WorldWideScience

Sample records for regulatory protein complex

  1. Pleiotropy constrains the evolution of protein but not regulatory sequences in a transcription regulatory network influencing complex social behaviours

    Directory of Open Access Journals (Sweden)

    Daria eMolodtsova

    2014-12-01

    Full Text Available It is increasingly apparent that genes and networks that influence complex behaviour are evolutionary conserved, which is paradoxical considering that behaviour is labile over evolutionary timescales. How does adaptive change in behaviour arise if behaviour is controlled by conserved, pleiotropic, and likely evolutionary constrained genes? Pleiotropy and connectedness are known to constrain the general rate of protein evolution, prompting some to suggest that the evolution of complex traits, including behaviour, is fuelled by regulatory sequence evolution. However, we seldom have data on the strength of selection on mutations in coding and regulatory sequences, and this hinders our ability to study how pleiotropy influences coding and regulatory sequence evolution. Here we use population genomics to estimate the strength of selection on coding and regulatory mutations for a transcriptional regulatory network that influences complex behaviour of honey bees. We found that replacement mutations in highly connected transcription factors and target genes experience significantly stronger negative selection relative to weakly connected transcription factors and targets. Adaptively evolving proteins were significantly more likely to reside at the periphery of the regulatory network, while proteins with signs of negative selection were near the core of the network. Interestingly, connectedness and network structure had minimal influence on the strength of selection on putative regulatory sequences for both transcription factors and their targets. Our study indicates that adaptive evolution of complex behaviour can arise because of positive selection on protein-coding mutations in peripheral genes, and on regulatory sequence mutations in both transcription factors and their targets throughout the network.

  2. Architecture of the 99 bp DNA-six-protein regulatory complex of the lambda att site.

    Science.gov (United States)

    Sun, Xingmin; Mierke, Dale F; Biswas, Tapan; Lee, Sang Yeol; Landy, Arthur; Radman-Livaja, Marta

    2006-11-17

    The highly directional and tightly regulated recombination reaction used to site-specifically excise the bacteriophage lambda chromosome out of its E. coli host chromosome requires the binding of six sequence-specific proteins to a 99 bp segment of the phage att site. To gain structural insights into this recombination pathway, we measured 27 FRET distances between eight points on the 99 bp regulatory DNA bound with all six proteins. Triangulation of these distances using a metric matrix distance-geometry algorithm provided coordinates for these eight points. The resulting path for the protein-bound regulatory DNA, which fits well with the genetics, biochemistry, and X-ray crystal structures describing the individual proteins and their interactions with DNA, provides a new structural perspective into the molecular mechanism and regulation of the recombination reaction and illustrates a design by which different families of higher-order complexes can be assembled from different numbers and combinations of the same few proteins.

  3. Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

    Science.gov (United States)

    Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

    2014-09-11

    The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with

  4. Drosophila Protein Kinase CK2: Genetics, Regulatory Complexity and Emerging Roles during Development

    Directory of Open Access Journals (Sweden)

    Mohna Bandyopadhyay

    2016-12-01

    Full Text Available CK2 is a Ser/Thr protein kinase that is highly conserved amongst all eukaryotes. It is a well-known oncogenic kinase that regulates vital cell autonomous functions and animal development. Genetic studies in the fruit fly Drosophila are providing unique insights into the roles of CK2 in cell signaling, embryogenesis, organogenesis, neurogenesis, and the circadian clock, and are revealing hitherto unknown complexities in CK2 functions and regulation. Here, we review Drosophila CK2 with respect to its structure, subunit diversity, potential mechanisms of regulation, developmental abnormalities linked to mutations in the gene encoding CK2 subunits, and emerging roles in multiple aspects of eye development. We examine the Drosophila CK2 “interaction map” and the eye-specific “transcriptome” databases, which raise the prospect that this protein kinase has many additional targets in the developing eye. We discuss the possibility that CK2 functions during early retinal neurogenesis in Drosophila and mammals bear greater similarity than has been recognized, and that this conservation may extend to other developmental programs. Together, these studies underscore the immense power of the Drosophila model organism to provide new insights and avenues to further investigate developmentally relevant targets of this protein kinase.

  5. Crystallization and preliminary X-ray diffraction analysis of iron regulatory protein 1 in complex with ferritin IRE RNA

    International Nuclear Information System (INIS)

    Selezneva, Anna I.; Cavigiolio, Giorgio; Theil, Elizabeth C.; Walden, William E.; Volz, Karl

    2006-01-01

    The iron regulatory protein IRP1 has been crystallized in a complex with ferritin IRE RNA and a complete data set has been collected to 2.8 Å resolution. Iron regulatory protein 1 (IRP1) is a bifunctional protein with activity as an RNA-binding protein or as a cytoplasmic aconitase. Interconversion of IRP1 between these mutually exclusive states is central to cellular iron regulation and is accomplished through iron-responsive assembly and disassembly of a [4Fe–4S] cluster. When in its apo form, IRP1 binds to iron responsive elements (IREs) found in mRNAs encoding proteins of iron storage and transport and either prevents translation or degradation of the bound mRNA. Excess cellular iron stimulates the assembly of a [4Fe–4S] cluster in IRP1, inhibiting its IRE-binding ability and converting it to an aconitase. The three-dimensional structure of IRP1 in its different active forms will provide details of the interconversion process and clarify the selective recognition of mRNA, Fe–S sites and catalytic activity. To this end, the apo form of IRP1 bound to a ferritin IRE was crystallized. Crystals belong to the monoclinic space group P2 1 , with unit-cell parameters a = 109.6, b = 80.9, c = 142.9 Å, β = 92.0°. Native data sets have been collected from several crystals with resolution extending to 2.8 Å and the structure has been solved by molecular replacement

  6. Bovine proteins containing poly-glutamine repeats are often polymorphic and enriched for components of transcriptional regulatory complexes

    LENUS (Irish Health Repository)

    Whan, Vicki

    2010-11-23

    Abstract Background About forty human diseases are caused by repeat instability mutations. A distinct subset of these diseases is the result of extreme expansions of polymorphic trinucleotide repeats; typically CAG repeats encoding poly-glutamine (poly-Q) tracts in proteins. Polymorphic repeat length variation is also apparent in human poly-Q encoding genes from normal individuals. As these coding sequence repeats are subject to selection in mammals, it has been suggested that normal variations in some of these typically highly conserved genes are implicated in morphological differences between species and phenotypic variations within species. At present, poly-Q encoding genes in non-human mammalian species are poorly documented, as are their functions and propensities for polymorphic variation. Results The current investigation identified 178 bovine poly-Q encoding genes (Q ≥ 5) and within this group, 26 genes with orthologs in both human and mouse that did not contain poly-Q repeats. The bovine poly-Q encoding genes typically had ubiquitous expression patterns although there was bias towards expression in epithelia, brain and testes. They were also characterised by unusually large sizes. Analysis of gene ontology terms revealed that the encoded proteins were strongly enriched for functions associated with transcriptional regulation and many contributed to physical interaction networks in the nucleus where they presumably act cooperatively in transcriptional regulatory complexes. In addition, the coding sequence CAG repeats in some bovine genes impacted mRNA splicing thereby generating unusual transcriptional diversity, which in at least one instance was tissue-specific. The poly-Q encoding genes were prioritised using multiple criteria for their likelihood of being polymorphic and then the highest ranking group was experimentally tested for polymorphic variation within a cattle diversity panel. Extensive and meiotically stable variation was identified

  7. The E1A proteins of all six human adenovirus subgroups target the p300/CBP acetyltransferases and the SAGA transcriptional regulatory complex

    International Nuclear Information System (INIS)

    Shuen, Michael; Avvakumov, Nikita; Torchia, Joe; Mymryk, Joe S.

    2003-01-01

    The N-terminal/conserved region 1 (CR1) portion of the human adenovirus (Ad) 5 E1A protein was previously shown to inhibit growth in the simple eukaryote Saccharomyces cerevisiae. We now demonstrate that the corresponding regions of the E1A proteins of Ad3,-4,-9,-12, and -40, which represent the remaining five Ad subgroups, also inhibit yeast growth. These results suggest that the E1A proteins of all six human Ad subgroups share a common cellular target(s) conserved in yeast. Growth inhibition induced by either full-length or the N-terminal/CR1 portion of Ad5 E1A was relieved by coexpression of the E1A binding portions of the mammalian p300, CBP, and pCAF acetyltransferases. Similarly, growth inhibition by the N-terminal/CR1 portions of the other Ad E1A proteins was suppressed by expression of the same regions of CBP or pCAF known to bind Ad5 E1A. The physical interaction of each of the different Ad E1A proteins with CBP, p300, and pCAF was confirmed in vitro. Furthermore, deletion of the gene encoding yGcn5, the yeast homolog of pCAF and a subunit of the SAGA transcriptional regulatory complex, restored growth in yeast expressing each of the different Ad E1A proteins. This indicates that the SAGA complex is a conserved target of all Ad E1A proteins. Our results demonstrate for the first time that the p300, CBP, and pCAF acetyltransferases are common targets for the E1A proteins of all six human Ad subgroups, highlighting the importance of these interactions for E1A function

  8. The adeno-associated virus major regulatory protein Rep78-c-Jun-DNA motif complex modulates AP-1 activity

    International Nuclear Information System (INIS)

    Prasad, C. Krishna; Meyers, Craig; Zhan Dejin; You Hong; Chiriva-Internati, Maurizio; Mehta, Jawahar L.; Liu Yong; Hermonat, Paul L.

