehicles in Switzerland, as well as diplomatic privileges, will continue to be dealt with by the Installation Service of HR Department (Building 33/1-011, tel. 73962). HR and GS Departments

  19. The S-layer homology domain-containing protein SlhA from Paenibacillus alvei CCM 2051(T is important for swarming and biofilm formation.

    Directory of Open Access Journals (Sweden)

    Bettina Janesch

    Full Text Available Swarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB.Paenibacillus alvei CCM 2051(T is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA.This study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T.

  20. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Nielsen, Morten Schallburg; Brejning, Jeanette; García, R.;

    1997-01-01

    Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C-terminal ......Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C...

  1. Importance of adult protein ingestion on the mating success of Ceratitis capitata Wiedemann males (Diptera: Tephritidae); Importancia da ingestao de proteina na fase adulta para o sucesso de acasalamento dos machos de Ceratitis capitata Wiedemann (Diptera: Tephritidae)

    Energy Technology Data Exchange (ETDEWEB)

    Silva Neto, Alberto M. da; Dias, Vanessa S.; Joachim-Bravo, Iara S., E-mail: bio.alberto@gmail.co, E-mail: vanessasidias@hotmail.co, E-mail: ibravo@ufba.b [Universidade Federal da Bahia (UFBA), Salvador, BA (Brazil). Inst. de Biologia. Dept. de Biologia Geral

    2010-04-15

    The importance of the protein ingestion during the adult stage on the mating success of males of Ceratitis capitata Wiedemann was evaluated in experiments of laboratory and fi eld cage. In laboratory, the effects of protein ingestion during the fi rst four or 12 days of the male adult life was assessed by the following parameters: mating success (capacity of being chosen by the female) and the number of males that give out pheromonal signals. Some experiments of mating success had been carried through with males in different ratios. In these tests, the number of males which had ingested protein (an unique male) was remained constant and the number of males fed without protein was gradually increased from 1:1 to 1:5. In the fi eld cages, the mating success experiments were done using a 1:1 ratio. The results showed that the protein ingestion in the fi rst four days of life did not influence any of the analyzed parameters. When the period of ingestion of protein was extended to 12 days, protein-fed males fed produced more pheromonal signals and had a higher mating success when at a 1:1 ratio in laboratory and fi eld cage assays. In laboratory, females randomly chose males in any other tested ratio (1:2, 1:3, 1:4 and 1:5), indicating that the female may lose the perception to identify the male who ingested protein in the fi rst 12 days. (author)

  2. Vaccination of mice with plasmids expressing processed capsid protein of foot-and-mouth disease virus - Importance of dominant and subdominant epitopes for antigenicity and protection

    DEFF Research Database (Denmark)

    Frimann, Tine; Barfoed, Annette Malene; Aasted, Bent

    2007-01-01

    example of a dominant and variable site. This variability is a problem when designing vaccines against this disease, because it necessitates a close match between vaccine strain and virus in an outbreak. We have introduced a series of mutations into viral capsid proteins with the aim of selectively...

  3. An integrated model of transcription factor diffusion shows the importance of intersegmental transfer and quaternary protein structure for target site finding.

    Directory of Open Access Journals (Sweden)

    Hugo G Schmidt

    Full Text Available We present a computational model of transcription factor motion that explains both the observed rapid target finding of transcription factors, and how this motion influences protein and genome structure. Using the Smoldyn software, we modelled transcription factor motion arising from a combination of unrestricted 3D diffusion in the nucleoplasm, sliding along the DNA filament, and transferring directly between filament sections by intersegmental transfer. This presents a fine-grain picture of the way in which transcription factors find their targets two orders of magnitude faster than 3D diffusion alone allows. Eukaryotic genomes contain sections of nucleosome free regions (NFRs around the promoters; our model shows that the presence and size of these NFRs can be explained as their acting as antennas on which transcription factors slide to reach their targets. Additionally, our model shows that intersegmental transfer may have shaped the quaternary structure of transcription factors: sequence specific DNA binding proteins are unusually enriched in dimers and tetramers, perhaps because these allow intersegmental transfer, which accelerates target site finding. Finally, our model shows that a 'hopping' motion can emerge from 3D diffusion on small scales. This explains the apparently long sliding lengths that have been observed for some DNA binding proteins observed in vitro. Together, these results suggest that transcription factor diffusion dynamics help drive the evolution of protein and genome structure.

  4. The germinlike protein GLP4 exhibits superoxide dismutase activity and is an important component of quantitative resistance in wheat and barley

    DEFF Research Database (Denmark)

    Christensen, Anders Bentsen; Thordal-Christensen, Hans; Zimmermann, Grit

    2004-01-01

    Germinlike proteins (GLP) are encoded in plants by a gene family with proposed functions in plant development and defense. Genes of GLP subfamily 4 of barley (HvGLP4, formerly referred to as HvOxOLP) and the wheat orthologue TaGLP4 (formerly referred to as TaGLP2a) were previously found to be exp...

  5. Butane-1,2,3,4-tetraol-based amphiphilic stereoisomers for membrane protein study: importance of chirality in the linker region

    DEFF Research Database (Denmark)

    Das, Manabendra; Du, Yang; Mortensen, Jonas S.;

    2016-01-01

    Amphiphile selection is a crucial step in membrane protein structural and functional study. As conventional detergents have limited scope and utility, novel agents with enhanced efficacy need to be developed. Although a large number of novel agents have been reported, so far there has been no sys...

  6. A pericentrin-related protein homolog in Aspergillus nidulans plays important roles in nucleus positioning and cell polarity by affecting microtubule organization.

    Science.gov (United States)

    Chen, Peiying; Gao, Rongsui; Chen, Shaochun; Pu, Li; Li, Pin; Huang, Ying; Lu, Ling

    2012-12-01

    Pericentrin is a large coiled-coil protein in mammalian centrosomes that serves as a multifunctional scaffold for anchoring numerous proteins. Recent studies have linked numerous human disorders with mutated or elevated levels of pericentrin, suggesting unrecognized contributions of pericentrin-related proteins to the development of these disorders. In this study, we characterized AnPcpA, a putative homolog of pericentrin-related protein in the model filamentous fungus Aspergillus nidulans, and found that it is essential for conidial germination and hyphal development. Compared to the hyphal apex localization pattern of calmodulin (CaM), which has been identified as an interactive partner of the pericentrin homolog, GFP-AnPcpA fluorescence dots are associated mainly with nuclei, while the accumulation of CaM at the hyphal apex depends on the function of AnPcpA. In addition, the depletion of AnPcpA by an inducible alcA promoter repression results in severe growth defects and abnormal nuclear segregation. Most interestingly, in mature hyphal cells, knockdown of pericentrin was able to significantly induce changes in cell shape and cytoskeletal remodeling; it resulted in some enlarged compartments with condensed nuclei and anucleate small compartments as well. Moreover, defects in AnPcpA significantly disrupted the microtubule organization and nucleation, suggesting that AnPcpA may affect nucleus positioning by influencing microtubule organization.

  7. Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

    Science.gov (United States)

    Barber-Zucker, Shiran; Uebe, René; Davidov, Geula; Navon, Yotam; Sherf, Dror; Chill, Jordan H; Kass, Itamar; Bitton, Ronit; Schüler, Dirk; Zarivach, Raz

    2016-08-23

    Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins' proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.

  8. C-Reactive Protein Is an Important Biomarker for Prognosis Tumor Recurrence and Treatment Response in Adult Solid Tumors: A Systematic Review.

    LENUS (Irish Health Repository)

    Shrotriya, Shiva

    2015-01-01

    A systematic literature review was done to determine the relationship between elevated CRP and prognosis in people with solid tumors. C-reactive protein (CRP) is a serum acute phase reactant and a well-established inflammatory marker. We also examined the role of CRP to predict treatment response and tumor recurrence.

  9. Overexpression of EphA2 correlates with epithelial-mesenchymal transition-related proteins in gastric cancer and their prognostic importance for postoperative patients.

    Science.gov (United States)

    Hou, Futao; Yuan, Weijie; Huang, Jin; Qian, Liyuan; Chen, Zhikang; Ge, Jie; Wu, Shaobin; Chen, Jinxiang; Wang, Jixu; Chen, Zihua

    2012-12-01

    The expression of EphA2 and three epithelial-mesenchymal transition-related proteins (E-cadherin, β-catenin and vimentin) was detected by immunohistochemistry in human gastric cancer and normal gastric mucosa. The expression of EphA2 and vimentin was significantly higher in gastric cancer tissues than in normal gastric mucosa tissues, and similar results were found for negative E-cadherin expression and ectopic β-catenin expression. Further analysis showed that the expression of EphA2 was closely correlated with the depth of tumor invasion, tumor-node-metastasis (TNM) stages and lymph node metastasis. Down-regulated expression of the epithelial protein E-cadherin, overexpression of the mesenchymal protein vimentin and ectopic expression of β-catenin were associated with the depth of tumor invasion, tumor differentiation, TNM stages and lymph node metastasis. The Spearman rank test indicated that the positive expression of EphA2 was negatively associated with E-cadherin expression and was positively correlated with β-catenin ectopic expression and vimentin expression. In addition, the Kaplan-Meier survival analysis showed that the overexpression of EphA2 and vimentin, ectopic expression of β-catenin and down-regulation of E-cadherin indicate a poor outcome. Moreover, multivariate Cox analysis showed that TNM stages, lymph node metastasis, EphA2 expression, E-cadherin expression and β-catenin ectopic expression were independent prognostic factors for postoperative gastric cancer. These findings indicate that the overexpression of EphA2 correlates with the loss of epithelial proteins and the appearance of mesenchymal proteins. Therefore, EphA2 may play a role in epithelial-mesenchymal transition in gastric cancer.

  10. Chromoplast-specific carotenoid-associated protein appears to be important for enhanced accumulation of carotenoids in hp1 tomato fruits.

    Science.gov (United States)

    Kilambi, Himabindu Vasuki; Kumar, Rakesh; Sharma, Rameshwar; Sreelakshmi, Yellamaraju

    2013-04-01

    Tomato (Solanum lycopersicum) high-pigment mutants with lesions in diverse loci such as DNA Damage-Binding Protein1 (high pigment1 [hp1]), Deetiolated1 (hp2), Zeaxanthin Epoxidase (hp3), and Intense pigment (Ip; gene product unknown) exhibit increased accumulation of fruit carotenoids coupled with an increase in chloroplast number and size. However, little is known about the underlying mechanisms exaggerating the carotenoid accumulation and the chloroplast number in these mutants. A comparison of proteome profiles from the outer pericarp of hp1 mutant and wild-type (cv Ailsa Craig) fruits at different developmental stages revealed at least 72 differentially expressed proteins during ripening. Hierarchical clustering grouped these proteins into three clusters. We found an increased abundance of chromoplast-specific carotenoid-associated protein (CHRC) in hp1 fruits at red-ripe stage that is also reflected in its transcript level. Western blotting using CHRC polyclonal antibody from bell pepper (Capsicum annuum) revealed a 2-fold increase in the abundance of CHRC protein in the red-ripe stage of hp1 fruits compared with the wild type. CHRC levels in hp2 were found to be similar to that of hp1, whereas hp3 and Ip showed intermediate levels to those in hp1, hp2, and wild-type fruits. Both CHRC and carotenoids were present in the isolated plastoglobules. Overall, our results suggest that loss of function of DDB1, DET1, Zeaxanthin Epoxidase, and Ip up-regulates CHRC levels. Increase in CHRC levels may contribute to the enhanced carotenoid content in these high-pigment fruits by assisting in the sequestration and stabilization of carotenoids.

  11. Exercise induced regulation of muscular Na+,K+ pump, FXYD1, and NHE1 mRNA and protein expression: importance of training status, intensity, and muscle type

    DEFF Research Database (Denmark)

    Rasmussen, Martin Krøyer; Juel, Carsten; Nordsborg, Nikolai Baastrup

    2011-01-01

    It is investigated if exercise induced mRNA changes cause similar protein expression changes of Na(+), K(+) pump isoforms (a1, a2, ß1, ß2), FXYD1 and NHE1 in rat skeletal muscle. Expression was evaluated (n=8 per group) in Soleus and EDL after 1 day, 3 days and 3 weeks (5 sessions per week......) of either sprint (4 x 3 min sprint + 1 min rest) or endurance (20 min) running. Two hours after exercise on day 1, no change in protein expression was apparent in either training group or muscle, whereas sprint exercise increased the mRNA of Soleus a2 (4.9±0.8 fold; P...

  12. Univariate and multivariate analysis of risk factors for severe clostridium difficile-associated diarrhoea Importance of co-morbidity and serum C-reactive protein

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate risk factors for severe clostridium difficile associated diarrhoea (CDAD) in hospitalised patients. METHODS: We analysed risk factors for severe CDAD (associated with systemic signs of hypovolemia) in 124 hospitalised patients by retrospective chart review. RESULTS: Severe CDAD was present in 27 patients (22%). Statistical analysis showed a significant association with a higher 30-d mortality (33% vs 4%, P < 0.001) and a higher proportion of longer hospital stay exceeding 14 d (74% vs 52%, P = 0.048). Charlson co-morbidity score (OR 1.29 for 1 point increment, P < 0.05) and serum C-reactive protein at diagnosis (OR 1.15 for 10 mg/L increment, P <0.001) were independent predictors of severe CDAD. CONCLUSION: Patients with a severe level of co- morbidity and high serum C-reactive protein levels at the time of diagnosis should receive particular attention.

  13. [The importance of recombinant proteins of West Siberian isolates of Borrelia burgdorferi S.L. for serological diagnostic of ixodic tick borreliosis].

    Science.gov (United States)

    Karavayev, V S; Genina, E S; Ryabchenko, A V; Beklemoschev, A B

    2014-06-01

    The structural proteins OspC, FlaB, FlaA and DbpB of agent of ixodic tick borreliosis are one of main antigens inducing humoral immunity at initial stages of disease. Owing to it, the task was stated to evaluate antigen activity of recombinant proteins OspC (OscP-Bg), fragment of FlaB (f-FlaB) and DbpB of genospecies B. garinii and OspC (OscC-Ba) of genospecies of B. afzelii of West Siberian isolates of Borrelia Burgdorferi S.L. for their possible application as antigens for serological diagnostic of ixodic tick borreliosis The recombinant proteins OscP-Bg, OscC-Ba, f-FlaB, FlaA and DbpB are analyzed using technique of enzymoimmunoassay to detect ability to bound antibodies of serums of patients with ixodic tick borreliosis with localized and disseminated stage of infection. The difference of their sensitivity as antigens during detection of specific antibodies in blood serum of patients with ixodic tick borreliosis was demonstrated In serum of patients with ixodic tick borreliosis with disseminated stage of infection the level of specific IgM and IgG antibodies reacting with OscP-Bg, OscC-Ba, f-FlaB, FlaA and DbpB is within the limits 15.7-52.6% for IgG. The results of enzymoimmunoassay applied to patients with ixodic tick borreliosis for detection of IgM and IgG in serum demonstrated that OscP-Bg and f-FlaB determined the highest antigen activity with antigens. The study results make it possible to consider these proteins as perspective components for development of immune enzyme test system of diagnostic of ixodic tick borreliosis.