    2003-01-01

    Multiple epidemiologic studies show that adeno-associated virus (AAV) is negatively associated with cervical cancer (CX CA), a cancer which is positively associated with human papillomavirus (HPV) infection. Mechanisms for this correlation may be by Rep78's (AAV's major regulatory protein) ability to bind the HPV-16 p97 promoter DNA and inhibit transcription, to bind and interfere with the functions of the E7 oncoprotein of HPV-16, and to bind a variety of HPV-important cellular transcription factors such as Sp1 and TBP. c-Jun is another important cellular factor intimately linked to the HPV life cycle, as well as keratinocyte differentiation and skin development. Skin is the natural host tissue for both HPV and AAV. In this article it is demonstrated that Rep78 directly interacts with c-Jun, both in vitro and in vivo, as analyzed by Western blot, yeast two-hybrid cDNA, and electrophoretic mobility shift-supershift assay (EMSA supershift). Addition of anti-Rep78 antibodies inhibited the EMSA supershift. Investigating the biological implications of this interaction, Rep78 inhibited the c-Jun-dependent c-jun promoter in transient and stable chloramphenicol acetyl-transferase (CAT) assays. Rep78 also inhibited c-Jun-augmented c-jun promoter as well as the HPV-16 p97 promoter activity (also c-Jun regulated) in in vitro transcription assays in T47D nuclear extracts. Finally, the Rep78-c-Jun interaction mapped to the amino-half of Rep78. The ability of Rep78 to interact with c-Jun and down-regulate AP-1-dependent transcription suggests one more mechanism by which AAV may modulate the HPV life cycle and the carcinogenesis process

  9. Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions.

    Science.gov (United States)

    Khan, Mateen A; Ma, Jia; Walden, William E; Merrick, William C; Theil, Elizabeth C; Goss, Dixie J

    2014-06-01

    Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Protein scaffolds and higher-order complexes in synthetic biology

    NARCIS (Netherlands)

    den Hamer, A.; Rosier, B.J.H.M.; Brunsveld, L.; de Greef, T.F.A.; Ryadnov, M.; Brunsveld, L.; Suga, H.

    2017-01-01

    Interactions between proteins control molecular functions such as signalling or metabolic activity. Assembly of proteins via scaffold proteins or in higher-order complexes is a key regulatory mechanism. Understanding and functionally applying this concept requires the construction, study, and

  11. Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes

    Energy Technology Data Exchange (ETDEWEB)

    Serrano, Pauline N. [Department of Chemistry, University of California, Davis CA 95616 USA; Wang, Hongxin [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA; Crack, Jason C. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Prior, Christopher [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Hutchings, Matthew I. [School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ UK; Thomson, Andrew J. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Kamali, Saeed [University of Tennessee Space Institute, Tullahome TN 37388-9700 USA; Yoda, Yoshitaka [Research and Utilization Division, SPring-8/JASRI, 1-1-1 Kouto, Sayo Hyogo 679-5198 Japan; Zhao, Jiyong [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Hu, Michael Y. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Alp, Ercan E. [Advanced Photon Source, Argonne National Laboratory, Argonne IL 60439 USA; Oganesyan, Vasily S. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Le Brun, Nick E. [Centre for Molecular and Structural Biochemistry, School of Chemistry, University of East Anglia, Norwich Research Park Norwich NR4 7TJ UK; Cramer, Stephen P. [Department of Chemistry, University of California, Davis CA 95616 USA; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley CA 94720 USA

    2016-10-25

    The reaction of protein-bound iron–sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2(NO)4(Cys)2]) and Roussin's Black Salt (RBS, [Fe4(NO)7S3]. In the latter case, the absence of 32S/34S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.

  12. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

  13. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  14. Neutron scattering with deuterium labeling reveals the nature of complexes formed by Ca{sup 2+}-binding proteins and their regulatory targets

    Energy Technology Data Exchange (ETDEWEB)

    Trewhella, J. [Los Alamos National Laboratory, NM (United States)

    1994-12-01

    Small-angle neutron scattering with deuterium labeling is extremely useful for studying the structures of complex biomolecular assemblies in solution. The different neutron scattering properties of their isotopes of hydrogen combines with the ability to uniformly label biomolecules with deuterium allow one to characterize the structures and relative dispositions of the individual components of an assembly using methods of {open_quotes}contrast variation.{close_quotes} We have applied these techniques to studies of the evolutionarily related dumbbell-shaped Ca{sup 2+}-binding proteins calmodulin and troponin C and their interactions with the target proteins whose activities they regulate. Ca{sup 2+} is one of the simplest of nature`s messengers used in the communication pathways between physiological stimulus and cellular response. The signaling mechanism generally involves Ca{sup 2+} binding to a protein and inducing a conformational change that transmits a signal to modify the activity of a specific target protein. Ca{sup 2+} is thus important in the regulation of a diverse array of intracellular responses, including neurotransmitter release, muscle contraction, the degradation of glycogen to glucose to generate energy, microtubule assembly, membrane phosphorylation, etc. It is the conformational language of the Ca{sup 2+} induced signal transduction that we have sought to understand because of its central importance to biochemical regulation and, hence, to healthy cellular function.

  15. Trans-acting translational regulatory RNA binding proteins.

    Science.gov (United States)

    Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

    2018-05-01

    The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

  16. Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

    Science.gov (United States)

    Hovingh, Elise S; van den Broek, Bryan; Jongerius, Ilse

    2016-01-01

    The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.

  17. Modeling complexes of modeled proteins.

    Science.gov (United States)

    Anishchenko, Ivan; Kundrotas, Petras J; Vakser, Ilya A

    2017-03-01

    Structural characterization of proteins is essential for understanding life processes at the molecular level. However, only a fraction of known proteins have experimentally determined structures. This fraction is even smaller for protein-protein complexes. Thus, structural modeling of protein-protein interactions (docking) primarily has to rely on modeled structures of the individual proteins, which typically are less accurate than the experimentally determined ones. Such "double" modeling is the Grand Challenge of structural reconstruction of the interactome. Yet it remains so far largely untested in a systematic way. We present a comprehensive validation of template-based and free docking on a set of 165 complexes, where each protein model has six levels of structural accuracy, from 1 to 6 Å C α RMSD. Many template-based docking predictions fall into acceptable quality category, according to the CAPRI criteria, even for highly inaccurate proteins (5-6 Å RMSD), although the number of such models (and, consequently, the docking success rate) drops significantly for models with RMSD > 4 Å. The results show that the existing docking methodologies can be successfully applied to protein models with a broad range of structural accuracy, and the template-based docking is much less sensitive to inaccuracies of protein models than the free docking. Proteins 2017; 85:470-478. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Secretory IgA in complex with Lactobacillus rhamnosus potentiates mucosal dendritic cell-mediated Treg cell differentiation via TLR regulatory proteins, RALDH2 and secretion of IL-10 and TGF-β.

    Science.gov (United States)

    Mikulic, Josip; Longet, Stéphanie; Favre, Laurent; Benyacoub, Jalil; Corthesy, Blaise

    2017-06-01

    The importance of secretory IgA in controlling the microbiota is well known, yet how the antibody affects the perception of the commensals by the local immune system is still poorly defined. We have previously shown that the transport of secretory IgA in complex with bacteria across intestinal microfold cells results in an association with dendritic cells in Peyer's patches. However, the consequences of such an interaction on dendritic cell conditioning have not been elucidated. In this study, we analyzed the impact of the commensal Lactobacillus rhamnosus, alone or associated with secretory IgA, on the responsiveness of dendritic cells freshly recovered from mouse Peyer's patches, mesenteric lymph nodes, and spleen. Lactobacillus rhamnosus-conditioned mucosal dendritic cells are characterized by increased expression of Toll-like receptor regulatory proteins [including single immunoglobulin interleukin-1 receptor-related molecule, suppressor of cytokine signaling 1, and Toll-interacting molecule] and retinaldehyde dehydrogenase 2, low surface expression of co-stimulatory markers, high anti- versus pro-inflammatory cytokine production ratios, and induction of T regulatory cells with suppressive function. Association with secretory IgA enhanced the anti-inflammatory/regulatory Lactobacillus rhamnosus-induced conditioning of mucosal dendritic cells, particularly in Peyer's patches. At the systemic level, activation of splenic dendritic cells exposed to Lactobacillus rhamnosus was partially dampened upon association with secretory IgA. These data suggest that secretory IgA, through coating of commensal bacteria, contributes to the conditioning of mucosal dendritic cells toward tolerogenic profiles essential for the maintenance of intestinal homeostasis.

  19. Complex Regulatory Networks Governing Production of the Glycopeptide A40926

    Directory of Open Access Journals (Sweden)

    Rosa Alduina

    2018-04-01

    Full Text Available Glycopeptides (GPAs are an important class of antibiotics, with vancomycin and teicoplanin being used in the last 40 years as drugs of last resort to treat infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus. A few new GPAs have since reached the market. One of them is dalbavancin, a derivative of A40926 produced by the actinomycete Nonomuraea sp. ATCC 39727, recently classified as N. gerenzanensis. This review summarizes what we currently know on the multilevel regulatory processes governing production of the glycopeptide A40926 and the different approaches used to increase antibiotic yields. Some nutrients, e.g., valine, l-glutamine and maltodextrin, and some endogenous proteins, e.g., Dbv3, Dbv4 and RpoBR, have a positive role on A40926 biosynthesis, while other factors, e.g., phosphate, ammonium and Dbv23, have a negative effect. Overall, the results available so far point to a complex regulatory network controlling A40926 in the native producing strain.