  14. Protein Kinase A Dependent Phosphorylation of Apical Membrane Antigen 1 Plays an Important Role in Erythrocyte Invasion by the Malaria Parasite

    OpenAIRE

    Kerstin Leykauf; Moritz Treeck; Gilson, Paul R.; Thomas Nebl; Thomas Braulke; Cowman, Alan F; Gilberger, Tim W; Brendan S Crabb

    2010-01-01

    Apicomplexan parasites are obligate intracellular parasites that infect a variety of hosts, causing significant diseases in livestock and humans. The invasive forms of the parasites invade their host cells by gliding motility, an active process driven by parasite adhesion proteins and molecular motors. A crucial point during host cell invasion is the formation of a ring-shaped area of intimate contact between the parasite and the host known as a tight junction. As the invasive zoite propels i...

  15. Signaling through the G-protein-coupled receptor Rickets is important for polarity, detachment, and migration of the border cells in Drosophila.

    Science.gov (United States)

    Anllo, Lauren; Schüpbach, Trudi

    2016-06-15

    Cell migration plays crucial roles during development. An excellent model to study coordinated cell movements is provided by the migration of border cell clusters within a developing Drosophila egg chamber. In a mutagenesis screen, we isolated two alleles of the gene rickets (rk) encoding a G-protein-coupled receptor. The rk alleles result in border cell migration defects in a significant fraction of egg chambers. In rk mutants, border cells are properly specified and express the marker Slbo. Yet, analysis of both fixed as well as live samples revealed that some single border cells lag behind the main border cell cluster during migration, or, in other cases, the entire border cell cluster can remain tethered to the anterior epithelium as it migrates. These defects are observed significantly more often in mosaic border cell clusters, than in full mutant clusters. Reduction of the Rk ligand, Bursicon, in the border cell cluster also resulted in migration defects, strongly suggesting that Rk signaling is utilized for communication within the border cell cluster itself. The mutant border cell clusters show defects in localization of the adhesion protein E-cadherin, and apical polarity proteins during migration. E-cadherin mislocalization occurs in mosaic clusters, but not in full mutant clusters, correlating well with the rk border cell migration phenotype. Our work has identified a receptor with a previously unknown role in border cell migration that appears to regulate detachment and polarity of the border cell cluster coordinating processes within the cells of the cluster themselves.

  16. The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species.

    Science.gov (United States)

    Alhebshi, Alawiah; Sideri, Theodora C; Holland, Sara L; Avery, Simon V

    2012-09-01

    Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary "Achilles' heel" of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster-defective Rli1p construct. The protein's FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily (55)FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress-related disease.

  17. Unveiling equal importance of two 14-3-3 proteins for morphogenesis, conidiation, stress tolerance and virulence of an insect pathogen.

    Science.gov (United States)

    Liu, Qian; Li, Jin-Gen; Ying, Sheng-Hua; Wang, Juan-Juan; Sun, Wen-Liang; Tian, Chao-Guang; Feng, Ming-Guang

    2015-04-01

    Two conserved 14-3-3 proteins orthologous to Saccharomyces cerevisiae Bmh1/2 are poorly understood in filamentous fungi. Here we show that Bmh1 and Bmh2 contribute equally to the fundamental biology and physiology of Beauveria bassiana by targeting many sets of proteins/enzymes. Single Bmh deletion caused similar upregulation of another. Excellent knockdown (∼91%) expressions of Bmh1 in ΔBmh2 and Bmh2 in ΔBmh1 resulted in equally more severe multiphenotypic defects than the single deletions, including G2 /M transition, blastospore size, carbon/nitrogen utilization, conidiation, germination and conidial tolerances to high osmolarity, oxidation, cell wall stress, high temperature and UV-B irradiation. All the deletion and deletion/knockdown mutants showed similar defects in blastospore yield and density, hyphal septation and cell size, hyphal responses to most chemical stresses and virulence. All the defects were evident with altered transcripts of phenotype-related genes and well restored by each Bmh complementation. Our Bmh1- and Bmh2-specific transcriptomes generated under osmotic and oxidative stresses revealed up to 6% genes differentially expressed by at least twofold in the fungal genome. Many of those were greatly depressed or co-depressed in ΔBmh1 and ΔBmh2. Our findings provide a thorough insight into the functions and complementary effects of the two 14-3-3 proteins in the filamentous entomopathogen.

  18. MOF and H4 K16 Acetylation Play Important Roles in DNA Damage Repair by Modulating Recruitment of DNA Damage Repair Protein Mdc1 ▿

    Science.gov (United States)

    Li, Xiangzhi; Corsa, Callie Ann Sprunger; Pan, Patricia W.; Wu, Lipeng; Ferguson, David; Yu, Xiaochun; Min, Jinrong; Dou, Yali

    2010-01-01

    MOF (MYST1) is the major enzyme to catalyze acetylation of histone H4 lysine 16 (K16) and is highly conserved through evolution. Using a conditional knockout mouse model and the derived mouse embryonic fibroblast cell lines, we showed that loss of Mof led to a global reduction of H4 K16 acetylation, severe G2/M cell cycle arrest, massive chromosome aberration, and defects in ionizing radiation-induced DNA damage repair. We further showed that although early DNA damage sensing and signaling by ATM were normal in Mof-null cells, the recruitment of repair mediator protein Mdc1 and its downstream signaling proteins 53bp1 and Brca1 to DNA damage foci was completely abolished. Mechanistic studies suggested that Mof-mediated H4 K16 acetylation and an intact acidic pocket on H2A.X were essential for the recruitment of Mdc1. Removal of Mof and its associated proteins phenocopied a charge-neutralizing mutant of H2A.X. Given the well-characterized H4-H2A trans interactions in regulating higher-order chromatin structure, our study revealed a novel chromatin-based mechanism that regulates the DNA damage repair process. PMID:20837706

  19. DNA Import into Mitochondria.

    Science.gov (United States)

    Konstantinov, Yu M; Dietrich, A; Weber-Lotfi, F; Ibrahim, N; Klimenko, E S; Tarasenko, V I; Bolotova, T A; Koulintchenko, M V

    2016-10-01

    In recent decades, it has become evident that the condition for normal functioning of mitochondria in higher eukaryotes is the presence of membrane transport systems of macromolecules (proteins and nucleic acids). Natural competence of the mitochondria in plants, animals, and yeasts to actively uptake DNA may be directly related to horizontal gene transfer into these organelles occurring at much higher rate compared to the nuclear and chloroplast genomes. However, in contrast with import of proteins and tRNAs, little is known about the biological role and molecular mechanism underlying import of DNA into eukaryotic mitochondria. In this review, we discuss current state of investigations in this area, particularly specificity of DNA import into mitochondria and its features in plants, animals, and yeasts; a tentative mechanism of DNA import across the mitochondrial outer and inner membranes; experimental data evidencing several existing, but not yet fully understood mechanisms of DNA transfer into mitochondria. Currently available data regarding transport of informational macromolecules (DNA, RNA, and proteins) into the mitochondria do not rule out that the mechanism of protein and tRNA import as well as tRNA and DNA import into the mitochondria may partially overlap.

  20. Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: Protein synthesis, cell proliferation, and T-cell activation

    Energy Technology Data Exchange (ETDEWEB)

    Navare, Arti T.; Sova, Pavel; Purdy, David E.; Weiss, Jeffrey M. [Department of Microbiology, University of Washington, Seattle, WA (United States); Wolf-Yadlin, Alejandro [Department of Genome Sciences, University of Washington, Seattle, WA (United States); Korth, Marcus J.; Chang, Stewart T.; Proll, Sean C. [Department of Microbiology, University of Washington, Seattle, WA (United States); Jahan, Tahmina A. [Proteomics Resource, UW Medicine at South Lake Union, Seattle, WA (United States); Krasnoselsky, Alexei L.; Palermo, Robert E. [Department of Microbiology, University of Washington, Seattle, WA (United States); Katze, Michael G., E-mail: honey@uw.edu [Department of Microbiology, University of Washington, Seattle, WA (United States); Washington National Primate Research Center, University of Washington, Seattle, WA (United States)

    2012-07-20

    Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value{<=}0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

  1. Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration

    Institute of Scientific and Technical Information of China (English)

    Takuya Watanabe; Masumi Tsuda; Yoshinori Makino; Tassos Konstantinou; Hiroshi Nishihara; Tokifumi Majima; Akio Minami; Stephan M Feller; Shinya Tanaka

    2009-01-01

    Upon growth factor stimulation, the scaffold protein, Gabl, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gabl. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extraceilular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of Crkll demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of Crkll, is critical for the induction of Gabl-Y307 phosphorylation. SH2 mutation of Crkll also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gabl, whereas Crk-SH3(N) interacted with the Gabl mutant, which lacks the clus-tered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gabl was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gabl. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gabl with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The GabI-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gabl-Y307F disturbed the localization of Crk, FAK, and paxiilin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphory-lation of Gabl-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.

  2. Envelope Protein Mutations L107F and E138K Are Important for Neurovirulence Attenuation for Japanese Encephalitis Virus SA14-14-2 Strain

    Directory of Open Access Journals (Sweden)

    Jian Yang

    2017-01-01

    Full Text Available The attenuated Japanese encephalitis virus (JEV strain SA14-14-2 has been successfully utilized to prevent JEV infection; however, the attenuation determinants have not been fully elucidated. The envelope (E protein of the attenuated JEV SA14-14-2 strain differs from that of the virulent parental SA14 strain at eight amino acid positions (E107, E138, E176, E177, E264, E279, E315, and E439. Here, we investigated the SA14-14-2-attenuation determinants by mutating E107, E138, E176, E177, and E279 in SA14-14-2 to their status in the parental virulent strain and tested the replication capacity, neurovirulence, neuroinvasiveness, and mortality associated with the mutated viruses in mice, as compared with those of JEV SA14-14-2 and SA14. Our findings indicated that revertant mutations at the E138 or E107 position significantly increased SA14-14-2 virulence, whereas other revertant mutations exhibited significant increases in neurovirulence only when combined with E138, E107, and other mutations. Revertant mutations at all eight positions in the E protein resulted in the highest degree of SA14-14-2 virulence, although this was still lower than that observed in SA14. These results demonstrated the critical role of the viral E protein in controlling JEV virulence and identified the amino acids at the E107 and E138 positions as the key determinants of SA14-14-2 neurovirulence.

  3. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    Science.gov (United States)

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  4. Towards an exact theory of linear absorbance and circular dichroism of pigment-protein complexes: Importance of non-secular contributions

    Energy Technology Data Exchange (ETDEWEB)

    Dinh, Thanh-Chung; Renger, Thomas, E-mail: thomas.renger@jku.at [Institut für Theoretische Physik, Johannes Kepler University Linz, Altenberger Str. 69, 4040 Linz (Austria)

    2015-01-21

    A challenge for the theory of optical spectra of pigment-protein complexes is the equal strength of the pigment-pigment and the pigment-protein couplings. Treating both on an equal footing so far can only be managed by numerically costly approaches. Here, we exploit recent results on a normal mode analysis derived spectral density that revealed the dominance of the diagonal matrix elements of the exciton-vibrational coupling in the exciton state representation. We use a cumulant expansion technique that treats the diagonal parts exactly, includes an infinite summation of the off-diagonal parts in secular and Markov approximations, and provides a systematic perturbative way to include non-secular and non-Markov corrections. The theory is applied to a model dimer and to chlorophyll (Chl) a and Chl b homodimers of the reconstituted water-soluble chlorophyll-binding protein (WSCP) from cauliflower. The model calculations reveal that the non-secular/non-Markov effects redistribute oscillator strength from the strong to the weak exciton transition in absorbance and they diminish the rotational strength of the exciton transitions in circular dichroism. The magnitude of these corrections is in a few percent range of the overall signal, providing a quantitative explanation of the success of time-local convolution-less density matrix theory applied earlier. A close examination of the optical spectra of Chl a and Chl b homodimers in WSCP suggests that the opening angle between Q{sub y} transition dipole moments in Chl b homodimers is larger by about 9{sup ∘} than for Chl a homodimers for which a crystal structure of a related WSCP complex exists. It remains to be investigated whether this change is due to a different mutual geometry of the pigments or due to the different electronic structures of Chl a and Chl b.

  5. Segments in the C-terminal folding domain of lipoprotein lipase important for binding to the low density lipoprotein receptor-related protein and to heparan sulfate proteoglycans

    DEFF Research Database (Denmark)

    Nielsen, Morten Schallburg; Brejning, Jeanette; García, R.

    1997-01-01

    Lipoprotein lipase (LpL) can mediate cellular uptake of chylomicron and VLDL remnants via binding to heparan sulfate proteoglycans (HSPG) and the endocytic alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR/LRP). Whereas it is established that the C......L-(347-448) in Escherichia coli. In addition to binding to alpha2MR/LRP, LpL-(313-448) displayed binding to heparin with an affinity similar to that of the LpL monomer, whereas it bound poorly to lipoprotein particles. Moreover, LpL-(313-448) displayed heparin sensitive binding to normal, but not to HSPG...

  6. Historical perspectives on the discovery and elucidation of autoantibodies to centromere proteins (CENP) and the emerging importance of antibodies to CENP-F.

    Science.gov (United States)

    Fritzler, Marvin J; Rattner, Jerome B; Luft, LeeAnne M; Edworthy, Steven M; Casiano, Carlos A; Peebles, Carol; Mahler, Michael

    2011-02-01

    Autoantibodies to the centromere proteins (CENP), which are major constituents of the primary constriction of metaphase chromosomes, were first described in 1980. In those seminal publications and 30 years of research that have followed, a number of CENP have been identified as autoantibody targets in human diseases. Historically, autoantibodies directed to CENP-A, -B and -C have been considered relatively specific biomarkers for limited cutaneous systemic sclerosis (lcSSc) or the calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) syndrome. These autoantibodies, found in up to 40% of SSc sera, can be identified by indirect immunofluorescence (IIF) on a variety of tissue culture cell lines as a discrete speckled staining pattern of both interphase nuclei and metaphase chromatin. Early in the investigation of anti-CENP, it became apparent that some autoantibodies had a similar IIF pattern wherein as cells entered into the cell cycle, speckled staining of the metaphase chromatin could be observed but, unlike conventional CENP staining, interphase nuclei were not stained. Subsequent studies identified one of the targets of these autoantibodies to be CENP-F, a kinesin binding protein essential for completion of the cell cycle. Early clinical studies found that, unlike antibodies to the earlier described CENP, lcSSc rarely expressed anti-CENP-F and approximately 50% of these patients had a malignancy. This review provides a historical perspective of CENP autoantibodies and focuses on an update of the information on CENP-F and their clinical associations.