  20. Complex Regulatory Networks Governing Production of the Glycopeptide A40926.

    Science.gov (United States)

    Alduina, Rosa; Sosio, Margherita; Donadio, Stefano

    2018-04-05

    Glycopeptides (GPAs) are an important class of antibiotics, with vancomycin and teicoplanin being used in the last 40 years as drugs of last resort to treat infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus . A few new GPAs have since reached the market. One of them is dalbavancin, a derivative of A40926 produced by the actinomycete Nonomuraea sp. ATCC 39727, recently classified as N. gerenzanensis . This review summarizes what we currently know on the multilevel regulatory processes governing production of the glycopeptide A40926 and the different approaches used to increase antibiotic yields. Some nutrients, e.g., valine, l-glutamine and maltodextrin, and some endogenous proteins, e.g., Dbv3, Dbv4 and RpoB R , have a positive role on A40926 biosynthesis, while other factors, e.g., phosphate, ammonium and Dbv23, have a negative effect. Overall, the results available so far point to a complex regulatory network controlling A40926 in the native producing strain.

  1. THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

    Science.gov (United States)

    Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

    2011-01-01

    The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

  2. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

  3. The fifth adaptor protein complex.

    Directory of Open Access Journals (Sweden)

    Jennifer Hirst

    2011-10-01

    Full Text Available Adaptor protein (AP complexes sort cargo into vesicles for transport from one membrane compartment of the cell to another. Four distinct AP complexes have been identified, which are present in most eukaryotes. We report the existence of a fifth AP complex, AP-5. Tagged AP-5 localises to a late endosomal compartment in HeLa cells. AP-5 does not associate with clathrin and is insensitive to brefeldin A. Knocking down AP-5 subunits interferes with the trafficking of the cation-independent mannose 6-phosphate receptor and causes the cell to form swollen endosomal structures with emanating tubules. AP-5 subunits can be found in all five eukaryotic supergroups, but they have been co-ordinately lost in many organisms. Concatenated phylogenetic analysis provides robust resolution, for the first time, into the evolutionary order of emergence of the adaptor subunit families, showing AP-3 as the basal complex, followed by AP-5, AP-4, and AP-1 and AP-2. Thus, AP-5 is an evolutionarily ancient complex, which is involved in endosomal sorting, and which has links with hereditary spastic paraplegia.

  4. Self-sustained oscillations of complex genomic regulatory networks

    International Nuclear Information System (INIS)

    Ye Weiming; Huang Xiaodong; Huang Xuhui; Li Pengfei; Xia Qinzhi; Hu Gang

    2010-01-01

    Recently, self-sustained oscillations in complex networks consisting of non-oscillatory nodes have attracted great interest in diverse natural and social fields. Oscillatory genomic regulatory networks are one of the most typical examples of this kind. Given an oscillatory genomic network, it is important to reveal the central structure generating the oscillation. However, if the network consists of large numbers of genes and interactions, the oscillation generator is deeply hidden in the complicated interactions. We apply the dominant phase-advanced driving path method proposed in Qian et al. (2010) to reduce complex genomic regulatory networks to one-dimensional and unidirectionally linked network graphs where negative regulatory loops are explored to play as the central generators of the oscillations, and oscillation propagation pathways in the complex networks are clearly shown by tree branches radiating from the loops. Based on the above understanding we can control oscillations of genomic networks with high efficiency.

  5. Dynamic SPR monitoring of yeast nuclear protein binding to a cis-regulatory element

    International Nuclear Information System (INIS)

    Mao, Grace; Brody, James P.

    2007-01-01

    Gene expression is controlled by protein complexes binding to short specific sequences of DNA, called cis-regulatory elements. Expression of most eukaryotic genes is controlled by dozens of these elements. Comprehensive identification and monitoring of these elements is a major goal of genomics. In pursuit of this goal, we are developing a surface plasmon resonance (SPR) based assay to identify and monitor cis-regulatory elements. To test whether we could reliably monitor protein binding to a regulatory element, we immobilized a 16 bp region of Saccharomyces cerevisiae chromosome 5 onto a gold surface. This 16 bp region of DNA is known to bind several proteins and thought to control expression of the gene RNR1, which varies through the cell cycle. We synchronized yeast cell cultures, and then sampled these cultures at a regular interval. These samples were processed to purify nuclear lysate, which was then exposed to the sensor. We found that nuclear protein binds this particular element of DNA at a significantly higher rate (as compared to unsynchronized cells) during G1 phase. Other time points show levels of DNA-nuclear protein binding similar to the unsynchronized control. We also measured the apparent association complex of the binding to be 0.014 s -1 . We conclude that (1) SPR-based assays can monitor DNA-nuclear protein binding and that (2) for this particular cis-regulatory element, maximum DNA-nuclear protein binding occurs during G1 phase

  6. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

    Directory of Open Access Journals (Sweden)

    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  7. Influence of endurance training on skeletal muscle mitophagy regulatory proteins in type 2 diabetic men.

    Science.gov (United States)

    Brinkmann, Christian; Przyklenk, Axel; Metten, Alexander; Schiffer, Thorsten; Bloch, Wilhelm; Brixius, Klara; Gehlert, Sebastian

    2017-11-01

    Mitophagy is a form of autophagy for the elimination of mitochondria. Mitochondrial content and function are reduced in the skeletal muscle of patients with type 2 diabetes mellitus (T2DM). Physical training has been shown to restore mitochondrial capacity in T2DM patients, but the role of mitophagy has not been examined in this context. This study analyzes the impact of a 3-month endurance training on important skeletal muscle mitophagy regulatory proteins and oxidative phosphorylation (OXPHOS) complexes in T2DM patients. Muscle biopsies were obtained from eight overweight/obese T2DM men (61±10 years) at T1 (6 weeks pre-training), T2 (1 week pre-training), and T3 (3 to 4 days post-training). Protein contents were determined by Western blotting. The training increased mitochondrial complex II significantly (T2-T3: +29%, p = 0.037). The protein contents of mitophagy regulatory proteins (phosphorylated form of forkhead box O3A (pFOXO3A), mitochondrial E3 ubiquitin protein ligase-1 (MUL1), Bcl-2/adenovirus E1B 19-kD interacting protein-3 (BNIP3), microtubule-associated protein 1 light chain-3B (the ratio LC3B-II/LC3B-I was determined)) did not differ significantly between T1, T2, and T3. The results imply that training-induced changes in OXPHOS subunits (significant increase in complex II) are not accompanied by changes in mitophagy regulatory proteins in T2DM men. Future studies should elucidate whether acute exercise might affect mitophagic processes in T2DM patients (and whether a transient regulation of mitophagy regulatory proteins is evident) to fully clarify the role of physical activity and mitophagy for mitochondrial health in this particular patient group.

  8. Dynamics in electron transfer protein complexes

    NARCIS (Netherlands)

    Bashir, Qamar

    2010-01-01

    Recent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We have used paramagnetic relaxation enhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize

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

    Directory of Open Access Journals (Sweden)

    Peng Liu

    2015-01-01

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

  10. Protein kinase A regulatory subunit distribution in medulloblastoma

    International Nuclear Information System (INIS)

    Mucignat-Caretta, Carla; Denaro, Luca; Redaelli, Marco; D'Avella, Domenico; Caretta, Antonio

    2010-01-01

    Previous studies showed a differential distribution of the four regulatory subunits of cAMP-dependent protein kinases inside the brain, that changed in rodent gliomas: therefore, the distribution of these proteins inside the brain can give information on the functional state of the cells. Our goal was to examine human brain tumors to provide evidence for a differential distribution of protein kinase A in different tumors. The distribution of detergent insoluble regulatory (R1 and R2) and catalytic subunits of cAMP dependent kinases was examined in pediatric brain tumors by immunohistochemistry and fluorescent cAMP analogues binding. R2 is organized in large single dots in medulloblastomas, while it has a different appearance in other tumors. Fluorescent cAMP labelling was observed only in medulloblastoma. A different distribution of cAMP dependent protein kinases has been observed in medulloblastoma

  11. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... In Arabidopsis,. At5g35220 gene being sterol regulatory element-binding protein site 2, protease and metalloendopeptidase activity were required for chloroplast development and play a role in regulation of endodermal plastid size and number that are involved in ethylene-dependent gravitropism of light-.

  12. CONSTRUCTION AND ANALYSIS OF IPBR/XYLS HYBRID REGULATORY PROTEINS

    Science.gov (United States)

    IpbR and XylS are related regulatory proteins (having 56% identity). IpbR responds to isopropylbenzene as well as to a variety of hydrophobic chemicals to activate expression of the isopropylbenzene catabolic pathway operon of pRE4 from ipbOP. XylS responds to substituted benzoic...

  13. Exploration of the dynamic properties of protein complexes predicted from spatially constrained protein-protein interaction networks.

    Directory of Open Access Journals (Sweden)

    Eric A Yen

    2014-05-01

    Full Text Available Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and

  14. 3D complex: a structural classification of protein complexes.

    Directory of Open Access Journals (Sweden)

    Emmanuel D Levy

    2006-11-01

    Full Text Available Most of the proteins in a cell assemble into complexes to carry out their function. It is therefore crucial to understand the physicochemical properties as well as the evolution of interactions between proteins. The Protein Data Bank represents an important source of information for such studies, because more than half of the structures are homo- or heteromeric protein complexes. Here we propose the first hierarchical classification of whole protein complexes of known 3-D structure, based on representing their fundamental structural features as a graph. This classification provides the first overview of all the complexes in the Protein Data Bank and allows nonredundant sets to be derived at different levels of detail. This reveals that between one-half and two-thirds of known structures are multimeric, depending on the level of redundancy accepted. We also analyse the structures in terms of the topological arrangement of their subunits and find that they form a small number of arrangements compared with all theoretically possible ones. This is because most complexes contain four subunits or less, and the large majority are homomeric. In addition, there is a strong tendency for symmetry in complexes, even for heteromeric complexes. Finally, through comparison of Biological Units in the Protein Data Bank with the Protein Quaternary Structure database, we identified many possible errors in quaternary structure assignments. Our classification, available as a database and Web server at http://www.3Dcomplex.org, will be a starting point for future work aimed at understanding the structure and evolution of protein complexes.

  15. Antidiabetic effects of glucokinase regulatory protein small-molecule disruptors

    Science.gov (United States)

    Lloyd, David J.; St Jean, David J.; Kurzeja, Robert J. M.; Wahl, Robert C.; Michelsen, Klaus; Cupples, Rod; Chen, Michelle; Wu, John; Sivits, Glenn; Helmering, Joan; Komorowski, Renée; Ashton, Kate S.; Pennington, Lewis D.; Fotsch, Christopher; Vazir, Mukta; Chen, Kui; Chmait, Samer; Zhang, Jiandong; Liu, Longbin; Norman, Mark H.; Andrews, Kristin L.; Bartberger, Michael D.; van, Gwyneth; Galbreath, Elizabeth J.; Vonderfecht, Steven L.; Wang, Minghan; Jordan, Steven R.; Véniant, Murielle M.; Hale, Clarence

    2013-12-01

    Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic β-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.

  16. Dynamics in electron transfer protein complexes

    OpenAIRE

    Bashir, Qamar

    2010-01-01

    Recent studies have provided experimental evidence for the existence of an encounter complex, a transient intermediate in the formation of protein complexes. We have used paramagnetic relaxation enhancement NMR spectroscopy in combination with Monte Carlo simulations to characterize and visualize the ensemble of encounter orientations in the short-lived electron transfer complex of yeast Cc and CcP. The complete conformational space sampled by the protein molecules during the dynamic part of ...

  17. Structural entanglements in protein complexes

    Science.gov (United States)

    Zhao, Yani; Chwastyk, Mateusz; Cieplak, Marek

    2017-06-01

    We consider multi-chain protein native structures and propose a criterion that determines whether two chains in the system are entangled or not. The criterion is based on the behavior observed by pulling at both termini of each chain simultaneously in the two chains. We have identified about 900 entangled systems in the Protein Data Bank and provided a more detailed analysis for several of them. We argue that entanglement enhances the thermodynamic stability of the system but it may have other functions: burying the hydrophobic residues at the interface and increasing the DNA or RNA binding area. We also study the folding and stretching properties of the knotted dimeric proteins MJ0366, YibK, and bacteriophytochrome. These proteins have been studied theoretically in their monomeric versions so far. The dimers are seen to separate on stretching through the tensile mechanism and the characteristic unraveling force depends on the pulling direction.

  18. Regulatory Mechanisms in the P4-ATPase Complex

    DEFF Research Database (Denmark)

    Costa, Sara

    . The functionality on the P4-ATPase complex is essential for several cellular processes, such as vesicle-mediated transport. However, the specific role of flippase activity in vesicle biogenesis and the regulatory mechanism behind this process is still poorly understood. In these studies, we identified...... affordable alternative using a microscope-based cytometer. This system can simultaneously provide information on flippase activity and expression levels. Taken together, the findings described in this thesis provide new tools for P4-ATPase characterization and valuable insights into the regulation...

  19. Myristoylated α subunits of guanine nucleotide-binding regulatory proteins

    International Nuclear Information System (INIS)

    Buss, J.E.; Mumby, S.M.; Casey, P.J.; Gilman, A.G.; Sefton, B.M.

    1987-01-01

    Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the α subunits of G/sub s/ (stimulatory) (α 45 and α 52 ), a 41-kDa subunit of G/sub i/ (inhibitory) (α 41 ), a 40-kDa protein (α 40 ), and the 36-kDa β subunit. No protein that comigrated with the α subunit of G 0 (unknown function) (α 39 ) was detected. In cells grown in the presence of [ 3 H]myristic acid, α 41 and α 40 contained 3 H label, while the β subunit did not. Chemical analysis of lipids attached covalently to purified α 41 and α 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein β and γ subunits and of G/sub t/ (Transducin) subunits (α, β, and γ) failed to reveal fatty acids. The fatty acid associated with α 41 , α 40 , and α 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins

  20. Identification of regulatory targets for the bacterial Nus factor complex.

    Science.gov (United States)

    Baniulyte, Gabriele; Singh, Navjot; Benoit, Courtney; Johnson, Richard; Ferguson, Robert; Paramo, Mauricio; Stringer, Anne M; Scott, Ashley; Lapierre, Pascal; Wade, Joseph T

    2017-12-11

    Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. Here, we identify an additional cellular function for the Nus factor complex in Escherichia coli: repression of the Nus factor-encoding gene, suhB. This repression occurs primarily by translation inhibition, followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Conservation of putative NusB/E binding sites upstream of Nus factor genes suggests that Nus factor autoregulation occurs in many bacterial species. Additionally, many putative NusB/E binding sites are also found upstream of other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

  1. Protein Kinase A Regulatory Subunits in Human Adipose Tissue

    Science.gov (United States)

    Mantovani, Giovanna; Bondioni, Sara; Alberti, Luisella; Gilardini, Luisa; Invitti, Cecilia; Corbetta, Sabrina; Zappa, Marco A.; Ferrero, Stefano; Lania, Andrea G.; Bosari, Silvano; Beck-Peccoz, Paolo; Spada, Anna

    2009-01-01

    OBJECTIVE—In human adipocytes, the cAMP-dependent pathway mediates signals originating from β-adrenergic activation, thus playing a key role in the regulation of important metabolic processes, i.e., lipolysis and thermogenesis. Cyclic AMP effects are mainly mediated by protein kinase A (PKA), whose R2B regulatory isoform is the most expressed in mouse adipose tissue, where it protects against diet-induced obesity and fatty liver development. The aim of the study was to investigate possible differences in R2B expression, PKA activity, and lipolysis in adipose tissues from obese and nonobese subjects. RESEARCH DESIGN AND METHODS—The expression of the different PKA regulatory subunits was evaluated by immunohistochemistry, Western blot, and real-time PCR in subcutaneous and visceral adipose tissue samples from 20 nonobese and 67 obese patients. PKA activity and glycerol release were evaluated in total protein extract and adipocytes isolated from fresh tissue samples, respectively. RESULTS—Expression techniques showed that R2B was the most abundant regulatory protein, both at mRNA and protein level. Interestingly, R2B mRNA levels were significantly lower in both subcutaneous and visceral adipose tissues from obese than nonobese patients and negatively correlated with BMI, waist circumference, insulin levels, and homeostasis model assessment of insulin resistance. Moreover, both basal and stimulated PKA activity and glycerol release were significantly lower in visceral adipose tissue from obese patients then nonobese subjects. CONCLUSIONS—Our results first indicate that, in human adipose tissue, there are important BMI-related differences in R2B expression and PKA activation, which might be included among the multiple determinants involved in the different lipolytic response to β-adrenergic activation in obesity. PMID:19095761

  2. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    International Nuclear Information System (INIS)

    Matsushita, Y.; Murakawa, T.; Shimamura, K.; Oishi, M.; Ohyama, T.; Kurita, N.

    2015-01-01

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA

  3. Specific interactions between DNA and regulatory protein controlled by ligand-binding: Ab initio molecular simulation

    Energy Technology Data Exchange (ETDEWEB)

    Matsushita, Y., E-mail: kurita@cs.tut.ac.jp; Murakawa, T., E-mail: kurita@cs.tut.ac.jp; Shimamura, K., E-mail: kurita@cs.tut.ac.jp; Oishi, M., E-mail: kurita@cs.tut.ac.jp; Ohyama, T., E-mail: kurita@cs.tut.ac.jp; Kurita, N., E-mail: kurita@cs.tut.ac.jp [Department of Computer Science and Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi, 441-8580 (Japan)

    2015-02-27

    The catabolite activator protein (CAP) is one of the regulatory proteins controlling the transcription mechanism of gene. Biochemical experiments elucidated that the complex of CAP with cyclic AMP (cAMP) is indispensable for controlling the mechanism, while previous molecular simulations for the monomer of CAP+cAMP complex revealed the specific interactions between CAP and cAMP. However, the effect of cAMP-binding to CAP on the specific interactions between CAP and DNA is not elucidated at atomic and electronic levels. We here considered the ternary complex of CAP, cAMP and DNA in solvating water molecules and investigated the specific interactions between them at atomic and electronic levels using ab initio molecular simulations based on classical molecular dynamics and ab initio fragment molecular orbital methods. The results highlight the important amino acid residues of CAP for the interactions between CAP and cAMP and between CAP and DNA.