  7. Exploring the Strength of the H-Bond in Synthetic Models for Heme Proteins: The Importance of the N-H Acidity of the Distal Base.

    Science.gov (United States)

    Alberti, Mariza N; Polyhach, Yevhen; Tzirakis, Manolis D; Tödtli, Laura; Jeschke, Gunnar; Diederich, François

    2016-07-11

    The distal hydrogen bond (H-bond) in dioxygen-binding proteins is crucial for the discrimination of O2 with respect to CO or NO. We report the preparation and characterization of a series of Zn(II) porphyrins, with one of three meso-phenyl rings bearing both an alkyl-tethered proximal imidazole ligand and a heterocyclic distal H-bond donor connected by a rigid acetylene spacer. Previously, we had validated the corresponding Co(II) complexes as synthetic model systems for dioxygen-binding heme proteins and demonstrated the structural requirements for proper distal H-bonding to Co(II) -bound dioxygen. Here, we systematically vary the H-bond donor ability of the distal heterocycles, as predicted based on pKa values. The H-bond in the dioxygen adducts of the Co(II) porphyrins was directly measured by Q-band Davies-ENDOR spectroscopy. It was shown that the strength of the hyperfine coupling between the dioxygen radical and the distal H-atom increases with enhanced acidity of the H-bond donor.

  8. The Importance of the KR-Rich Region of the Coat Protein of Ourmia melon virus for Host Specificity, Tissue Tropism, and Interference With Antiviral Defense.

    Science.gov (United States)

    Rossi, Marika; Vallino, Marta; Abbà, Simona; Ciuffo, Marina; Balestrini, Raffaella; Genre, Andrea; Turina, Massimo

    2015-01-01

    The N-terminal region of the Ourmia melon virus (OuMV) coat protein (CP) contains a short lysine/arginine-rich (KR) region. By alanine scanning mutagenesis, we showed that the KR region influences pathogenicity and virulence of OuMV without altering viral particle assembly. A mutant, called OuMV6710, with three basic residue substitutions in the KR region, was impaired in the ability to maintain the initial systemic infection in Nicotiana benthamiana and to infect both cucumber and melon plants systemically. The integrity of this protein region was also crucial for encapsidation of viral genomic RNA; in fact, certain mutations within the KR region partially compromised the RNA encapsidation efficiency of the CP. In Arabidopsis thaliana Col-0, OuMV6710 was impaired in particle accumulation; however, this phenotype was abolished in dcl2/dcl4 and dcl2/dcl3/dcl4 Arabidopsis mutants defective for antiviral silencing. Moreover, in contrast to CPwt, in situ immunolocalization experiments indicated that CP6710 accumulates efficiently in the spongy mesophyll tissue of infected N. benthamiana and A. thaliana leaves but only occasionally infects palisade tissues. These results provided strong evidence of a crucial role for OuMV CP during viral infection and highlighted the relevance of the KR region in determining tissue tropism, host range, pathogenicity, and RNA affinity, which may be all correlated with a possible CP silencing-suppression activity.

  9. Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.

    Science.gov (United States)

    Merlino, Antonello; Russo Krauss, Irene; Castellano, Immacolata; Ruocco, Maria Rosaria; Capasso, Alessandra; De Vendittis, Emmanuele; Rossi, Bianca; Sica, Filomena

    2014-03-01

    A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. The interactions between rat-adipose-derived stromal cells, recombinant human bone morphogenetic protein-2, and beta-tricalcium phosphate play an important role in bone tissue engineering.

    Science.gov (United States)

    E, Ling-Ling; Xu, Lu-Lu; Wu, Xia; Wang, Dong-Sheng; Lv, Yan; Wang, Jia-Zhu; Liu, Hong-Chen

    2010-09-01

    Cells, scaffolds, and growth factors are the three main factors for creating a stem-cell-based tissue-engineered construct, but the interactions between three factors are not very clear. We hereby explored the interactions between rat-adipose-derived stromal cells (rASCs), recombinant human bone morphogenetic protein-2 (rhBMP-2), and beta-tricalcium phosphate (beta-TCP) to provide evidence for their application in bone tissue engineering by evaluating the protein adsorption of beta-TCP, the cell attachment, alkaline phosphatase (ALP) activity/protein, osteocalcin (OCN) content, mineral formation, calcium content, phosphonium content, cell vitality, gene expression, and implantation in the backs of severe combined immunodeficient mice of rhBMP-2 preinducing rASCs seeded onto beta-TCP. The results showed that beta-TCP could adsorb the proteins from the media. The attachment, proliferation, and osteogenic properties of rASCs were supported by beta-TCP, as revealed using scanning electron microscopy. Compared with rASCs cultured on the culture plate, rASCs cultured on beta-TCP had significantly higher ALP activity/protein, OCN content, and mineral formation. These values for rASCs cultured on beta-TCP with rhBMP-2 increased most significantly. The rhBMP-2 significantly increased the calcium content, phosphonium content, and ALP, type I collagen, and OCN mRNA levels of rASCs cultured on beta-TCP. The methylthiazol tetrazolium method revealed that the vitality of rASCs cultured on beta-TCP with or without rhBMP-2 for 4, 7, and 28 days in vitro was insignificantly different. After 8 and 12 weeks of implantation, each group displayed increased bone formation over the 12-week period. The percentage of the new bone formed areas for beta-TCP/rhBMP-2 and beta-TCP was not significantly different. This value for rASCs/beta-TCP construct was significantly higher than that for beta-TCP group, but the maximal and robust bone formation was presented in rASCs/beta-TCP with rhBMP-2

  11. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay

    2013-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a...... in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly...

  12. [Imported histoplasmosis].

    Science.gov (United States)

    Stete, Katarina; Kern, Winfried V; Rieg, Siegbert; Serr, Annerose; Maurer, Christian; Tintelnot, Kathrin; Wagner, Dirk

    2015-06-01

    Infections with Histoplasma capsulatum are rare in Germany, and mostly imported from endemic areas. Infections can present as localized or disseminated diseases in immunocompromised as well as immunocompetent hosts. A travel history may be a major clue for diagnosing histoplasmosis. Diagnostic tools include histology, cultural and molecular detection as well as serology. Here we present four cases of patients diagnosed and treated in Freiburg between 2004 and 2013 that demonstrate the broad range of clinical manifestations of histoplasmosis: an immunocompetent patient with chronic basal meningitis; a patient with HIV infection and fatal disseminated disease; a patient with pulmonary and cutaneous disease and mediastinal and cervical lymphadenopathy; and an immunosuppressed patient with disseminated involvement of lung, bone marrow and adrenal glands.

  13. C-Reactive Protein Is an Important Biomarker for Prognosis Tumor Recurrence and Treatment Response in Adult Solid Tumors: A Systematic Review

    Science.gov (United States)

    Shrotriya, Shiva; Walsh, Declan; Bennani-Baiti, Nabila; Thomas, Shirley; Lorton, Cliona

    2015-01-01

    Purpose A systematic literature review was done to determine the relationship between elevated CRP and prognosis in people with solid tumors. C-reactive protein (CRP) is a serum acute phase reactant and a well-established inflammatory marker. We also examined the role of CRP to predict treatment response and tumor recurrence. Methods MeSH (Medical Subject Heading) terms were used to search multiple electronic databases (PubMed, EMBASE, Web of Science, SCOPUS, EBM-Cochrane). Two independent reviewers selected research papers. We also included a quality Assessment (QA) score. Reports with QA scores lung, pancreas, hepatocellular cancer, and bladder) an elevated CRP also predicted prognosis. In addition there is also evidence to support the use of CRP to help decide treatment response and identify tumor recurrence. Better designed large scale studies should be conducted to examine these issues more comprehensively. PMID:26717416

  14. The importance of monitoring metabolic recovery in the coral Acropora cervicornis after short-term exposure to drilling muds: Calcification rate and protein concentration

    Science.gov (United States)

    Kendall, J. J.; Powell, E. N.; Connor, S. J.; Bright, T. J.; Zastrow, C. E.

    1984-04-01

    The effect of used drilling muds on coral health was examined by monitoring changes in calcification rate and soluble tissue protein concentration in the coral Acropora cervicornis. Exposure to 25 ppm (v/v) of one mud for 24 h reduced calcification rate in the growing tips by as much as 62%. In recovery experiments, corals were exposed to drilling muds for 24 h; some of them were allowed to recover in clean seawater for 48 h. After the 24-hour exposure, calcification rates were significantly less than those of the controls. After a 48-hour recovery period, calcification rates returned to control levels for one mud but were still significantly below control levels for another. The results indicate that the capacity for recovery after exposure cannot be predicted from the results of experiments on exposure only. Recovery capacity must be independently verified for all studies on the effects of short-term exposure to drilling muds.

  15. Positive Regulation of Staphylococcal Enterotoxin H by Rot (Repressor of Toxin) Protein and Its Importance in Clonal Complex 81 Subtype 1 Lineage-Related Food Poisoning.

    Science.gov (United States)

    Sato'o, Yusuke; Hisatsune, Junzo; Nagasako, Yuria; Ono, Hisaya K; Omoe, Katsuhiko; Sugai, Motoyuki

    2015-11-01

    We previously demonstrated the clonal complex 81 (CC81) subtype 1 lineage is the major staphylococcal food poisoning (SFP)-associated lineage in Japan (Y. Sato'o et al., J Clin Microbiol 52:2637-2640, 2014, http://dx.doi.org/10.1128/JCM.00661-14). Strains of this lineage produce staphylococcal enterotoxin H (SEH) in addition to SEA. However, an evaluation of the risk for the recently reported SEH has not been sufficiently conducted. We first searched for staphylococcal enterotoxin (SE) genes and SE proteins in milk samples that caused a large SFP outbreak in Japan. Only SEA and SEH were detected, while there were several SE genes detected in the samples. We next designed an experimental model using a meat product to assess the productivity of SEs and found that only SEA and SEH were detectably produced in situ. Therefore, we investigated the regulation of SEH production using a CC81 subtype 1 isolate. Through mutant analysis of global regulators, we found the repressor of toxin (Rot) functioned oppositely as a stimulator of SEH production. SEA production was not affected by Rot. seh mRNA expression correlated with rot both in media and on the meat product, and the Rot protein was shown to directly bind to the seh promoter. The seh promoter sequence was predicted to form a loop structure and to hide the RNA polymerase binding sequences. We propose Rot binds to the promoter sequence of seh and unfolds the secondary structure that may lead the RNA polymerase to bind the promoter, and then seh mRNA transcription begins. This alternative Rot regulation for SEH may contribute to sufficient toxin production by the CC81 subtype 1 lineage in foods to induce SFP.

  16. The Importance of Non-accessible Crosslinks and Solvent Accessible Surface Distance in Modeling Proteins with Restraints From Crosslinking Mass Spectrometry.

    Science.gov (United States)

    Matthew Allen Bullock, Joshua; Schwab, Jannik; Thalassinos, Konstantinos; Topf, Maya

    2016-07-01

    Crosslinking mass spectrometry (XL-MS) is becoming an increasingly popular technique for modeling protein monomers and complexes. The distance restraints garnered from these experiments can be used alone or as part of an integrative modeling approach, incorporating data from many sources. However, modeling practices are varied and the difference in their usefulness is not clear. Here, we develop a new scoring procedure for models based on crosslink data-Matched and Nonaccessible Crosslink score (MNXL). We compare its performance with that of other commonly-used scoring functions (Number of Violations and Sum of Violation Distances) on a benchmark of 14 protein domains, each with 300 corresponding models (at various levels of quality) and associated, previously published, experimental crosslinks (XLdb). The distances between crosslinked lysines are calculated either as Euclidean distances or Solvent Accessible Surface Distances (SASD) using a newly-developed method (Jwalk). MNXL takes into account whether a crosslink is nonaccessible, i.e. an experimentally observed crosslink has no corresponding SASD in a model due to buried lysines. This metric alone is shown to have a significant impact on modeling performance and is a concept that is not considered at present if only Euclidean distances are used. Additionally, a comparison between modeling with SASD or Euclidean distance shows that SASD is superior, even when factoring out the effect of the nonaccessible crosslinks. Our benchmarking also shows that MNXL outperforms the other tested scoring functions in terms of precision and correlation to Cα-RMSD from the crystal structure. We finally test the MNXL at different levels of crosslink recovery (i.e. the percentage of crosslinks experimentally observed out of all theoretical ones) and set a target recovery of ∼20% after which the performance plateaus.

  17. Binding of an RNA aptamer and a partial peptide of a prion protein: crucial importance of water entropy in molecular recognition

    Science.gov (United States)

    Hayashi, Tomohiko; Oshima, Hiraku; Mashima, Tsukasa; Nagata, Takashi; Katahira, Masato; Kinoshita, Masahiro

    2014-01-01

    It is a central issue to elucidate the new type of molecular recognition accompanied by a global structural change of a molecule upon binding to its targets. Here we investigate the driving force for the binding of R12 (a ribonucleic acid aptamer) and P16 (a partial peptide of a prion protein) during which P16 exhibits the global structural change. We calculate changes in thermodynamic quantities upon the R12–P16 binding using a statistical-mechanical approach combined with molecular models for water which is currently best suited to studies on hydration of biomolecules. The binding is driven by a water-entropy gain originating primarily from an increase in the total volume available to the translational displacement of water molecules in the system. The energy decrease due to the gain of R12–P16 attractive (van der Waals and electrostatic) interactions is almost canceled out by the energy increase related to the loss of R12–water and P16–water attractive interactions. We can explain the general experimental result that stacking of flat moieties, hydrogen bonding and molecular-shape and electrostatic complementarities are frequently observed in the complexes. It is argued that the water-entropy gain is largely influenced by the geometric characteristics (overall shapes, sizes and detailed polyatomic structures) of the biomolecules. PMID:24803670

  18. The status and importance of crude protein and macro minerals in native pastures growing on Vertisols of the central highlands of Ethiopia.

    Science.gov (United States)

    Gizachew, Lemma; Smit, G N

    2012-01-01

    The effects of pasture management, season and soil nutrient status on crude protein (CP) and macro mineral concentration of native pasture was studied in the Vertisol areas of the central Ethiopian highland. Soil and herbage samples from 18 continuously grazed (CG) and 12 seasonally grazed (SG) pasture sites were analyzed for N, P, Ca, Mg, K and Na. Soil and dry season CG pasture samples were collected in January and February 2001 (dry season: November-February), while wet season CG and SG pasture samples were collected during September 2001 (wet season: April-October). The Potassium concentration (2.55%) of mixed herbage samples from SG pasture exceeded the K values (1.80%) from CG pasture (P macro minerals concentrations were noted among forage species. The mean CP and K concentrations of herbage from CG pasture were higher (P macro minerals and the corresponding herbage macro mineral concentrations, significant (P macro mineral composition. The levels of CP in CG pasture and that of P and Na in both CG and SG pastures may fall below the requirements of grazing livestock. Resting at critical stages of the growth cycle of the forage species encouraged the recovery of desirable species. For this reason resting of pasture can contribute significantly to the quality of the native pastures of the Vertisols of the central Ethiopian highlands and should be encouraged.