  4. Characterization of Mediator Complex and its Associated Proteins from Rice.

    Science.gov (United States)

    Samanta, Subhasis; Thakur, Jitendra Kumar

    2017-01-01

    The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

  5. Peroxisome protein import: a complex journey.

    Science.gov (United States)

    Baker, Alison; Lanyon-Hogg, Thomas; Warriner, Stuart L

    2016-06-15

    The import of proteins into peroxisomes possesses many unusual features such as the ability to import folded proteins, and a surprising diversity of targeting signals with differing affinities that can be recognized by the same receptor. As understanding of the structure and function of many components of the protein import machinery has grown, an increasingly complex network of factors affecting each step of the import pathway has emerged. Structural studies have revealed the presence of additional interactions between cargo proteins and the PEX5 receptor that affect import potential, with a subtle network of cargo-induced conformational changes in PEX5 being involved in the import process. Biochemical studies have also indicated an interdependence of receptor-cargo import with release of unloaded receptor from the peroxisome. Here, we provide an update on recent literature concerning mechanisms of protein import into peroxisomes. © 2016 The Author(s).

  6. Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

    Science.gov (United States)

    Meier, Daniel; Schindler, Detlev

    2011-01-01

    The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

  7. Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

    Directory of Open Access Journals (Sweden)

    Daniel Meier

    Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

  8. Nanoscale Dewetting Transition in Protein Complex Folding

    Science.gov (United States)

    Hua, Lan; Huang, Xuhui; Liu, Pu; Zhou, Ruhong; Berne, Bruce J.

    2011-01-01

    In a previous study, a surprising drying transition was observed to take place inside the nanoscale hydrophobic channel in the tetramer of the protein melittin. The goal of this paper is to determine if there are other protein complexes capable of displaying a dewetting transition during their final stage of folding. We searched the entire protein data bank (PDB) for all possible candidates, including protein tetramers, dimers, and two-domain proteins, and then performed the molecular dynamics (MD) simulations on the top candidates identified by a simple hydrophobic scoring function based on aligned hydrophobic surface areas. Our large scale MD simulations found several more proteins, including three tetramers, six dimers, and two two-domain proteins, which display a nanoscale dewetting transition in their final stage of folding. Even though the scoring function alone is not sufficient (i.e., a high score is necessary but not sufficient) in identifying the dewetting candidates, it does provide useful insights into the features of complex interfaces needed for dewetting. All top candidates have two features in common: (1) large aligned (matched) hydrophobic areas between two corresponding surfaces, and (2) large connected hydrophobic areas on the same surface. We have also studied the effect on dewetting of different water models and different treatments of the long-range electrostatic interactions (cutoff vs PME), and found the dewetting phenomena is fairly robust. This work presents a few proteins other than melittin tetramer for further experimental studies of the role of dewetting in the end stages of protein folding. PMID:17608515

  9. The 20S proteasome as an assembly platform for the 19S regulatory complex

    DEFF Research Database (Denmark)

    Hendil, Klaus Aksel Bjørner; Kriegenburg, Franziska; Tanaka, Keiji

    2009-01-01

    26S proteasomes consist of cylindrical 20S proteasomes with 19S regulatory complexes attached to the ends. Treatment with high concentrations of salt causes the regulatory complexes to separate into two sub-complexes, the base, which is in contact with the 20S proteasome, and the lid, which...... is the distal part of the 19S complex. Here, we describe two assembly intermediates of the human regulatory complex. One is a dimer of the two ATPase subunits, Rpt3 and Rpt6. The other is a complex of nascent Rpn2, Rpn10, Rpn11, Rpn13, and Txnl1, attached to preexisting 20S proteasomes. This early assembly...... complex does not yet contain Rpn1 or any of the ATPase subunits of the base. Thus, assembly of 19S regulatory complexes takes place on preexisting 20S proteasomes, and part of the lid is assembled before the base....

  10. Detection of protein complex from protein-protein interaction network using Markov clustering

    International Nuclear Information System (INIS)

    Ochieng, P J; Kusuma, W A; Haryanto, T

    2017-01-01

    Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks. (paper)

  11. Enzymatic Mercury Detoxification: The Regulatory Protein MerR

    CERN Multimedia

    Ctortecka, B; Walsh, C T; Comess, K M

    2002-01-01

    Mercury ions and organomercurial reagents are extremely toxic due to their affinity for thiol groups. Many bacteria contain an elaborate detoxification system for a metabolic conversion of toxic Hg$^{2+}$ or organomercurials to less toxic elemental Hg$^0$. The main components of the enzymatic mercury detoxification (see Fig. 1) are the regulatory protein MerR (mercury responsive genetic switch), the organomercurial lyase MerB (cleavage of carbon mercury bonds), and the mercuric ion reductase MerA (reduction of mercuric ions). In these proteins Hg$^{2+}$ is usually coordinated by the thiol groups of cysteines. We utilize the nuclear quadrupole interaction (NQI) of ${\\rm^{199m}}$Hg detected by time differential perturbed angular correlation (TDPAC) to identify the Hg metal site geometries in these proteins in order to elucidate the molecular origin of the ultrasensitivity, selectivity and reaction mechanism of this detoxification system. The short lived TDPAC probe ${\\rm^{199m}}$Hg ($\\tau_{1/2} =$ 43 min) is su...

  12. A conserved regulatory mechanism in bifunctional biotin protein ligases.

    Science.gov (United States)

    Wang, Jingheng; Beckett, Dorothy

    2017-08-01

    Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization. Like E. coli BirA (EcBirA), Staphylococcus aureus, and Bacillus subtilis BirA (Sa and BsBirA) repress transcription in vivo in a biotin-dependent manner. In this work, sedimentation equilibrium measurements were performed to investigate the molecular basis of this biotin-responsive transcription regulation. The results reveal that, as observed for EcBirA, Sa, and BsBirA dimerization reactions are significantly enhanced by bio-5'-AMP binding. Thus, the molecular mechanism of the Biotin Regulatory System is conserved in the biotin repressors from these three organisms. © 2017 The Protein Society.

  13. Characterising antimicrobial protein-membrane complexes

    International Nuclear Information System (INIS)

    Xun, Gloria; Dingley, Andrew; Tremouilhac, Pierre

    2009-01-01

    Full text: Antimicrobial proteins (AMPs) are host defence molecules that protect organisms from microbial infection. A number of hypotheses for AMP activity have been proposed which involve protein membrane interactions. However, there is a paucity of information describing AMP-membrane complexes in detail. The aim of this project is to characterise the interactions of amoebapore-A (APA-1) with membrane models using primarily solution-state NMR spectroscopy. APA-1 is an AMP which is regulated by a pH-dependent dimerisation event. Based on the atomic resolution solution structure of monomeric APA-1, it is proposed that this dimerisation is a prerequisite for ring-like hexameric pore formation. Due to the cytotoxicity of APA-1, we have developed a cell-free system to produce this protein. To facilitate our studies, we have adapted the cell-free system to isotope label APA-1. 13 C /15 N -enriched APA-1 sample was achieved and we have begun characterising APA-1 dimerisation and membrane interactions using NMR spectroscopy and other biochemical/biophysical methods. Neutron reflectometry is a surface-sensitive technique and therefore represents an ideal technique to probe how APA-1 interacts with membranes at the molecular level under different physiological conditions. Using Platypus, the pH-induced APA-1-membrane interactions should be detectable as an increase of the amount of protein adsorbed at the membrane surface and changes in the membrane properties. Specifically, detailed information of the structure and dimensions of the protein-membrane complex, the position and amount of the protein in the membrane, and the perturbation of the membrane phospholipids on protein incorporation can be extracted from the neutron reflectometry measurement. Such information will enable critical assessment of current proposed mechanisms of AMP activity in bacterial membranes and complement our NMR studies

  14. Solubilization and reconstitution of the formylmethionylleucylphenylalanine receptor coupled to guanine nucleotide regulatory protein

    International Nuclear Information System (INIS)

    Williamson, K.; Dickey, B.F.; Pyun, H.Y.; Navarro, J.

    1988-01-01

    The authors describe the solubilization, resolution, and reconstitution of the formylmethionylleucylphenylalanine (fMet-Leu-Phe) receptor and guanine nucleotide regulatory proteins (G-proteins). The receptor was solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Guanine nucleotides decreased the number of high-affinity binding sites and accelerated the rate of dissociation of the receptor-ligand complex, suggesting that the solubilized receptor remained coupled to endogenous G-proteins. The solubilized receptor was resolved from endogenous G-proteins by fractionation on a wheat germ agglutinin (WGA)-Sepharose 4B column. High-affinity [ 3 H]fMet-Leu-Phe binding to the WGA-purified receptor was diminished and exhibited reduced guanine nucleotide sensitivity. High-affinity [ 3 H]fMET-Leu-Phe binding and guanine nucleotide sensitivity were reconstituted upon the addition of purified brain G-proteins. Similar results were obtained when the receptor was reconstituted with brain G-proteins into phospholipid vesicles by gel filtration chromatography. In addition, they also demonstrated fMET-Leu-Phe-dependent GTP hydrolysis in the reconstituted vesicles. The results of this work indicate that coupling of the fMet-Leu-Phe receptor to G-proteins converts the receptor to a high-affinity binding state and that agonist produces activation of G-proteins. The resolution and functional reconstitution of this receptor should provide an important step toward the elucidation of the molecular mechanism of the fMet-Leu-Phe transduction system in neutrophils

  15. Protein complex prediction in large ontology attributed protein-protein interaction networks.

    Science.gov (United States)

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian; Li, Yanpeng; Xu, Bo

    2013-01-01

    Protein complexes are important for unraveling the secrets of cellular organization and function. Many computational approaches have been developed to predict protein complexes in protein-protein interaction (PPI) networks. However, most existing approaches focus mainly on the topological structure of PPI networks, and largely ignore the gene ontology (GO) annotation information. In this paper, we constructed ontology attributed PPI networks with PPI data and GO resource. After constructing ontology attributed networks, we proposed a novel approach called CSO (clustering based on network structure and ontology attribute similarity). Structural information and GO attribute information are complementary in ontology attributed networks. CSO can effectively take advantage of the correlation between frequent GO annotation sets and the dense subgraph for protein complex prediction. Our proposed CSO approach was applied to four different yeast PPI data sets and predicted many well-known protein complexes. The experimental results showed that CSO was valuable in predicting protein complexes and achieved state-of-the-art performance.

  16. Radiolysis of DNA-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Begusova, Marie [Department of Radiation Dosimetry, Nuclear Physics Institute, Na Truhlarce 39/64, CZ-18086, Prague 8 (Czech Republic)]. E-mail: begusova@ujf.cas.cz; Gillard, Nathalie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Sy, Denise [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Castaing, Bertrand [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Charlier, Michel [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France); Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire, CNRS, rue Charles-Sadron, F-45071 Orleans Cedex 2 (France)

    2005-02-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK.

  17. Radiolysis of DNA-protein complexes

    International Nuclear Information System (INIS)

    Begusova, Marie; Gillard, Nathalie; Sy, Denise; Castaing, Bertrand; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-01-01

    We discuss here modifications of DNA and protein radiolysis due to the interaction of these two partners in specific complexes. Experimental patterns of frank strand breaks (FSB) and alkali revealed breaks (ARB) obtained for DNA lac operator bound to the lac repressor and for a DNA containing an abasic site analog bound to the formamidopyrimidine-DNA glycosylase are reported. Experimental data are compared to predicted damage distribution obtained using the theoretical model RADACK

  18. Protein complex prediction based on k-connected subgraphs in protein interaction network

    OpenAIRE

    Habibi, Mahnaz; Eslahchi, Changiz; Wong, Limsoon

    2010-01-01

    Abstract Background Protein complexes play an important role in cellular mechanisms. Recently, several methods have been presented to predict protein complexes in a protein interaction network. In these methods, a protein complex is predicted as a dense subgraph of protein interactions. However, interactions data are incomplete and a protein complex does not have to be a complete or dense subgraph. Results We propose a more appropriate protein complex prediction method, CFA, that is based on ...

  19. Alpha complexes in protein structure prediction

    DEFF Research Database (Denmark)

    Winter, Pawel; Fonseca, Rasmus

    2015-01-01

    Reducing the computational effort and increasing the accuracy of potential energy functions is of utmost importance in modeling biological systems, for instance in protein structure prediction, docking or design. Evaluating interactions between nonbonded atoms is the bottleneck of such computations......-complexes from scratch for every configuration encountered during the search for the native structure would make this approach hopelessly slow. However, it is argued that kinetic a-complexes can be used to reduce the computational effort of determining the potential energy when "moving" from one configuration...... to a neighboring one. As a consequence, relatively expensive (initial) construction of an a-complex is expected to be compensated by subsequent fast kinetic updates during the search process. Computational results presented in this paper are limited. However, they suggest that the applicability of a...

  20. Critical protein GAPDH and its regulatory mechanisms in cancer cells

    International Nuclear Information System (INIS)

    Zhang, Jin-Ying; Zhang, Fan; Hong, Chao-Qun; Giuliano, Armando E.; Cui, Xiao-Jiang; Zhou, Guang-Ji; Zhang, Guo-Jun; Cui, Yu-Kun

    2015-01-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and posttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 (HIF-1), p53, nitric oxide (NO), and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications (PTMs) occurring in GAPDH in cancer cells result in new activities unrelated to the original glycolytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described

  1. BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements.

    Science.gov (United States)

    Bhagwat, Anand S; Roe, Jae-Seok; Mok, Beverly Y L; Hohmann, Anja F; Shi, Junwei; Vakoc, Christopher R

    2016-04-19

    The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  2. The Evolution of the Secreted Regulatory Protein Progranulin.

    Directory of Open Access Journals (Sweden)

    Roger G E Palfree

    Full Text Available Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i: the origins of metazoan progranulins (ii: the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii: the evolution of granulin module architectures of vertebrate progranulins (iv: the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl

  3. The Evolution of the Secreted Regulatory Protein Progranulin.

    Science.gov (United States)

    Palfree, Roger G E; Bennett, Hugh P J; Bateman, Andrew

    2015-01-01

    Progranulin is a secreted growth factor that is active in tumorigenesis, wound repair, and inflammation. Haploinsufficiency of the human progranulin gene, GRN, causes frontotemporal dementia. Progranulins are composed of chains of cysteine-rich granulin modules. Modules may be released from progranulin by proteolysis as 6kDa granulin polypeptides. Both intact progranulin and some of the granulin polypeptides are biologically active. The granulin module occurs in certain plant proteases and progranulins are present in early diverging metazoan clades such as the sponges, indicating their ancient evolutionary origin. There is only one Grn gene in mammalian genomes. More gene-rich Grn families occur in teleost fish with between 3 and 6 members per species including short-form Grns that have no tetrapod counterparts. Our goals are to elucidate progranulin and granulin module evolution by investigating (i): the origins of metazoan progranulins (ii): the evolutionary relationships between the single Grn of tetrapods and the multiple Grn genes of fish (iii): the evolution of granulin module architectures of vertebrate progranulins (iv): the conservation of mammalian granulin polypeptide sequences and how the conserved granulin amino acid sequences map to the known three dimensional structures of granulin modules. We report that progranulin-like proteins are present in unicellular eukaryotes that are closely related to metazoa suggesting that progranulin is among the earliest extracellular regulatory proteins still employed by multicellular animals. From the genomes of the elephant shark and coelacanth we identified contemporary representatives of a precursor for short-from Grn genes of ray-finned fish that is lost in tetrapods. In vertebrate Grns pathways of exon duplication resulted in a conserved module architecture at the amino-terminus that is frequently accompanied by an unusual pattern of tandem nearly identical module repeats near the carboxyl-terminus. Polypeptide

  4. The expression of cytoskeleton regulatory protein Mena in colorectal lesions.

    Science.gov (United States)

    Gurzu, Simona; Jung, I; Prantner, I; Ember, I; Pávai, Z; Mezei, T

    2008-01-01

    The actin regulatory proteins Ena/VASP (Enabled/Vasodilator stimulated phosphoprotein) family is involved in the control of cell motility and adhesion. They are important in the actin-dependent processes where dynamic actin reorganization it is necessary. The deregulation of actin cycle could have an important role in the cells' malignant transformation, tumor invasion or metastasis. Recently studies revealed that the human orthologue of murine Mena is modulated during the breast carcinogenesis. In our study, we tried to observe the immunohistochemical expression of mammalian Ena (Mena) in the colorectal polyps and carcinomas. We analyzed 10 adenomatous polyps (five with dysplasia) and 36 adenocarcinomas. We used the indirect immunoperoxidase staining. BD Biosciences have provided the Mena antibody. We observed that Mena was not expressed in the normal colorectal mucosa neither in polyps without dysplasia, but its expression was very high in polyps with high dysplasia. In colorectal carcinomas, Mena marked the tumoral cells in 80% of cases. In 25% of positive cases, the intensity was 3+, in 60% 2+ and in the other 15% 1+. The Mena intensity was higher in the microsatellite stable tumors (MSS) and was correlated with vascular invasion, with intensity of angiogenesis marked with CD31 and CD105 and with c-erbB-2 and p53 expression. This is the first study in the literature about Mena expression in colorectal lesions.

  5. Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

    OpenAIRE

    Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

    2014-01-01

    Homologous recombination is a conserved pathway for repairing double?stranded breaks, which are processed to yield single?stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single?molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA?ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 b...

  6. Pro-protein convertases control the maturation and processing of the iron-regulatory protein, RGMc/hemojuvelin

    Directory of Open Access Journals (Sweden)

    Rotwein Peter

    2008-04-01

    Full Text Available Abstract Background Repulsive guidance molecule c (RGMc or hemojuvelin, a glycosylphosphatidylinositol-linked glycoprotein expressed in liver and striated muscle, plays a central role in systemic iron balance. Inactivating mutations in the RGMc gene cause juvenile hemochromatosis (JH, a rapidly progressing iron storage disorder with severe systemic manifestations. RGMc undergoes complex biosynthetic steps leading to membrane-bound and soluble forms of the protein, including both 50 and 40 kDa single-chain species. Results We now show that pro-protein convertases (PC are responsible for conversion of 50 kDa RGMc to a 40 kDa protein with a truncated COOH-terminus. Unlike related molecules RGMa and RGMb, RGMc encodes a conserved PC recognition and cleavage site, and JH-associated RGMc frame-shift mutants undergo COOH-terminal cleavage only if this site is present. A cell-impermeable peptide PC inhibitor blocks the appearance of 40 kDa RGMc in extra-cellular fluid, as does an engineered mutation in the conserved PC recognition sequence, while the PC furin cleaves 50 kDa RGMc in vitro into a 40 kDa molecule with an intact NH2-terminus. Iron loading reduces release of RGMc from the cell membrane, and diminishes accumulation of the 40 kDa species in cell culture medium. Conclusion Our results define a role for PCs in the maturation of RGMc that may have implications for the physiological actions of this critical iron-regulatory protein.