  19. A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Francia, Maria E.; Wicher, Sarah [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Pace, Douglas A. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Sullivan, Jack [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Moreno, Silvia N.J. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Arrizabalaga, Gustavo, E-mail: gustavo@uidaho.edu [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States)

    2011-06-10

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  20. A Toxoplasma gondii protein with homology to intracellular type Na⁺/H⁺ exchangers is important for osmoregulation and invasion.

    Science.gov (United States)

    Francia, Maria E; Wicher, Sarah; Pace, Douglas A; Sullivan, Jack; Moreno, Silvia N J; Arrizabalaga, Gustavo

    2011-06-10

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na(+)/H(+) exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca(2+) concentration [Ca(2+)](i), and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  1. Disrupting LIN28 in atypical teratoid rhabdoid tumors reveals the importance of the mitogen activated protein kinase pathway as a therapeutic target.

    Science.gov (United States)

    Weingart, Melanie F; Roth, Jacquelyn J; Hutt-Cabezas, Marianne; Busse, Tracy M; Kaur, Harpreet; Price, Antoinette; Maynard, Rachael; Rubens, Jeffrey; Taylor, Isabella; Mao, Xing-Gang; Xu, Jingying; Kuwahara, Yasumichi; Allen, Sariah J; Erdreich-Epstein, Anat; Weissman, Bernard E; Orr, Brent A; Eberhart, Charles G; Biegel, Jaclyn A; Raabe, Eric H

    2015-02-20

    Atypical teratoid rhabdoid tumor (AT/RT) is among the most fatal of all pediatric brain tumors. Aside from loss of function mutations in the SMARCB1 (BAF47/INI1/SNF5) chromatin remodeling gene, little is known of other molecular drivers of AT/RT. LIN28A and LIN28B are stem cell factors that regulate thousands of RNAs and are expressed in aggressive cancers. We identified high-levels of LIN28A and LIN28B in AT/RT primary tumors and cell lines, with corresponding low levels of the LIN28-regulated microRNAs of the let-7 family. Knockdown of LIN28A by lentiviral shRNA in the AT/RT cell lines CHLA-06-ATRT and BT37 inhibited growth, cell proliferation and colony formation and induced apoptosis. Suppression of LIN28A in orthotopic xenograft models led to a more than doubling of median survival compared to empty vector controls (48 vs 115 days). LIN28A knockdown led to increased expression of let-7b and let-7g microRNAs and a down-regulation of KRAS mRNA. AT/RT primary tumors expressed increased mitogen activated protein (MAP) kinase pathway activity, and the MEK inhibitor selumetinib (AZD6244) decreased AT/RT growth and increased apoptosis. These data implicate LIN28/RAS/MAP kinase as key drivers of AT/RT tumorigenesis and indicate that targeting this pathway may be a therapeutic option in this aggressive pediatric malignancy.

  2. Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

    Energy Technology Data Exchange (ETDEWEB)

    Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: yxh0@cdc.gov [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

    2014-05-15

    Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

  3. Role of Human Breast Cancer Related Protein versus P-Glycoprotein as an Efflux Transporter for Benzylpenicillin: Potential Importance at the Blood-Brain Barrier.

    Directory of Open Access Journals (Sweden)

    Yangfang Li

    Full Text Available While the blood-brain barrier (BBB protects the brain by controlling the access of solutes and toxic substances to brain, it also limits drug entry to treat central nervous system disorders. Many drugs are substrates for ATP-binding cassette (ABC transporters at the BBB that limit their entry into the brain. The role of those transporters in limiting the entry of the widely prescribed therapeutic, benzylpenicillin, has produced conflicting results. This study investigated the possible potential involvement of P-glycoprotein (P-gp and breast cancer resistance protein (BCRP, two ABC transporters, in benzylpenicillin transport at BBB in human using MDCKII cells overexpressing those transporters as well as pharmacological inhibition. MDCKII cells overexpressing human BCRP (MDCKII-BCRP but not those overexpressing human P-gp (MDCKII-MDR cells had reduced [3H]benzylpenicillin uptake. Similarly, inhibiting BCRP increased [3H]benzylpenicillin uptake in MDCKII-BCRP cells, while inhibiting P-gp in MDCKII-MDR cells had no effect on uptake although there was evidence that benzylpenicillin is a substrate for canine P-gp. While inhibiting BCRP affected [3H]benzylpenicillin cell concentrations it did not affect transepithelial flux in MDCKII-BCRP cells. In summary, the results indicate that human BCRP and not human P-gp is involved in benzylpenicillin transport. However, targeting BCRP alone was not sufficient to alter transepithelial flux in MDCKII cells. Whether it would be sufficient to alter blood-to-brain flux at the human BBB remains to be investigated.

  4. Chemically optimized antimyosin Fab conjugates with chelating polymers: importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarction localization.

    Science.gov (United States)

    Trubetskoy, V S; Narula, J; Khaw, B A; Torchilin, V P

    1993-01-01

    Murine antimyosin Fab fragment was conjugated with 111In-labeled N-terminal-modified DTPA-polylysine using three bifunctional reagents: N-hydroxysuccinimide esters of 3-(2-pyridyldithio)propionic acid (SPDP conjugate), 4-(maleimidomethyl)cyclohexanecarboxylic acid (SMCC conjugate) and bromoacetic acid (BrAc conjugate) for potential localization of experimental myocardial infarction. Using various antibody preparations and a rabbit acute myocardial infarction model the following parameters were observed: (1) an in vitro antigen binding activity of SPDP conjugate = SMCC conjugate > BrAc conjugate, (2) a blood clearance rate of SPDP conjugate > BrAc conjugate > SMCC conjugate, (3) a liver and splenic accumulation of SPDP conjugate > BrAc conjugate > SMCC conjugate, and (4) the infarcted tissue activity showed an accumulation of SMCC conjugate > SPDP conjugate > BrAc conjugate. This study exemplifies the importance of rational chemical design of antimyosin Fab-chelating polymer conjugate for improved target tissue localization in vivo.

  5. Centrosomal protein Cep63, an important protein involved in cell division%中心体蛋白Cep63——参与细胞分裂的重要蛋白

    Institute of Scientific and Technical Information of China (English)

    徐朝阳; 孙莹璞

    2013-01-01

    电子显微镜下,中心体由中心粒和中心粒周围物质组成.中心粒周围物质由一系列纤维和蛋白质组成,这些蛋白质是中心体执行其功能的基础.作为中心体蛋白家族的一名成员,Cep63的研究近年取得了很大的进展.在有丝分裂周期的各个阶段,Cep63都定位在中心体内,其与Cep152和Cep57一起,在中心粒近端形成一个环状结构,对中心体复制、纺锤体组装和G2/M转换起到重要作用.在多种肿瘤组织内发现Cep63表达的异常,其缺陷还会导致小头畸形的发生.鉴于Cep63在有丝分裂中的重要地位,研究其在减数分裂中的定位和功能,探讨卵母细胞体外成熟机制,对生殖医学的发展具有重要意义.%Under the electron microscope,the eentrosome consists of centriole and peri-centriolar material (PCM) . PCM is composed of a series of proteins and fibers. Most of the centrosome function are carried out by these proteins.As a member of centrosomal proteins family,the Cep63 has been studied with great progress in recent years.The Cep63 is ahnost exclusively localized to centrosomes throughout the cell cycle.Together with Cep152 and Cep57,they form a ring-like structure,which lies around the proximal end of centriole,and functions in centrosome duplication,spindle assemble and G2 / M transition mechanism.Abnormal expression of Cep63 has been reported to be associated with several kinds of tumors and primary microcephaly.In view of the role of Cep63 in mitosis,the study of its location and function in meiosis will favor the exploration of the oocyte maturation mechanism in vitro,and eventually may help for the development of reproductive medicine in the future.

  6. Histidine at Residue 99 and the Transmembrane Region of the Precursor Membrane prM Protein Are Important for the prM-E Heterodimeric Complex Formation of Japanese Encephalitis Virus

    OpenAIRE

    Lin, Ying-Ju; Wu, Suh-Chin

    2005-01-01

    The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucro...

  7. The Lysine Residues within the Human Ribosomal Protein S17 Sequence Naturally Inserted into the Viral Nonstructural Protein of a Unique Strain of Hepatitis E Virus Are Important for Enhanced Virus Replication

    OpenAIRE

    Kenney, Scott P.; Meng, Xiang-Jin

    2015-01-01

    Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Due largely to the lack of an efficient cell culture system for HEV, the molecular mechanisms of HEV replication and pathogenesis are poorly understood. Recently, a unique genotype 3 strain of HEV recovered from a chronically infected patient was adapted for growth in HepG2C3A human hepatoma cells. The adaptation of the Kernow C-1 P6 HEV to propagate in HepG2C3A cells selected for a rare virus recombinant that ...

  8. Mutations in Streptococcus pneumoniae penicillin-binding protein 2x: importance of the C-terminal penicillin-binding protein and serine/threonine kinase-associated domains for beta-lactam binding.

    Science.gov (United States)

    Maurer, Patrick; Todorova, Katya; Sauerbier, Julia; Hakenbeck, Regine

    2012-06-01

    Penicillin-binding protein 2x (PBP2x) mutations that occur during the selection with beta-lactams are located within the central penicillin-binding/transpeptidase (TP) domain, and are believed to mediate resistance by interfering with the formation of a covalent complex of the active site serine with the antibiotic. We now investigated the effect of two point mutations found in two independently obtained laboratory mutants that are located at the surface of the TP domain with their side chains facing outside (G422D respectively R426C). They have no significant effect on resistance to cefotaxime in vivo or on binding to Bocillin™FL to the active site in vitro using purified PBP2x derivatives, thus apparently do not affect the active site directly. In contrast, in silico modeling revealed that they affect van der Waal's interactions with the PASTA1 (PBP and serine/threonine kinase associated) domain of the C-terminal extension and a noncovalent cefuroxime molecule found in the X-ray structure of an acylated PBP2x, suggesting some effect of the mutations on the interaction of the TP domain with PASTA1 and/or with the antibiotic associated with PASTA1. The effect of the PASTA domains on covalent binding of PBP2x to Bocillin FL was then investigated using a series of soluble truncated PBP2x derivatives. Deletion of 127 C-terminal residues, that is, of both PASTA domains, decreased binding dramatically by ∼90%. Surprisingly, deletion of only 40 amino acids resulted in the same phenotype, whereas the absence of 30 amino acids affected binding marginally by 10%, documenting a crucial role of the C-terminal domain for beta-lactam binding.

  9. Expression of the plastid-located glutamine synthetase of Medicago truncatula. Accumulation of the precursor in root nodules reveals an in vivo control at the level of protein import into plastids.

    Science.gov (United States)

    Melo, Paula M; Lima, Lígia M; Santos, Isabel M; Carvalho, Helena G; Cullimore, Julie V

    2003-05-01

    In this paper, we report the cloning and characterization of the plastid-located glutamine synthetase (GS) of Medicago truncatula Gaertn (MtGS2). A cDNA was isolated encoding a GS2 precursor polypeptide of 428 amino acids composing an N-terminal transit peptide of 49 amino acids. Expression analysis, by Westerns and by northern hybridization, revealed that MtGS2 is expressed in both photosynthetic and non-photosynthetic organs. Both transcripts and proteins of MtGS2 were detected in substantial amounts in root nodules, suggesting that the enzyme might be performing some important role in this organ. Surprisingly, about 40% of the plastid GS in nodules occurred in the non-processed precursor form (preGS2). This precursor was not detected in any other organ studied and moreover was not observed in non-fixing nodules. Cellular fractionation of nodule extracts revealed that preGS2 is associated with the plastids and that it is catalytically inactive. Immunogold electron microscopy revealed a frequent coincidence of GS with the plastid envelope. Taken together, these results suggest a nodule-specific accumulation of the GS2 precursor at the surface of the plastids in nitrogen-fixing nodules. These results may reflect a regulation of GS2 activity in relation to nitrogen fixation at the level of protein import into nodule plastids.

  10. Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

    Science.gov (United States)

    Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

    2010-02-15

    The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

  11. A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis

    KAUST Repository

    Chen, Tao

    2013-10-17

    Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges. © 2013 Chen et al.

  12. Histidine at residue 99 and the transmembrane region of the precursor membrane prM protein are important for the prM-E heterodimeric complex formation of Japanese encephalitis virus.

    Science.gov (United States)

    Lin, Ying-Ju; Wu, Suh-Chin

    2005-07-01

    The formation of the flavivirus prM-E complex is an important step for the biogenesis of immature virions, which is followed by a subsequent cleavage of prM to M protein through cellular protease to result in the production and release of mature virions. In this study, the intracellular formation of the prM-E complex of Japanese encephalitis virus was investigated by baculovirus coexpression of prM and E in trans in Sf9 insect cells as analyzed by anti-E antibody immunoprecipitation and sucrose gradient sedimentation analysis. A series of carboxyl-terminally truncated prM mutant baculoviruses was constructed to demonstrate that the truncations of the transmembrane (TM) region resulted in a reduction of the formation of the stable prM-E complex by approximately 40% for the TM1 (at residues 130 to 147 [prM130-147]) truncation and 20% for TM2 (at prM153-167) truncation. Alanine-scanning site-directed mutagenesis on the prM99-103 region indicated that the His99 residue was the critical prM binding element for stable prM-E heterodimeric complex formation. The single amino acid mutation at the His99 residue of prM abolishing the prM-E interaction was not due to reduced expression or different subcellular location of the mutant prM protein involved in prM-E interactions as characterized by pulse-chase labeling and confocal scanning microscopic analysis. Recombinant subviral particles were detected in the Sf9 cell culture supernatants by baculovirus coexpression of prM and E proteins but not with the prM H99A mutant. Sequence alignment analysis was further conducted with different groups of flaviviruses to show that the prM H99 residues are generally conserved. Our findings are the first report to characterize the minimum binding elements of the prM protein that are involved in prM-E interactions of flaviviruses. This information, concerning a molecular framework for the prM protein, is considered to elucidate the structure/function relationship of the prM-E complex

  13. Simian-Human immunodeficiency viruses expressing chimeric subtype B/C Vpu proteins demonstrate the importance of the amino terminal and transmembrane domains in the rate of CD4(+) T cell loss in macaques.