  7. CCAAT/enhancer-binding proteins regulate expression of the human steroidogenic acute regulatory protein (StAR) gene.

    Science.gov (United States)

    Christenson, L K; Johnson, P F; McAllister, J M; Strauss, J F

    1999-09-10

    Two putative CCAAT/enhancer-binding protein (C/EBP) response elements were identified in the proximal promoter of the human steroidogenic acute regulatory protein (StAR) gene, which encodes a key protein-regulating steroid hormone synthesis. Expression of C/EBPalpha and -beta increased StAR promoter activity in COS-1 and HepG2 cells. Cotransfection of C/EBPalpha or -beta and steroidogenic factor 1, a transcription factor required for cAMP regulation of StAR expression, into COS-1 augmented 8-bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP)-stimulated promoter activity. When the putative C/EBP response elements were mutated, individually or together, a pronounced decline in basal StAR promoter activity in human granulosa-lutein cells resulted, but the fold stimulation of promoter activity by 8-Br-cAMP was unaffected. Recombinant C/EBPalpha and -beta bound to the two identified sequences but not the mutated elements. Human granulosa-lutein cell nuclear extracts also bound these elements but not the mutated sequences. An antibody to C/EBPbeta, but not C/EBPalpha, supershifted the nuclear protein complex associated with the more distal element. The complex formed by nuclear extracts with the proximal element was not supershifted by either antibody. Western blot analysis revealed the presence of C/EBPalpha and C/EBPbeta in human granulosa-lutein cell nuclear extracts. C/EBPbeta levels were up-regulated 3-fold by 8-Br-cAMP treatment. Our studies demonstrate a role for C/EBPbeta as well as yet to be identified proteins, which can bind to C/EBP response elements, in the regulation of StAR gene expression and suggest a mechanism by which C/EBPbeta participates in the cAMP regulation of StAR gene transcription.

  8. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    Energy Technology Data Exchange (ETDEWEB)

    Mena, Natalia P. [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Bulteau, Anne Laure [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Salazar, Julio [Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile); Hirsch, Etienne C. [UPMC Univ Paris 06, UMRS 975 - UMR 7725, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); Inserm, U 975, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); CNRS, UMR 7225, Centre de Recherche en Neurosciences, ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, F-75005 Paris (France); ICM, Therapeutique Experimentale de la Neurodegenerescence, Hopital de la Salpetriere, Paris 75013 (France); Nunez, Marco T., E-mail: mnunez@uchile.cl [Department of Biology, Faculty of Sciences, Universidad de Chile, Las Palmeras 3425, Santiago (Chile); Millennium Institute of Cell Dynamics and Biotechnology, Santiago (Chile)

    2011-06-03

    Highlights: {yields} Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. {yields} Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. {yields} Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. {yields} Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that

  9. Effect of mitochondrial complex I inhibition on Fe-S cluster protein activity

    International Nuclear Information System (INIS)

    Mena, Natalia P.; Bulteau, Anne Laure; Salazar, Julio; Hirsch, Etienne C.; Nunez, Marco T.

    2011-01-01

    Highlights: → Mitochondrial complex I inhibition resulted in decreased activity of Fe-S containing enzymes mitochondrial aconitase and cytoplasmic aconitase and xanthine oxidase. → Complex I inhibition resulted in the loss of Fe-S clusters in cytoplasmic aconitase and of glutamine phosphoribosyl pyrophosphate amidotransferase. → Consistent with loss of cytoplasmic aconitase activity, an increase in iron regulatory protein 1 activity was found. → Complex I inhibition resulted in an increase in the labile cytoplasmic iron pool. -- Abstract: Iron-sulfur (Fe-S) clusters are small inorganic cofactors formed by tetrahedral coordination of iron atoms with sulfur groups. Present in numerous proteins, these clusters are involved in key biological processes such as electron transfer, metabolic and regulatory processes, DNA synthesis and repair and protein structure stabilization. Fe-S clusters are synthesized mainly in the mitochondrion, where they are directly incorporated into mitochondrial Fe-S cluster-containing proteins or exported for cytoplasmic and nuclear cluster-protein assembly. In this study, we tested the hypothesis that inhibition of mitochondrial complex I by rotenone decreases Fe-S cluster synthesis and cluster content and activity of Fe-S cluster-containing enzymes. Inhibition of complex I resulted in decreased activity of three Fe-S cluster-containing enzymes: mitochondrial and cytosolic aconitases and xanthine oxidase. In addition, the Fe-S cluster content of glutamine phosphoribosyl pyrophosphate amidotransferase and mitochondrial aconitase was dramatically decreased. The reduction in cytosolic aconitase activity was associated with an increase in iron regulatory protein (IRP) mRNA binding activity and with an increase in the cytoplasmic labile iron pool. Since IRP activity post-transcriptionally regulates the expression of iron import proteins, Fe-S cluster inhibition may result in a false iron deficiency signal. Given that inhibition of complex

  10. Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

    Science.gov (United States)

    Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

    2015-02-01

    sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.

  11. Evolution of context dependent regulation by expansion of feast/famine regulatory proteins.

    Science.gov (United States)

    Plaisier, Christopher L; Lo, Fang-Yin; Ashworth, Justin; Brooks, Aaron N; Beer, Karlyn D; Kaur, Amardeep; Pan, Min; Reiss, David J; Facciotti, Marc T; Baliga, Nitin S

    2014-11-14

    Expansion of transcription factors is believed to have played a crucial role in evolution of all organisms by enabling them to deal with dynamic environments and colonize new environments. We investigated how the expansion of the Feast/Famine Regulatory Protein (FFRP) or Lrp-like proteins into an eight-member family in Halobacterium salinarum NRC-1 has aided in niche-adaptation of this archaeon to a complex and dynamically changing hypersaline environment. We mapped genome-wide binding locations for all eight FFRPs, investigated their preference for binding different effector molecules, and identified the contexts in which they act by analyzing transcriptional responses across 35 growth conditions that mimic different environmental and nutritional conditions this organism is likely to encounter in the wild. Integrative analysis of these data constructed an FFRP regulatory network with conditionally active states that reveal how interrelated variations in DNA-binding domains, effector-molecule preferences, and binding sites in target gene promoters have tuned the functions of each FFRP to the environments in which they act. We demonstrate how conditional regulation of similar genes by two FFRPs, AsnC (an activator) and VNG1237C (a repressor), have striking environment-specific fitness consequences for oxidative stress management and growth, respectively. This study provides a systems perspective into the evolutionary process by which gene duplication within a transcription factor family contributes to environment-specific adaptation of an organism.

  12. Identification and analysis of multi-protein complexes in placenta.

    Directory of Open Access Journals (Sweden)

    Fuqiang Wang

    Full Text Available Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE and Liquid chromatography-tandem mass spectrometry (LC-MS/MS were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.

  13. Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element

    Directory of Open Access Journals (Sweden)

    Dudley Jaquelin P

    2009-02-01

    Full Text Available Abstract Background Mouse mammary tumor virus (MMTV encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV, human T-cell leukemia virus (HTLV, and human endogenous retrovirus type K (HERV-K. In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE. Results MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export. Conclusion These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.

  14. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    Proper healing of mucosal wounds requires careful orchestration of epithelial cell migration and proliferation. To elucidate the molecular basis of the lack of cellular proliferation in the migrating 'epithelial tongue' during the re-epithelialization of oral mucosal wounds, the expression of cell......-cycle regulators critical for G(1)-phase progression and S-phase entry was here analysed immunohistochemically. Compared to normal human mucosa, epithelia migrating to cover 2- or 3-day-old wounds made either in vivo or in an organotypic cell culture all showed loss of the proliferation marker Ki67 and cyclins D(1......) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  15. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    Science.gov (United States)

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  16. SUCROSE SYNTHASE: ELUCIDATION OF COMPLEX POST-TRANSLATIONAL REGULATORY MECHANISMS

    Energy Technology Data Exchange (ETDEWEB)

    Steven C. Huber

    2009-05-12

    Studies have focused on the enzyme sucrose synthase, which plays an important role in the metabolism of sucrose in seeds and tubers. There are three isoforms of SUS in maize, referred to as SUS1, SUS-SH1, and SUS2. SUS is generally considered to be tetrameric protein but recent evidence suggests that SUS can also occur as a dimeric protein. The formation of tetrameric SUS is regulated by sucrose concentration in vitro and this could also be an important factor in the cellular localization of the protein. We found that high sucrose concentrations, which promote tetramer formation, also inhibit the binding of SUS1 to actin filaments in vitro. Previously, high sucrose concentrations were shown to promote SUS association with the plasma membrane. The specific regions of the SUS molecule involved in oligomerization are not known, but we identified a region of the SUS1 moelcule by bioinformatic analysis that was predicted to form a coiled coil. We demonstrated that this sequence could, in fact, self-associate as predicted for a coiled coil, but truncation analysis with the full-length recombinant protein suggested that it was not responsible for formation of dimers or tetramers. However, the coiled coil may function in binding of other proteins to SUS1. Overall, sugar availability may differentially influence the binding of SUS to cellular structures, and these effects may be mediated by changes in the oligomeric nature of the enzyme.

  17. HKC: An Algorithm to Predict Protein Complexes in Protein-Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Xiaomin Wang

    2011-01-01

    Full Text Available With the availability of more and more genome-scale protein-protein interaction (PPI networks, research interests gradually shift to Systematic Analysis on these large data sets. A key topic is to predict protein complexes in PPI networks by identifying clusters that are densely connected within themselves but sparsely connected with the rest of the network. In this paper, we present a new topology-based algorithm, HKC, to detect protein complexes in genome-scale PPI networks. HKC mainly uses the concepts of highest k-core and cohesion to predict protein complexes by identifying overlapping clusters. The experiments on two data sets and two benchmarks show that our algorithm has relatively high F-measure and exhibits better performance compared with some other methods.