    Science.gov (United States)

    Ruiz, Autumn; Schmitt, Kimberly; Culley, Nathan; Stephens, Edward B

    2013-01-20

    Previously, we reported that simian-human immunodeficiency viruses expressing either the lab adapted subtype B (SHIV(KU-1bMC33)) or subtype C (SHIV(SCVpu)) Vpu proteins of human immunodeficiency virus type 1 (HIV-1) had different rates of CD4(+) T cell loss following inoculation into macaques. In this study, we have generated SHIVs that express either the subtype B or subtype C N-terminal (NTD) and transmembrane (TMD) domains and the opposing cytoplasmic domain (SHIV(VpuBC), SHIV(VpuCB)). In culture systems, SHIV(VpuBC) replicated faster than SHIV(VpuCB) while both proteins exhibited similar ability to down-modulate CD4 surface expression. Following inoculation into macaques, SHIV(VpuBC) resulted in rapid CD4(+) T cell loss similar to the parental SHIV(KU-1bMC33), while the rate of CD4(+) T cell loss in those inoculated with SHIV(VpuCB) was intermediate of SHIV(SCVpu) and SHIV(KU-1bMC33). These results emphasize the importance of the Vpu NTD/TMD region in the rate of CD4(+) T cell loss in the pathogenic X4 SHIV/macaque model.

  14. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  15. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein i

  16. 酿酒酵母线粒体蛋白转运系统在立克次体中的同源分析%The homology analysis of Saccharomyces cerevisiae mitochondrial protein import machinery in Rickettsia prowazekii

    Institute of Scientific and Technical Information of China (English)

    刘震; 李校; 赵培虎; 桂俊鸿; 张义正

    2011-01-01

    进化分析表明,与线粒体亲缘关系最近的原核生物是立克次体(Rickettsia prowazekii).搜索酿酒酵母(Saccharom ycescerevisia)线粒体蛋白转运系统的35个亚基在立克次体中的同源蛋白质,仅发现了5个同源蛋白,且它们只属于转运系统在线粒体外膜和基质上的模块,没有发现转运系统在膜间隙和内膜模块的同源蛋白.对大肠杆菌(Escherichia coli K-12 MG1655)的分析也有类似的结果.因此线粒体蛋白转运系统应该不是直接起源于内共生的原核生物.%ConA from 0 μg/mL to 100 μg/mL was added into Tca8113 cells, and MTT, cellular mor phology observation, LDH assay, flow cytometry detection and caspase assay were performed. MTT was applied to detect the inhibitory ratio of Tca8113 cells, and-morphologic changes, LDH, flow cytom etry detection were applied to detect apoptosis. In addition, caspase assy was also applied to observe whether caspase was involved in ConA-induced apoptosis in Tca8113 cells. After 24 h incubation with 25 μg/mL ConA, the inhibitory rate reached nearly 50%. Typical morphologic changes like membrane blebbing, chromatin condensation and nuclei fragmentation were confirmed. LDH assay and flow cytom etry detection showed that with the progressively increasing the concentration of ConA, more and more apoptotic cells appeared. And, caspase assay demonstrated that ConA could induce apoptosis in Tca8113 Phylogenetic analyses identified that Rickettsia prowazekii is the closest relatives of modern mitochondria. The homology analysis results from the 35 subunits of mitochondrial protein import mechanism of Saccharomyces cerevisiae, with thoce of Rickettsia prowazekii, suggest that only 5 sub units of the machinery have orthologous sequences from Rickettsia prowazekii. These 5 subunits are lo cated in the outer membrane and the matrix of mitochondria, while no subunit located in the inner mem brane and intermembrane space. Analysis of the Escherichia coli K

  17. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    Science.gov (United States)

    Hernández-Rodríguez, Carmen Sara; Hernández-Martínez, Patricia; Van Rie, Jeroen; Escriche, Baltasar; Ferré, Juan

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV) from last instar larval midguts were used in competition binding assays with (125)I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  18. A karyopherin alpha2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells.

    Science.gov (United States)

    Cassany, Aurélia; Guillemain, Ghislaine; Klein, Christophe; Dalet, Véronique; Brot-Laroche, Edith; Leturque, Armelle

    2004-01-01

    We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

  19. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda.

    Directory of Open Access Journals (Sweden)

    Carmen Sara Hernández-Rodríguez

    Full Text Available First generation of insect-protected transgenic corn (Bt-corn was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A.105 is a chimeric protein with domains I and II and the C-terminal half of the protein from Cry1Ac, and domain III almost identical to Cry1Fa. The aim of the present study was to determine whether the chimeric Cry1A.105 has shared binding sites either with Cry1A proteins, with Cry1Fa, or with both, in O. nubilalis and in S. frugiperda. Brush-border membrane vesicles (BBMV from last instar larval midguts were used in competition binding assays with (125I-labeled Cry1A.105, Cry1Ab, and Cry1Fa, and unlabeled Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, Cry2Ab and Cry2Ae. The results showed that Cry1A.105, Cry1Ab, Cry1Ac and Cry1Fa competed with high affinity for the same binding sites in both insect species. However, Cry2Ab and Cry2Ae did not compete for the binding sites of Cry1 proteins. Therefore, according to our results, the development of cross-resistance among Cry1Ab/Ac, Cry1A.105, and Cry1Fa proteins is possible in these two insect species if the alteration of shared binding sites occurs. Conversely, cross-resistance between these proteins and Cry2A proteins is very unlikely in such case.

  20. Shared midgut binding sites for Cry1A.105, Cry1Aa, Cry1Ab, Cry1Ac and Cry1Fa proteins from Bacillus thuringiensis in two important corn pests, Ostrinia nubilalis and Spodoptera frugiperda

    OpenAIRE

    Carmen Sara Hernández-Rodríguez; Patricia Hernández-Martínez; Jeroen Van Rie; Baltasar Escriche; Juan Ferré

    2013-01-01

    First generation of insect-protected transgenic corn (Bt-corn) was based on the expression of Cry1Ab or Cry1Fa proteins. Currently, the trend is the combination of two or more genes expressing proteins that bind to different targets. In addition to broadening the spectrum of action, this strategy helps to delay the evolution of resistance in exposed insect populations. One of such examples is the combination of Cry1A.105 with Cry1Fa and Cry2Ab to control O. nubilalis and S. frugiperda. Cry1A....

  1. Use of a Spreadsheet to Calculate the Net Charge of Peptides and Proteins as a Function of pH: An Alternative to Using "Canned" Programs to Estimate the Isoelectric Point of These Important Biomolecules

    Science.gov (United States)

    Sims, Paul A.

    2010-01-01

    An approach is presented that utilizes a spreadsheet to allow students to explore different means of calculating and visualizing how the charge on peptides and proteins varies as a function of pH. In particular, the concept of isoelectric point is developed to allow students to compare the results of their spreadsheet calculations with those of…

  2. Use of a Spreadsheet to Calculate the Net Charge of Peptides and Proteins as a Function of pH: An Alternative to Using "Canned" Programs to Estimate the Isoelectric Point of These Important Biomolecules

    Science.gov (United States)

    Sims, Paul A.

    2010-01-01

    An approach is presented that utilizes a spreadsheet to allow students to explore different means of calculating and visualizing how the charge on peptides and proteins varies as a function of pH. In particular, the concept of isoelectric point is developed to allow students to compare the results of their spreadsheet calculations with those of…

  3. Import of periplasmic bacteriocins targeting the murein.

    Science.gov (United States)

    Braun, Volkmar; Helbig, Stephanie; Patzer, Silke I

    2012-12-01

    Colicins are the only proteins imported by Escherichia coli and thus serve as tools to study the protein import mechanism. Most of the colicins studied degrade DNA, 16S RNA or tRNA in the cytoplasm, or form pores in the cytoplasmic membrane. Two bacteriocins, Cma (colicin M) and Pst (pesticin), affect the murein structure in the periplasm. These two bacteriocins must be imported only across the outer membrane and therefore represent the simplest system for studying protein import. Cma can be reversibly translocated across the outer membrane. Cma and Pst unfold during import. The crystal structure of Pst reveals a phage T4L (T4 lysozyme) fold of the activity domain. Both bacteriocins require energy for import which is translocated from the cytoplasmic membrane into the outer membrane by the Ton system. Cma kills cells only when the periplasmic FkpA PPIase (peptidylprolyl cis-trans isomerase)/chaperone is present.

  4. Activity of Protein Kinase C is Important for 3α,5α-THP’s Actions at Dopamine Type 1-like and/or GABAA receptors in the Ventral Tegmental Area for Lordosis of Rats

    Science.gov (United States)

    Frye, Cheryl A.; Walf, Alicia A.

    2008-01-01

    In the ventral tegmental area, progestogens facilitate sexual receptivity of rodents via actions at dopamine type 1-like and/or γ-aminobutyric type A receptors and activation of downstream signal transduction molecules. In the present study, we investigated whether effects of progesterone’s metabolite, 3α,5α-THP, to enhance lordosis via actions at these receptors in the ventral tegmental area requires phospholipase C-dependent protein kinase C. The objective of this study was to test the hypothesis that: if progestogens’ actions through dopamine type 1-like and/or γ-aminobutyric type A receptors in the ventral tegmental area for lordosis require protein kinase C, then inhibiting protein kinase C in the ventral tegmental area should reduce 3α,5α-THP-facilitated lordosis and its enhancement by dopamine type 1-like or γ-aminobutyric type A receptor agonists. Ovariectomized, E2 (10 μg s.c. at hr 0)-primed rats were tested for their baseline lordosis responses and then received a series of three infusions to the ventral tegmental area: first, bisindolylmaleimide (75 nM/side) or vehicle; second, SKF38393 (100 ng/side), muscimol (100 ng/side), or vehicle; third, 3α,5α-THP (100, 200 ng) or vehicle. Rats were pre-tested for lordosis and motor behavior and then tested for lordosis after each infusion and 10 and 60 mins after the last infusion. Rats were tested for motor behavior following their last lordosis test. As has been previously demonstrated, 3α,5α-THP infusions to the ventral tegmental area increased lordosis and effects were further enhanced by infusions of SKF38393 and muscimol. Infusions of bisindolylmaleimide to the ventral tegmental area attenuated 3α,5α-THP-, SKF38393-, and/or muscimol-facilitated lordosis. Effects on lordosis were not solely due to changes in general motor behavior. Thus, 3α,5α-THP’s actions in the ventral tegmental area through membrane receptors may require activity of protein kinase C. PMID:18675324

  5. Ser⁄ Thr residues at α3⁄β5 loop of Gαs are important in morphine-induced adenylyl cyclase sensitization but not mitogen-activated protein kinase phosphorylation

    Science.gov (United States)

    Seyedabadi, Mohammad; Ostad, Seyed Nasser; Albert, Paul R.; Dehpour, Ahmad R.; Rahimian, Reza; Ghazi-Khansari, Mahmoud; Ghahremani, Mohammad H.

    2015-01-01

    The signaling switch of β2-adrenergic and μ1-opioid receptors from stimulatory G-protein (Gαs) to inhibitory G-protein (Gαi) (and vice versa) influences adenylyl cyclase (AC) and extracellular-regulated kinase (ERK)1 ⁄ 2 activation. Post-translational modifications, including dephosphorylation of Gαs, enhance opioid receptor coupling to Gαs. In the present study, we substituted the Ser ⁄ Thr residues of Gαs at the α3 ⁄ β5 and α4 ⁄ β6 loops aiming to study the role of Gαs lacking Ser ⁄ Thr phosphorylation with respect to AC sensitization and mitogen-activated protein kinase activation. Isoproterenol increased the cAMP concentration (EC50 = 22.8 ± 3.4 μM) in Gαs-transfected S49 cyc– cells but not in nontransfected cells. However, there was no significant difference between the Gαs-wild-type (wt) and mutants. Morphine (10 μM) inhibited AC activity more efficiently in cyc– compared to Gαs-wt introduced cells (P < 0.05); however, we did not find a notable difference between Gαs-wt and mutants. Interestingly, Gαs-wt transfected cells showed more sensitization with respect to AC after chronic morphine compared to nontransfected cells (101 ± 12% versus 34 ± 6%; P < 0.001); μ1-opioid receptor interacted with Gαs, and both co-immunoprecipitated after chronic morphine exposure. Furthermore, mutation of T270A and S272A (P < 0.01), as well as T270A, S272A and S261A (P < 0.05), in α3 ⁄ β5, resulted in a higher level of AC supersensitization. ERK1⁄ 2 phosphorylation was rapidly induced by isoproterenol (by 9.5 ± 2.4-fold) and morphine (22 ± 2.2-fold) in Gαs-transfected cells; mutations of α3 ⁄ β5 and α4 ⁄ β6 did not affect the pattern or extent of mitogen-activated protein kinase activation. The findings of the present study show that Gαs interacts with the μ1-opioid receptor, and the Ser ⁄ Thr mutation to Ala at the α3 ⁄ β5 loop of Gαs enhances morphine-induced AC sensitization. In addition, Gαs was required for

  6. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  7. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  8. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  9. Molecular principles of protein stability and protein-protein interactions

    OpenAIRE

    Lendel, Christofer

    2005-01-01

    Proteins with highly specific binding properties constitute the basis for many important applications in biotechnology and medicine. Immunoglobulins have so far been the obvious choice but recent advances in protein engineering have provided several novel constructs that indeed challenge antibodies. One class of such binding proteins is based on the 58 residues three-helix bundle Z domain from staphylococcal protein A (SPA). These so-called affibodies are selected from libraries containing Z ...

  10. Importance of shearing fat emulsification and protein matrix in meat batter stability%脂肪剪切乳化和蛋白基质对肉糜乳化稳定性的重要作用

    Institute of Scientific and Technical Information of China (English)

    汪张贵; 闫利萍; 彭增起; 周光宏

    2011-01-01

    Finely comminuted meat products were a mixture of proteins,fat particles,water,salt and carbohydrates,and so on.During the process of preparing meat batter,fat was chopped into solid particles and/or liquid fat droplets of different shapes and sizes.Nowadays,the challenge is how to minimize and/or prevent fat and moisture separation from the product.There were two alternative theories for meat batter stabilization:the oil-in-water emulsion theory,which was based on the formation of an interfacial protein film(IPF)around fat globules,and the physical entrapment theory,which centered on the entrapment of the fat within the protein matrix.The exact mechanism by which finely comminuted meat batters stabilized was still uncertain.Arguments presented to support both research thrusts were discussed.Based on this,several pieces of suggestions were also put forward.%完全粉碎肉制品由肌肉蛋白质、脂肪颗粒、水、盐和碳水化合物等物质组成。在肉糜生产过程中,脂肪被剪切成形状和大小不同的固态脂肪颗粒或液态脂肪滴,如何防止脂肪和水分从产品中分离成为挑战。在过去几十年里,涌现出两种学说用于解释肉糊乳化稳定性:水包油型乳化学说和蛋白基质物理镶嵌固定学说,前者强调脂肪球周围形成界面蛋白膜作用,后者强调蛋白基质对脂肪的固定作用。本文讨论了支持这两个研究方向的成果,并在此基础上提出几点建议。

  11. Emergy and Its Importance

    Science.gov (United States)

    Emergy is an important quantity needed for public policy analysis that is based on a complex methodology. The intent of this Environmental Research Brief is to define emergy and its importance in a manner that is accessible to everyone with at least a high school education. Emer...

  12. Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Ahring, Philip K; Olsen, Jeppe A

    2015-01-01

    residues, H142, Q150, and T152, were demonstrated to be involved in the distinct pharmacology of the α4-α4 versus α4-β2 binding sites. To obtain insight into the three-dimensional structure of the α4-α4 binding site, a surrogate protein reproducing α4-α4 binding characteristics was constructed...... structure of the mutated Ls-AChBP with the high-efficacy ligand NS3920, the corresponding histidine forms an intersubunit bridge that reinforces the ligand-mediated interactions between subunits. The structures further reveal that the binding site residues gain different and ligand-dependent interactions...... that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting...

  13. Functional importance of the Ala(116)-Pro(136) region in the calcium-sensing receptor. Constitutive activity and inverse agonism in a family C G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, T A; Burstein, E S

    2000-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein-coupled receptor superfamily. To date 14 activating mutations in CaR showing increased sensitivity to Ca(2+) have been identified in humans with autosomal dominant hypocalcemia. Four of these activating mutations are found...... in the Ala(116)-Pro(136) region of CaR, indicating that this part of the receptor is particularly sensitive to mutation-induced activation. This region was subjected to random saturation mutagenesis, and 219 mutant receptor clones were isolated and screened pharmacologically in a high throughput screening...... when the mutations were transferred to a chimeric receptor Ca/1a consisting of the amino-terminal domain of the CaR and the 7 transmembrane and intracellular domains of the metabotropic glutamate receptor mGluR1a. CPCCOEt, a noncompetitive antagonist acting at the 7 transmembrane domain of mGluR1a...

  14. A Motif in the F Homomorph of Rabbit Haemorrhagic Disease Virus Polymerase Is Important for the Subcellular Localisation of the Protein and Its Ability to Induce Redistribution of Golgi Membranes

    Directory of Open Access Journals (Sweden)

    Nadya Urakova

    2017-08-01

    Full Text Available Rabbit haemorrhagic disease virus (RHDV is a calicivirus that infects and frequently kills rabbits. Previously, we showed that the RHDV RNA-dependent RNA polymerase (RdRp is associated with distinct, but yet uncharacterised subcellular structures and is capable of inducing a redistribution of Golgi membranes. In this study, we identified a partially hidden hydrophobic motif that determines the subcellular localisation of recombinant RHDV RdRp in transfected cells. This novel motif, 189LLWGCDVGVAVCAAAVFHNICY210, is located within the F homomorph, between the conserved F3 and A motifs of the core RdRp domain. Amino acid substitutions that decrease the hydrophobicity of this motif reduced the ability of the protein to accumulate in multiple subcellular foci and to induce a rearrangement of the Golgi network. Furthermore, preliminary molecular dynamics simulations suggest that the RHDV RdRp could align with the negatively charged surfaces of biological membranes and undergo a conformational change involving the F homomorph. These changes would expose the newly identified hydrophobic motif so it could immerse itself into the outer leaflet of intracellular membranes.

  15. New approach for predicting protein-protein interactions

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ Protein-protein interactions (PPIs) are of vital importance for virtually all processes of a living cell. The study of these associations of protein molecules could improve people's understanding of diseases and provide basis for therapeutic approaches.

  16. Importância prognóstica da invasão neural no câncer colorretal: estudo imunoistoquímico com a proteína S-100 Prognostic importance of neural invasion in colorectal cancer: an immunohistochemical study with the S-100 protein

    Directory of Open Access Journals (Sweden)

    José Vinícius Cruz

    2006-09-01

    ções prognósticas adicionais nos doentes classificados nos estádios B, C e C2 das classificações de Dukes e Astler-Coller, respectivamente.Among the anatomopathological variables related to the prognosis for patients with colorectal cancer, neural invasion remains little studied. OBJECTIVE: To evaluate whether neural invasion in patients with colorectal cancer in Dukes stages B and C could be considered to be an independent prognostic factor, by means of univariate and multivariate analysis. METHODS: Ninety-seven patients who underwent operations with curative intent by the same surgical team were followed up for a minimum period of five years and were studied. Patients who received adjuvant treatment were excluded. The surgical specimens were stained with hematoxylin-eosin (HE and immunohistochemical techniques for S-100 protein analysis, with the aim of comparing the two techniques for detecting neural invasion. Accuracy, specificity, sensitivity and positive and negative predictive values were analyzed for HE in relation to S-100 protein. Comparison between the incidence of neural invasion and tumor recurrence was made by using the chi-squared test. Survival and disease-free survival were studied by univariate analysis. A significance level of 5% (p £ 0.05 was established for all the tests adopted. RESULTS: The HE technique presented weak ability to detect neural invasion and was inadequate for this analysis in colorectal cancer patients. The survival and disease-free survival curves for patients with neural invasion, investigated by means of immunostaining for S-100 protein, were significantly worse, thus identifying this histological characteristic as having independent prognostic value (p = 0.0003 and p = 0.0002, respectively. There was significantly more tumor recurrence among patients who presented neural invasion (p = 0.0010. CONCLUSION: The results from the present study allow the conclusion that, among colorectal cancer patients, neural invasion was shown to

  17. American coal imports 2015

    Energy Technology Data Exchange (ETDEWEB)

    Frank Kolojeski [TransGlobal Ventures Corp. (United States)

    2007-09-15

    As 2007 ends, the US coal industry passes two major milestones - the ending of the Synfuel tax break, affecting over 100M st annually, and the imposition of tighter and much more expensive safety measures, particularly in deep mines. Both of these issues, arriving at a time of wretched steam coal price levels, promise to result in a major shake up in the Central Appalachian mining sector. The report utilizes a microeconomic regional approach to determine whether either of these two schools of thought have any validity. Transport, infrastructure, competing fuels and regional issues are examined in detail and this forecasts estimates coal demand and imports on a region by region basis for the years 2010 and 2015. Some of the major highlights of the forecast are: Import growth will be driven by steam coal demand in the eastern and southern US; Transport will continue to be the key driver - we believe that inland rail rates will deter imports from being railed far inland and that the great majority of imports will be delivered directly by vessel, barge or truck to end users; Colombian coal will be the overwhelmingly dominant supply source and possesses a costs structure to enable it to compete with US-produced coal in any market conditions; Most of the growth will come from existing power plants - increasing capacity utilization at existing import facilities and other plants making investments to add imports to the supply portfolio - the growth is not dependent upon a lot of new coal fired capacity being built. Contents of the report are: Key US market dynamics; International supply dynamics; Structure of the US coal import market; and Geographic analysis.

  18. Scaffolds for blocking protein-protein interactions.

    Science.gov (United States)

    Hershberger, Stefan J; Lee, Song-Gil; Chmielewski, Jean

    2007-01-01

    Due to the pivotal roles that protein-protein interactions play in a plethora of biological processes, the design of therapeutic agents targeting these interactions has become an attractive and important area of research. The development of such agents is faced with a variety of challenges. Nevertheless, considerable progress has been made in the design of proteomimetics capable of disrupting protein-protein interactions. Those inhibitors based on molecular scaffold designs hold considerable interest because of the ease of variation in regard to their displayed functionality. In particular, protein surface mimetics, alpha-helical mimetics, beta-sheet/beta-strand mimetics, as well as beta-turn mimetics have successfully modulated protein-protein interactions involved in such diseases as cancer and HIV. In this review, current progress in the development of molecular scaffolds designed for the disruption of protein-protein interactions will be discussed with an emphasis on those active against biological targets.

  19. The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis.

    Science.gov (United States)

    Samejima, Kumiko; Platani, Melpomeni; Wolny, Marcin; Ogawa, Hiromi; Vargiu, Giulia; Knight, Peter J; Peckham, Michelle; Earnshaw, William C

    2015-08-28

    The chromosome passenger complex (CPC) is a master regulator of mitosis. Inner centromere protein (INCENP) acts as a scaffold regulating CPC localization and activity. During early mitosis, the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled coil domain acting as a spacer between the N- and C-terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ∼ 32-nm-long single α-helix (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ∼ 80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible "dog leash," allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the heterochromatin of the inner centromere. Furthermore, by achieving this flexibility via an SAH domain, the CPC avoids a need for dimerization (required for coiled coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation.

  20. Similarity measures for protein ensembles

    DEFF Research Database (Denmark)

    Lindorff-Larsen, Kresten; Ferkinghoff-Borg, Jesper

    2009-01-01

    Analyses of similarities and changes in protein conformation can provide important information regarding protein function and evolution. Many scores, including the commonly used root mean square deviation, have therefore been developed to quantify the similarities of different protein conformatio...

  1. Protein Foods

    Science.gov (United States)

    ... Text Size: A A A Listen En Español Protein Foods Foods high in protein such as fish, ... for the vegetarian proteins, whether they have carbohydrate. Protein Choices Plant-Based Proteins Plant-based protein foods ...

  2. Penicillin Binding Protein 1 Is Important in the Compensatory Response of Staphylococcus aureus to Daptomycin-Induced Membrane Damage and Is a Potential Target for β-Lactam–Daptomycin Synergy

    Science.gov (United States)

    Berti, Andrew D.; Theisen, Erin; Sauer, John-Demian; Nonejuie, Poochit; Olson, Joshua; Pogliano, Joseph; Sakoulas, George; Nizet, Victor; Proctor, Richard A.

    2015-01-01

    The activity of daptomycin (DAP) against methicillin-resistant Staphylococcus aureus (MRSA) is enhanced in the presence of β-lactam antibiotics. This effect is more pronounced with β-lactam antibiotics that exhibit avid binding to penicillin binding protein 1 (PBP1). Here, we present evidence that PBP1 has a significant role in responding to DAP-induced stress on the cell. Expression of the pbpA transcript, encoding PBP1, was specifically induced by DAP exposure whereas expression of pbpB, pbpC, and pbpD, encoding PBP2, PBP3, and PBP4, respectively, remained unchanged. Using a MRSA COL strain with pbpA under an inducible promoter, increased pbpA transcription was accompanied by reduced susceptibility to, and killing by, DAP in vitro. Exposure to β-lactams that preferentially inactivate PBP1 was not associated with increased DAP binding, suggesting that synergy in the setting of anti-PBP1 pharmacotherapy results from increased DAP potency on a per-molecule basis. Combination exposure in an in vitro pharmacokinetic/pharmacodynamic model system with β-lactams that preferentially inactivate PBP1 (DAP-meropenem [MEM] or DAP-imipenem [IPM]) resulted in more-rapid killing than did combination exposure with DAP-nafcillin (NAF) (nonselective), DAP-ceftriaxone (CRO) or DAP-cefotaxime (CTX) (PBP2 selective), DAP-cefaclor (CEC) (PBP3 selective), or DAP-cefoxitin (FOX) (PBP4 selective). Compared to β-lactams with poor PBP1 binding specificity, exposure of S. aureus to DAP plus PBP1-selective β-lactams resulted in an increased frequency of septation and cell wall abnormalities. These data suggest that PBP1 activity may contribute to survival during DAP-induced metabolic stress. Therefore, targeted inactivation of PBP1 may enhance the antimicrobial efficiency of DAP, supporting the use of DAP–β-lactam combination therapy for serious MRSA infections, particularly when the β-lactam undermines the PBP1-mediated compensatory response. PMID:26525797

  3. The Importance of Education.

    Science.gov (United States)

    Echohawk, John

    1994-01-01

    The executive director of the Native American Rights Fund recounts his education experiences in the area of Indian law. Encourages native youth to pursue higher education even under financial pressures, noting the importance of education for native youth and their tribes in this era of Indian self-determination. (HTH)

  4. Minimal Pairs: Minimal Importance?

    Science.gov (United States)

    Brown, Adam

    1995-01-01

    This article argues that minimal pairs do not merit as much attention as they receive in pronunciation instruction. There are other aspects of pronunciation that are of greater importance, and there are other ways of teaching vowel and consonant pronunciation. (13 references) (VWL)

  5. The Importance of Ethos.

    Science.gov (United States)

    Oft-Rose, Nancy

    1989-01-01

    Discusses how studying the 1988 Presidential Debates reveals the importance of ethos in speaking and being elected to office. Asserts that the debates also offer an excellent opportunity to teach propaganda devices or fallacies. Provides two examples from the second Presidential Debate. (MM)

  6. Partner Performs More Important

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Reporter: Would you please offer an introduction to exhibition development of Frankfurt? Michael von Zitzewitz: Mess Frankfurt GmbH has made great achievements in despite of dissatisfying economy in Europe. 364 million Euro of sales volume has made Mess Frankfurt GmbH become the 2nd largest world’s exhibition corporation, overseas sales is an important fortune resource for

  7. Exports to Imports

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    China covers ground as it attempts to balance imports and exports after a trade deficit in the first quarter As China gears up to rebalance its economy,a key factor will be weaning its reliance on exports.The vigorous efforts are paying off as the country reported a trade deficit in the first

  8. Import and nuclear size

    OpenAIRE

    Cohen-Fix, Orna

    2010-01-01

    The size of a cell’s nucleus is usually proportional to the size of the cell itself. How are the two linked? The answer lies, at least in part, in the import of one or more cytoplasmic cargoes into the nucleus.

  9. Import, Offshoring and Wages

    DEFF Research Database (Denmark)

    Fosse, Henrik Barslund; Maitra, Madhura

    exports - as positive exogenous shocks to the incentive to offshore to China. Both skill composition and rent sharing effects are found to be important in explaining the resultant gain in wages. We also show that the firm’s timing in the offshoring process determines the relative importance of a channel...... of skill composition and rent sharing available in typical firm level datasets - like ratio of educated to uneducated workers and sales per employee.......Offshoring firms are found to pay higher average wages than purely domestic firms. We provide a unifying empirical approach by capturing the different channels through which offshoring may explain this wage difference: (i) due to change in the composition of workers (skill composition effect) (ii...

  10. Importance of Mangrove Ecosystem

    OpenAIRE

    Kathiresan, K.

    2012-01-01

    Mangrove forests are extremely important coastal resources, which are vital to our socio-economic development. Much of the ecological service of mangroves lies in protecting the coast from solar UV-B radiation, “green house” effects, and fury of cyclones, floods, sea level rise, wave action and coastal erosion. Mangroves contribute significantly to the global carbon cycle and produce large amounts of litter in the form of falling leaves, branches and other debris. Besides, mangrove habitats c...

  11. Imported Wines Widen Appeal

    Institute of Scientific and Technical Information of China (English)

    WAN LIXIN

    2006-01-01

    @@ "There are many factors contributing to the healthy growth of the wine market in China,"says Don St. Pierre, Jr., managing partner of ASC Fine Wines (Shanghai) Co Ltd, one of China's largest importers and distributors of fine wines. "The reduction in tariffs has helped - it has made wines more affordable. But also driving growth is the rise in Chinese people's incomes and their increasing interest in enjoying what is perceived as a socially and culturally sophisticated lifestyle."

  12. Calcium is important forus.

    Institute of Scientific and Technical Information of China (English)

    高利平

    2005-01-01

    Calcium is important for our health.We must have it in our diet to stay well.A good place to get it is from dairy products like milk, cheese and ice cream.One pound of cheese has fifty times the calcium we should have every day.Other foods have less.For example,a pound of beans also has calcium.But it has only three times the amount we ought to have daily.

  13. GLOBALIZATION AND IMPORT RISKS

    Directory of Open Access Journals (Sweden)

    Popa Ioan

    2014-07-01

    Full Text Available Delocalization of production and diversification of the sources of offer in the global market place the issue of protection of consumer rights in major consumption centres, namely the European Union in a new light. A review of policies for the protection of consumer rights in the EU, USA and China, reveals major differences regarding the protection of consumer rights and the existence of gaps, and in particular the implementation of effective legislation in this regard. As such, the risks associated with imports have become a major concern in the European Union. The consumer has – one can say – a central role in the globalization process, which justifies the measures aimed at its protection. Although worldwide there are major differences in the degree of market regulation in matters of protection of consumer rights, the trend is the continuous adaptation of the offer to the requirements of global demand. However, one can still find significant gaps which translate into risks specific to the consumers in developed countries, namely in the EU. An important issue arises from this radical change of the localization of production centres in relation to the main consumption centres. While in the developed world, consumer rights protection has reached high levels both by creating an appropriate legislative framework and through consumer awareness and activism regarding their rights, in areas where much of the offer comes from the Western market (China, India, etc. modern mentality on the protection of consumer rights is just emerging. A major requirement is therefore the provision of a status of the consumer compatible with the benefits and risks of globalization, a status defined by safety and protection of imports. This paper confirms the thesis that, ultimately, the main factor counteracting the risks in matters of protection of consumer rights is the consumer, its awareness of its rights.

  14. Why is Astronomy Important?

    CERN Document Server

    Rosenberg, Marissa; Bladon, Georgia; Christensen, Lars Lindberg

    2013-01-01

    For a long time astronomers and other scientists believed that the importance of their work was evident to society. But in these difficult days of financial austerity, even the most obvious benefits of science have to undergo careful scrutiny. Eradicating poverty and hunger is a worldwide priority, and activities that do not directly attempt to resolve these issues can be hard to justify and support. However, several studies have told us that investing in science education, research and technology provides a great return (not only economically, but culturally and indirectly for the population in general and has helped countries to face and overcome crises. The scientific and technological development of a country or region is closely linked to its human development index) a statistic that is a measure of life expectancy, education and income.

  15. Most important human aluminoses

    Energy Technology Data Exchange (ETDEWEB)

    Avtsyn, A.P.

    1986-01-01

    Aluminoses are widespread forms of trace element deficiency of various severity. The organism is protected against a possible pathogenic influence of aluminum by a number of physiological mechanisms the most important among which is a regular urine excretion. A complex of pathological symptoms develops if serious homeostasis disturbances take place e. g. when renal deficiency or direct penetration of the trace element occur. The following forms of aluminosis should be distinguished: 1) simple accumulation in central nervous system which occurs in persons over 65 years; 2) aluminum accumulation in Alzheimer disease, in severe form of presenile and senile dementia; 3) dialysis aluminum encephalopathy; 4) non-dialysis infantile encephalopathy; 5) aluminum encephalopathy in total parenteral nutrition; 6) iatrogenic dialysis aluminum osteodystrophy; 7) jatrogenic peritoneal aluminosis; 8) aluminum pneumoconiosis of an occupational origin; 9) bronchospastic syndrome in aluminum smelter workers.

  16. Importance of binaural hearing.

    Science.gov (United States)

    Avan, Paul; Giraudet, Fabrice; Büki, Béla

    2015-01-01

    An essential task for the central auditory pathways is to parse the auditory messages sent by the two cochleae into auditory objects, the segregation and localisation of which constitute an important means of separating target signals from noise and competing sources. When hearing losses are too asymmetric, the patients face a situation in which the monaural exploitation of sound messages significantly lessens their performance compared to what it should be in a binaural situation. Rehabilitation procedures must aim at restoring as many binaural advantages as possible. These advantages encompass binaural redundancy, head shadow effect and binaural release from masking, the principles and requirements of which make up the topic of this short review. Notwithstanding the complete understanding of their neuronal mechanisms, empirical data show that binaural advantages can be restored even in situations in which faultless symmetry is inaccessible. © 2015 S. Karger AG, Basel.

  17. Import vs. Imitation?

    DEFF Research Database (Denmark)

    Kölcze, Zsófia

    2012-01-01

    of our analysis and interpretation more explicit.In archaeology there has been a tradition of distinguishing between original objects and imitations. However, this distinction does not provide us with much information about the social function and symbolic meaning of an object in a cultural context......In recent years archaeological research has developed a radically new theoretical approach to prehistoric material culture. Objects are no longer regarded as simple products of human behavior, but rather as agents interacting with people on multiple levels. As such, artifacts play an active role...... in producing, maintaining and reproducing social identities, communicating new ideas and technological innovations and creating ideologies and cosmologies. Our understanding of material culture has obtained a social dimension, and we as archaeologists have become aware of the importance of making this aspect...

  18. Protein Crystallization

    Science.gov (United States)

    Chernov, Alexander A.

    2005-01-01

    Nucleation, growth and perfection of protein crystals will be overviewed along with crystal mechanical properties. The knowledge is based on experiments using optical and force crystals behave similar to inorganic crystals, though with a difference in orders of magnitude in growing parameters. For example, the low incorporation rate of large biomolecules requires up to 100 times larger supersaturation to grow protein, rather than inorganic crystals. Nucleation is often poorly reproducible, partly because of turbulence accompanying the mixing of precipitant with protein solution. Light scattering reveals fluctuations of molecular cluster size, its growth, surface energies and increased clustering as protein ages. Growth most often occurs layer-by-layer resulting in faceted crystals. New molecular layer on crystal face is terminated by a step where molecular incorporation occurs. Quantitative data on the incorporation rate will be discussed. Rounded crystals with molecularly disordered interfaces will be explained. Defects in crystals compromise the x-ray diffraction resolution crucially needed to find the 3D atomic structure of biomolecules. The defects are immobile so that birth defects stay forever. All lattice defects known for inorganics are revealed in protein crystals. Contribution of molecular conformations to lattice disorder is important, but not studied. This contribution may be enhanced by stress field from other defects. Homologous impurities (e.g., dimers, acetylated molecules) are trapped more willingly by a growing crystal than foreign protein impurities. The trapped impurities induce internal stress eliminated in crystals exceeding a critical size (part of mni for ferritin, lysozyme). Lesser impurities are trapped from stagnant, as compared to the flowing, solution. Freezing may induce much more defects unless quickly amorphysizing intracrystalline water.

  19. 27 CFR 478.112 - Importation by a licensed importer.

    Science.gov (United States)

    2010-04-01

    ... importer. 478.112 Section 478.112 Alcohol, Tobacco Products, and Firearms BUREAU OF ALCOHOL, TOBACCO... Importation § 478.112 Importation by a licensed importer. (a) No firearm, firearm barrel, or ammunition shall be imported or brought into the United States by a licensed importer (as defined in § 478.11)...

  20. Do proteomics analyses provide insights into reduced oxidative stress in the brain of an Alzheimer disease transgenic mouse model with an M631L amyloid precursor protein substitution and thereby the importance of amyloid-beta-resident methionine 35 in Alzheimer disease pathogenesis?

    Science.gov (United States)

    Sultana, Rukhsana; Robinson, Renã A S; Lange, Miranda Bader; Fiorini, Ada; Galvan, Veronica; Fombonne, Joanna; Baker, Austin; Gorostiza, Olivia; Zhang, Junli; Cai, Jian; Pierce, William M; Bredesen, Dale E; Butterfield, D Allan

    2012-12-01

    The single methionine (Met/M) residue of amyloid-beta (Aβ) peptide, at position 35 of the 42-mer, has important relevance for Aβ-induced oxidative stress and neurotoxicity. Recent in vivo brain studies in a transgenic (Tg) Alzheimer disease (AD) mouse model with Swedish and Indiana familial AD mutations in human amyloid precursor protein (APP) (referred to as the J20 Tg mouse) demonstrated increased levels of oxidative stress. However, the substitution of the Met631 residue of APP to leucine (Leu/L) (M631L in human APP numbering, referred to as M631L Tg and corresponding to residue 35 of Aβ1-42) resulted in no significant in vivo oxidative stress levels, thereby supporting the hypothesis that Met-35 of Aβ contributes to oxidative insult in the AD brain. It is conceivable that oxidative stress mediated by Met-35 of Aβ is important in regulating numerous downstream effects, leading to differential levels of relevant biochemical pathways in AD. Therefore, in the current study using proteomics, we tested the hypothesis that several brain proteins involved in pathways such as energy and metabolism, antioxidant activity, proteasome degradation, and pH regulation are altered in J20Tg versus M631L Tg AD mice.

  1. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  2. Protein-protein interactions

    DEFF Research Database (Denmark)

    Byron, Olwyn; Vestergaard, Bente

    2015-01-01

    Responsive formation of protein:protein interaction (PPI) upon diverse stimuli is a fundament of cellular function. As a consequence, PPIs are complex, adaptive entities, and exist in structurally heterogeneous interplays defined by the energetic states of the free and complexed protomers....... The biophysical and structural investigations of PPIs consequently demand hybrid approaches, implementing orthogonal methods and strategies for global data analysis. Currently, impressive developments in hardware and software within several methodologies define a new era for the biostructural community. Data can...

  3. Challenges of protein extraction from recalcitrant plant tissues for proteomics

    Science.gov (United States)

    Proteins play an important role in several biological processes. Proteomics encompasses basically four principal applications, namely protein mining, protein expression profiling, protein-network mapping and mapping of protein modifications. The results in these applications depend mostly on the c...

  4. 两种麦类土传花叶病毒外壳蛋白叶绿体离体跨膜运输%Analysis on the Import of Coat Proteins of Chinese Wheat Mosaic Virus and Barley Yellow Mosaic Virus into Intact Chloroplasts in Vitro

    Institute of Scientific and Technical Information of China (English)

    任春梅; 程兆榜; 魏利辉; 范永坚; 周益军

    2012-01-01

    根据Banerjees等(1992)的方法,建立了中国小麦花叶病毒(CWMV)和大麦黄花叶病毒(BaYMV)外壳蛋白(CP)叶绿体离体跨膜运输体系,研究了孵育时间、CP浓度对跨膜运输效率的影响。结果表明,CWMV-CP和BaYMV-CP可分别快速进入离体的小麦与大麦叶绿体中,其跨膜运输所需的孵育时间均最低为5min,孵育时间超过15min后进入叶绿体中CP的量不受影响;加入跨膜体系中CP的浓度与跨膜后进入叶绿体的CP浓度呈正相关,能够实现跨膜的最低CWMV-CP和BaYMV-CP浓度分别为4.2和37.8μg.mL^-1。%Referred to Banerjee's method, the systems of importing the coat proteins of Chinese wheat mosaic virus and Barley yellow mosaic virus (CWMV CP and BaYMV-CP) into intact chloroplasts in vitro were established, the effects of incubation duration, the concentration of CWMV-CP and BaYMV-CP on import efficiency were studied. The results showed that both CWMV-CP and BaYMV- CP was able to rapidly import into isolated chloroplasts of wheat and barley. In the test, the lowest incubation duration of CWMV-CP and BaYMV-CP importing into the chloroplasts were both 5 min, the amounts of CWMV-CP and BaYMV-CP imported into the chloroplasts were not increased with the extension of incubation time after 15 min. Besides, the relation between the amounts of CWMV-CP and BaYMV-CP imported into the chloroplasts and the amounts of CWMV-CP and BaYMV-CP added in the import system was significantly positive, the lowest concentrations of CWMV CP and BaYMV- CP in the import system was 4.2 and 37.8 μg. mL^-1 , respectively. The establishment of CWMV-CP and BaYMV-CP importing into intact chloroplasts in vitro could supply foundation for studying the pathogenesis of CWMV and BaYMV.

  5. Cold gelation of globular proteins

    NARCIS (Netherlands)

    Alting, A.C.

    2003-01-01

    Keywords : globular proteins, whey protein, ovalbumin, cold gelation, disulfide bonds, texture, gel hardnessProtein gelation in food products is important to obtain desirable sensory and textural properties. Cold gelation is a novel method to produce protein-based gels. It is a two step process in w

  6. Cold gelation of globular proteins

    NARCIS (Netherlands)

    Alting, A.C.

    2003-01-01

    Keywords : globular proteins, whey protein, ovalbumin, cold gelation, disulfide bonds, texture, gel hardnessProtein gelation in food products is important to obtain desirable sensory and textural properties. Cold gelation is a novel method to produce protein-based gels. It is a two step process in w

  7. Automatic Extraction of Protein Interaction in Literature

    OpenAIRE

    Liu, Peilei; Wang, Ting

    2014-01-01

    Protein-protein interaction extraction is the key precondition of the construction of protein knowledge network, and it is very important for the research in the biomedicine. This paper extracted directional protein-protein interaction from the biological text, using the SVM-based method. Experiments were evaluated on the LLL05 corpus with good results. The results show that dependency features are import for the protein-protein interaction extraction and features related to the interaction w...

  8. AND/OR Importance Sampling

    OpenAIRE

    Gogate, Vibhav; Dechter, Rina

    2012-01-01

    The paper introduces AND/OR importance sampling for probabilistic graphical models. In contrast to importance sampling, AND/OR importance sampling caches samples in the AND/OR space and then extracts a new sample mean from the stored samples. We prove that AND/OR importance sampling may have lower variance than importance sampling; thereby providing a theoretical justification for preferring it over importance sampling. Our empirical evaluation demonstrates that AND/OR importance sampling is ...

  9. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  10. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  11. On a matter of seminal importance

    Science.gov (United States)

    McGraw, Lisa A.; Suarez, Susan S.; Wolfner, Mariana F.

    2015-01-01

    Egg and sperm have, understandably, been the “stars” of mammalian fertilization biology, particularly because artificial reproductive technologies allow for fertilization to occur outside of the female reproductive tract without other apparent contributions from either sex. Yet, recent research, including an exciting new paper, reveals unexpected and important contributions of seminal plasma to fertility. For example, seminal plasma proteins play critical roles in modulating female reproductive physiology, and a new study in mice demonstrates that effects of some of these proteins on the female can even affect the health of her progeny. Furthermore, although several actions of seminal plasma have been conserved across taxa, male accessory glands and their products are diverse — even among mammals. Taken together, these studies suggest that the actions of seminal plasma components are important to understand, and also to consider in future development of assisted reproductive technologies for humans, farm species, and endangered species of mammals. PMID:25379987

  12. Current importance of oleochemistry in the indusindustrial

    OpenAIRE

    Gómez Herrera, Carlos

    2009-01-01

    Oleochemistry is an industrial sector with various applications and important developments in the field of surfactans. In this article different parts are dedicated to: A) Sources of oleochemical raw materials; B) Valorization of the oleochemistry in the field of surfactants; C) Hydrophilic heads based on carbohydrates or proteins, D) Examples of surfactants currently prepared at industrial scale, such as esters of polyols, alkylpolyglucosides, glucamides and lipoaminoacids. Collaboration bet...

  13. Controllability in protein interaction networks.

    Science.gov (United States)

    Wuchty, Stefan

    2014-05-13

    Recently, the focus of network research shifted to network controllability, prompting us to determine proteins that are important for the control of the underlying interaction webs. In particular, we determined minimum dominating sets of proteins (MDSets) in human and yeast protein interaction networks. Such groups of proteins were defined as optimized subsets where each non-MDSet protein can be reached by an interaction from an MDSet protein. Notably, we found that MDSet proteins were enriched with essential, cancer-related, and virus-targeted genes. Their central position allowed MDSet proteins to connect protein complexes and to have a higher impact on network resilience than hub proteins. As for their involvement in regulatory functions, MDSet proteins were enriched with transcription factors and protein kinases and were significantly involved in bottleneck interactions, regulatory links, phosphorylation events, and genetic interactions.

  14. Evolution studied through protein structural domains

    OpenAIRE

    YANG, SONG

    2007-01-01

    A protein structural domain is defined as a compact, spatially distinct part of a protein that can fold independently of neighboring sequences. Since the number of protein domains is limited, and protein domains are evolutionarily more conserved than protein sequences, protein domains play an important role in our understanding of the structure, function and evolution of proteins. As fundamental evolutionary units, protein domains are associated with a variety of evolutionary processes such a...

  15. Role of Dietary Soy Protein in Obesity

    OpenAIRE

    Manuel T. Velasquez, Sam J. Bhathena

    2007-01-01

    Soy protein is an important component of soybeans and provides an abundant source of dietary protein. Among the dietary proteins, soy protein is considered a complete protein in that it contains ample amounts of all the essential amino acids plus several other macronutrients with a nutritional value roughly equivalent to that of animal protein of high biological value. Soy protein is unique among the plant-based proteins because it is associated with isoflavones, a group of compounds with a v...

  16. Acanthamoeba castellanii STAT Protein

    OpenAIRE

    Anna Kicinska; Jacek Leluk; Wieslawa Jarmuszkiewicz

    2014-01-01

    STAT (signal transducers and activators of transcription) proteins are one of the important mediators of phosphotyrosine-regulated signaling in metazoan cells. We described the presence of STAT protein in a unicellular, free-living amoebae with a simple life cycle, Acanthamoeba castellanii. A. castellanii is the only, studied to date, Amoebozoan that does not belong to Mycetozoa but possesses STATs. A sequence of the A. castellanii STAT protein includes domains similar to those of the Dictyos...

  17. Protein Condensation

    Science.gov (United States)

    Gunton, James D.; Shiryayev, Andrey; Pagan, Daniel L.

    2014-07-01

    Preface; 1. Introduction; 2. Globular protein structure; 3. Experimental methods; 4. Thermodynamics and statistical mechanics; 5. Protein-protein interactions; 6. Theoretical studies of equilibrium; 7. Nucleation theory; 8. Experimental studies of nucleation; 9. Lysozyme; 10. Some other globular proteins; 11. Membrane proteins; 12. Crystallins and cataracts; 13. Sickle hemoglobin and sickle cell anemia; 14, Alzheimer's disease; Index.

  18. Prediction of Protein-Protein Interactions Using Protein Signature Profiling

    Institute of Scientific and Technical Information of China (English)

    Mahmood A. Mahdavi; Yen-Han Lin

    2007-01-01

    Protein domains are conserved and functionally independent structures that play an important role in interactions among related proteins. Domain-domain inter- actions have been recently used to predict protein-protein interactions (PPI). In general, the interaction probability of a pair of domains is scored using a trained scoring function. Satisfying a threshold, the protein pairs carrying those domains are regarded as "interacting". In this study, the signature contents of proteins were utilized to predict PPI pairs in Saccharomyces cerevisiae, Caenorhabditis ele- gans, and Homo sapiens. Similarity between protein signature patterns was scored and PPI predictions were drawn based on the binary similarity scoring function. Results show that the true positive rate of prediction by the proposed approach is approximately 32% higher than that using the maximum likelihood estimation method when compared with a test set, resulting in 22% increase in the area un- der the receiver operating characteristic (ROC) curve. When proteins containing one or two signatures were removed, the sensitivity of the predicted PPI pairs in- creased significantly. The predicted PPI pairs are on average 11 times more likely to interact than the random selection at a confidence level of 0.95, and on aver- age 4 times better than those predicted by either phylogenetic profiling or gene expression profiling.

  19. Pengaruh Self-Acceptance Importance, Affiliation Importance, dan Community Feeling Importance terhadap Compulsive Buying

    Directory of Open Access Journals (Sweden)

    Euis Soliha

    2011-03-01

    Full Text Available This study focused on phenomenon behavior of compulsive buying. The study examined how Self-Acceptance Importance, Affiliation Importance, and Community Feeling Importance influenced on Compulsive Buying. Population in this research was students in Kota Semarang, and 104 students become samples. To answer problem that is accurate, researcher applies econometrics Logit model. Result of research indicates that there were negativity influence Self-Acceptance Importance, Affiliation Importance and Community Feeling to Compulsive Buying. Result of this supports all hypothesis and consistent with theory.Keywords:    compulsive buying, self-acceptance Importance, affiliation Importance, community feeling Importance, Logit Model

  20. Protein C

    Science.gov (United States)

    ... have an unexplained blood clot, or a family history of blood clots. Protein C helps control blood clotting. A lack of this protein or problem with the function of this protein may cause blood clots to ...

  1. Protein S

    Science.gov (United States)

    ... have an unexplained blood clot, or a family history of blood clots. Protein S helps control blood clotting. A lack of this protein or problem with the function of this protein may cause blood clots to ...

  2. How important is importance for prospective memory? A review

    Directory of Open Access Journals (Sweden)

    Stefan eWalter

    2014-06-01

    Full Text Available Forgetting to carry out an intention as planned can have serious consequences in everyday life. People sometimes even forget intentions that they consider as very important. Here, we review the literature on the impact of importance on prospective memory performance. We highlight different methods used to manipulate the importance of a prospective memory task such as providing rewards, importance relative to other ongoing activities, absolute importance, and providing social motives. Moreover, we address the relationship between importance and other factors known to affect prospective memory and ongoing task performance such as type of prospective memory task (time-, event- or activity-based, cognitive loads, and cue focality. Finally, we provide a connection to motivation, we summarize the effects of task importance and we identify important venues for future research.

  3. Proteins Are the Body's Worker Molecules

    Science.gov (United States)

    ... PDF Chapter 1: Proteins are the Body's Worker Molecules You've probably heard that proteins are important ... are much more than that. Proteins are worker molecules that are necessary for virtually every activity in ...

  4. Import Security: Assessing the Risks of Imported Food.

    Science.gov (United States)

    Welburn, Jonathan; Bier, Vicki; Hoerning, Steven

    2016-11-01

    We use data on food import violations from the FDA Operational and Administrative System for Import Support (OASIS) to address rising concerns associated with imported food, quantify import risks by product and by country of origin, and explore the usefulness of OASIS data for risk assessment. In particular, we assess whether there are significant trends in violations, whether import violations can be used to quantify risks by country and by product, and how import risks depend on economic factors of the country of origin. The results show that normalizing import violations by volume of imports provides a meaningful indicator of risk. We then use regression analysis to characterize import risks.  Using this model, we analyze import risks by product type, violation type, and economic factors of the country of origin.  We find that OASIS data are useful in quantifying food import risks, and that the rate of refusals provides a useful decision tool for risk management.  Furthermore, we find that some economic factors are significant indicators of food import risk by country. © 2016 Society for Risk Analysis.

  5. An evaluation of in vitro protein-protein interaction techniques: assessing contaminating background proteins.

    Science.gov (United States)

    Howell, Jenika M; Winstone, Tara L; Coorssen, Jens R; Turner, Raymond J

    2006-04-01

    Determination of protein-protein interactions is an important component in assigning function and discerning the biological relevance of proteins within a broader cellular context. In vitro protein-protein interaction methodologies, including affinity chromatography, coimmunoprecipitation, and newer approaches such as protein chip arrays, hold much promise in the detection of protein interactions, particularly in well-characterized organisms with sequenced genomes. However, each of these approaches attracts certain background proteins that can thwart detection and identification of true interactors. In addition, recombinant proteins expressed in Escherichia coli are also extensively used to assess protein-protein interactions, and background proteins in these isolates can thus contaminate interaction studies. Rigorous validation of a true interaction thus requires not only that an interaction be found by alternate techniques, but more importantly that researchers be aware of and control for matrix/support dependence. Here, we evaluate these methods for proteins interacting with DmsD (an E. coli redox enzyme maturation protein chaperone), in vitro, using E. coli subcellular fractions as prey sources. We compare and contrast the various in vitro interaction methods to identify some of the background proteins and protein profiles that are inherent to each of the methods in an E. coli system.

  6. High throughput recombinant protein production of fungal secreted proteins

    DEFF Research Database (Denmark)

    Vala, Andrea Lages Lino; Roth, Doris; Grell, Morten Nedergaard

    2011-01-01

    Secreted proteins are important for both symbiotic and pathogenic interactions between fungi and their hosts. Our research group uses screens and genomic mining to discover novel proteins involved in these processes. To efficiently study the large number of candidate proteins, we are establishing...

  7. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Amarnath Chtterjee; Ashutosh Kumar; Jeetender Chugh; Sudha Srivastava; Neel S Bhavesh; Ramakrishna V Hosur

    2005-01-01

    In the post-genomic era, as more and more genome sequences are becoming known and hectic efforts are underway to decode the information content in them, it is becoming increasingly evident that flexibility in proteins plays a crucial role in many of the biological functions. Many proteins have intrinsic disorder either wholly or in specific regions. It appears that this disorder may be important for regulatory functions of the proteins, on the one hand, and may help in directing the folding process to reach the compact native state, on the other. Nuclear magnetic resonance (NMR) has over the last two decades emerged as the sole, most powerful technique to help characterize these disordered protein systems. In this review, we first discuss the significance of disorder in proteins and then describe the recent developments in NMR methods for their characterization. A brief description of the results obtained on several disordered proteins is presented at the end.

  8. Protein Models Comparator

    CERN Document Server

    Widera, Paweł

    2011-01-01

    The process of comparison of computer generated protein structural models is an important element of protein structure prediction. It has many uses including model quality evaluation, selection of the final models from a large set of candidates or optimisation of parameters of energy functions used in template free modelling and refinement. Although many protein comparison methods are available online on numerous web servers, their ability to handle a large scale model comparison is often very limited. Most of the servers offer only a single pairwise structural comparison, and they usually do not provide a model-specific comparison with a fixed alignment between the models. To bridge the gap between the protein and model structure comparison we have developed the Protein Models Comparator (pm-cmp). To be able to deliver the scalability on demand and handle large comparison experiments the pm-cmp was implemented "in the cloud". Protein Models Comparator is a scalable web application for a fast distributed comp...

  9. Packing in protein cores

    Science.gov (United States)

    Gaines, J. C.; Clark, A. H.; Regan, L.; O'Hern, C. S.

    2017-07-01

    Proteins are biological polymers that underlie all cellular functions. The first high-resolution protein structures were determined by x-ray crystallography in the 1960s. Since then, there has been continued interest in understanding and predicting protein structure and stability. It is well-established that a large contribution to protein stability originates from the sequestration from solvent of hydrophobic residues in the protein core. How are such hydrophobic residues arranged in the core; how can one best model the packing of these residues, and are residues loosely packed with multiple allowed side chain conformations or densely packed with a single allowed side chain conformation? Here we show that to properly model the packing of residues in protein cores it is essential that amino acids are represented by appropriately calibrated atom sizes, and that hydrogen atoms are explicitly included. We show that protein cores possess a packing fraction of φ ≈ 0.56 , which is significantly less than the typically quoted value of 0.74 obtained using the extended atom representation. We also compare the results for the packing of amino acids in protein cores to results obtained for jammed packings from discrete element simulations of spheres, elongated particles, and composite particles with bumpy surfaces. We show that amino acids in protein cores pack as densely as disordered jammed packings of particles with similar values for the aspect ratio and bumpiness as found for amino acids. Knowing the structural properties of protein cores is of both fundamental and practical importance. Practically, it enables the assessment of changes in the structure and stability of proteins arising from amino acid mutations (such as those identified as a result of the massive human genome sequencing efforts) and the design of new folded, stable proteins and protein-protein interactions with tunable specificity and affinity.

  10. Reconstitution of nuclear import in permeabilized cells.

    Science.gov (United States)

    Cassany, Aurelia; Gerace, Larry

    2009-01-01

    The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin-cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin alpha, importin beta, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.

  11. Protein and protein hydrolysates in sports nutrition.

    Science.gov (United States)

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  12. A simple dependence between protein evolution rate and the number of protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Hirsh Aaron E

    2003-05-01

    Full Text Available Abstract Background It has been shown for an evolutionarily distant genomic comparison that the number of protein-protein interactions a protein has correlates negatively with their rates of evolution. However, the generality of this observation has recently been challenged. Here we examine the problem using protein-protein interaction data from the yeast Saccharomyces cerevisiae and genome sequences from two other yeast species. Results In contrast to a previous study that used an incomplete set of protein-protein interactions, we observed a highly significant correlation between number of interactions and evolutionary distance to either Candida albicans or Schizosaccharomyces pombe. This study differs from the previous one in that it includes all known protein interactions from S. cerevisiae, and a larger set of protein evolutionary rates. In both evolutionary comparisons, a simple monotonic relationship was found across the entire range of the number of protein-protein interactions. In agreement with our earlier findings, this relationship cannot be explained by the fact that proteins with many interactions tend to be important to yeast. The generality of these correlations in other kingdoms of life unfortunately cannot be addressed at this time, due to the incompleteness of protein-protein interaction data from organisms other than S. cerevisiae. Conclusions Protein-protein interactions tend to slow the rate at which proteins evolve. This may be due to structural constraints that must be met to maintain interactions, but more work is needed to definitively establish the mechanism(s behind the correlations we have observed.

  13. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  14. PARALLEL IMPORT: REALITY FOR RUSSIA

    Directory of Open Access Journals (Sweden)

    Т. А. Сухопарова

    2014-01-01

    Full Text Available Problem of parallel import is urgent question at now. Parallel import legalization in Russia is expedient. Such statement based on opposite experts opinion analysis. At the same time it’s necessary to negative consequences consider of this decision and to apply remedies to its minimization.Purchase on Elibrary.ru > Buy now

  15. Textile Machinery: Imports Rebound Again

    Institute of Scientific and Technical Information of China (English)

    Zheng Yan

    2007-01-01

    @@ In the year of 2006, the general situation of China's textile machinery equipment imports had shown a clear sign of revival from the downward trend of two years ago, with a total annual import of 4.1 billion USD, an increase of 19.05% against the same period of 2005.