  18. Ethical and legal challenges in bioenergy governance: Coping with value disagreement and regulatory complexity

    International Nuclear Information System (INIS)

    Gamborg, Christian; Anker, Helle Tegner; Sandøe, Peter

    2014-01-01

    The article focuses on the interplay between two factors giving rise to friction in bioenergy governance: profound value disagreements (e.g. the prioritizing of carbon concerns like worries over GHG emissions savings over non-carbon related concerns) and regulatory complexity (in terms of regulatory measures and options). We present ethical and legal analyses of the current stalemate on bioenergy governance in the EU using two illustrative cases: liquid biofuels for transport and solid biomass-based bioenergy. The two cases disclose some similarities between these two factors, but the remaining differences may partly explain, or justify, contrasting forms of governance. While there seems to be no easy way in which the EU and national governments can deal with the multiple sustainability issues raised by bioenergy, it is argued that failure to deal explicitly with the underlying value disagreements, or to make apparent the regulatory complexity, clouds the issue of how to move forward with governance of bioenergy. We suggest that governance should be shaped with greater focus on the role of value disagreements and regulatory complexity. There is a need for more openness and transparency about such factors, and about the inherent trade-offs in bioenergy governance. - Highlights: • Ethical and legal challenges in governance of liquid biofuels and wood pellets. • EU sustainability criteria legal and ethical analysis—EU bioenergy policy options. • Analysis of interplay between carbon and non-carbon concerns and regulatory options. • Governance must cope with value disagreement and regulatory complexity

  19. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

    Directory of Open Access Journals (Sweden)

    Kathrin Roderer

    Full Text Available The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS. The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

  20. Functional mapping of protein-protein interactions in an enzyme complex by directed evolution.

    Science.gov (United States)

    Roderer, Kathrin; Neuenschwander, Martin; Codoni, Giosiana; Sasso, Severin; Gamper, Marianne; Kast, Peter

    2014-01-01

    The shikimate pathway enzyme chorismate mutase converts chorismate into prephenate, a precursor of Tyr and Phe. The intracellular chorismate mutase (MtCM) of Mycobacterium tuberculosis is poorly active on its own, but becomes >100-fold more efficient upon formation of a complex with the first enzyme of the shikimate pathway, 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (MtDS). The crystal structure of the enzyme complex revealed involvement of C-terminal MtCM residues with the MtDS interface. Here we employed evolutionary strategies to probe the tolerance to substitution of the C-terminal MtCM residues from positions 84-90. Variants with randomized positions were subjected to stringent selection in vivo requiring productive interactions with MtDS for survival. Sequence patterns identified in active library members coincide with residue conservation in natural chorismate mutases of the AroQδ subclass to which MtCM belongs. An Arg-Gly dyad at positions 85 and 86, invariant in AroQδ sequences, was intolerant to mutation, whereas Leu88 and Gly89 exhibited a preference for small and hydrophobic residues in functional MtCM-MtDS complexes. In the absence of MtDS, selection under relaxed conditions identifies positions 84-86 as MtCM integrity determinants, suggesting that the more C-terminal residues function in the activation by MtDS. Several MtCM variants, purified using a novel plasmid-based T7 RNA polymerase gene expression system, showed that a diminished ability to physically interact with MtDS correlates with reduced activatability and feedback regulatory control by Tyr and Phe. Mapping critical protein-protein interaction sites by evolutionary strategies may pinpoint promising targets for drugs that interfere with the activity of protein complexes.

  1. Complex and unexpected dynamics in simple genetic regulatory networks

    Science.gov (United States)

    Borg, Yanika; Ullner, Ekkehard; Alagha, Afnan; Alsaedi, Ahmed; Nesbeth, Darren; Zaikin, Alexey

    2014-03-01

    One aim of synthetic biology is to construct increasingly complex genetic networks from interconnected simpler ones to address challenges in medicine and biotechnology. However, as systems increase in size and complexity, emergent properties lead to unexpected and complex dynamics due to nonlinear and nonequilibrium properties from component interactions. We focus on four different studies of biological systems which exhibit complex and unexpected dynamics. Using simple synthetic genetic networks, small and large populations of phase-coupled quorum sensing repressilators, Goodwin oscillators, and bistable switches, we review how coupled and stochastic components can result in clustering, chaos, noise-induced coherence and speed-dependent decision making. A system of repressilators exhibits oscillations, limit cycles, steady states or chaos depending on the nature and strength of the coupling mechanism. In large repressilator networks, rich dynamics can also be exhibited, such as clustering and chaos. In populations of Goodwin oscillators, noise can induce coherent oscillations. In bistable systems, the speed with which incoming external signals reach steady state can bias the network towards particular attractors. These studies showcase the range of dynamical behavior that simple synthetic genetic networks can exhibit. In addition, they demonstrate the ability of mathematical modeling to analyze nonlinearity and inhomogeneity within these systems.

  2. Construction of ontology augmented networks for protein complex prediction.

    Science.gov (United States)

    Zhang, Yijia; Lin, Hongfei; Yang, Zhihao; Wang, Jian

    2013-01-01

    Protein complexes are of great importance in understanding the principles of cellular organization and function. The increase in available protein-protein interaction data, gene ontology and other resources make it possible to develop computational methods for protein complex prediction. Most existing methods focus mainly on the topological structure of protein-protein interaction networks, and largely ignore the gene ontology annotation information. In this article, we constructed ontology augmented networks with protein-protein interaction data and gene ontology, which effectively unified the topological structure of protein-protein interaction networks and the similarity of gene ontology annotations into unified distance measures. After constructing ontology augmented networks, a novel method (clustering based on ontology augmented networks) was proposed to predict protein complexes, which was capable of taking into account the topological structure of the protein-protein interaction network, as well as the similarity of gene ontology annotations. Our method was applied to two different yeast protein-protein interaction datasets and predicted many well-known complexes. The experimental results showed that (i) ontology augmented networks and the unified distance measure can effectively combine the structure closeness and gene ontology annotation similarity; (ii) our method is valuable in predicting protein complexes and has higher F1 and accuracy compared to other competing methods.

  3. Protein complex finding and ranking: An application to Alzheimer's

    Indian Academy of Sciences (India)

    Protein complexes are known to play a major role in controlling cellular activity in a living being. Identifying complexesfrom raw protein–protein interactions (PPIs) is an important area of research. Earlier work has been limited mostly to yeastand a few other model organisms. Such protein complex identification methods, ...

  4. Improved understanding of protein complex offers insight into DNA

    Science.gov (United States)

    Summer Science Writing Internship Improved understanding of protein complex offers insight into DNA clearer understanding of the origin recognition complex (ORC) - a protein complex that directs DNA replication - through its crystal structure offers new insight into fundamental mechanisms of DNA replication

  5. Equivalence of complex drug products: advances in and challenges for current regulatory frameworks.

    Science.gov (United States)

    Hussaarts, Leonie; Mühlebach, Stefan; Shah, Vinod P; McNeil, Scott; Borchard, Gerrit; Flühmann, Beat; Weinstein, Vera; Neervannan, Sesha; Griffiths, Elwyn; Jiang, Wenlei; Wolff-Holz, Elena; Crommelin, Daan J A; de Vlieger, Jon S B

    2017-11-01

    Biotechnology and nanotechnology provide a growing number of innovator-driven complex drug products and their copy versions. Biologics exemplify one category of complex drugs, but there are also nonbiological complex drug products, including many nanomedicines, such as iron-carbohydrate complexes, drug-carrying liposomes or emulsions, and glatiramoids. In this white paper, which stems from a 1-day conference at the New York Academy of Sciences, we discuss regulatory frameworks in use worldwide (e.g., the U.S. Food and Drug Administration, the European Medicines Agency, the World Health Organization) to approve these complex drug products and their follow-on versions. One of the key questions remains how to assess equivalence of these complex products. We identify a number of points for which consensus was found among the stakeholders who were present: scientists from innovator and generic/follow-on companies, academia, and regulatory bodies from different parts of the world. A number of topics requiring follow-up were identified: (1) assessment of critical attributes to establish equivalence for follow-on versions, (2) the need to publish scientific findings in the public domain to further progress in the field, (3) the necessity to develop worldwide consensus regarding nomenclature and labeling of these complex products, and (4) regulatory actions when substandard complex drug products are identified. © 2017 The Authors. Annals of the New York Academy of Sciences published by Wiley Periodicals, Inc. on behalf of New York Academy of Sciences.

  6. Structural studies of Ca2+-ATPase ligand and regulatory complexes

    DEFF Research Database (Denmark)

    Drachmann, Nikolaj Düring

    2015-01-01

    , the surrounding membrane itself has a huge influence on SERCA structure and function. Changes in the membrane thickness can alter the activity of the ATPase significantly, and even cause changes in the stoichiometry of ion transport. Structural studies on SERCA in the presence of four different phosphatidyl...... choline lipids with different aliphatic chain length and saturation show three specific lipid binding sites. The four different lipids analysed bind to the same binding sites with varying degrees of disorder. The study contributes to understanding the complex interplay between the surrounding membrane...... to explore the possibilities for an efficient screening of ligand-bound SERCA structures, serial femtosecond crystallography experiments of microcrystals of SERCA1a in the Ca2+ bound state and in a vanadate stabilised E2 state was conducted. A structure obtained at 2.8 Å maximum resolution of the proof...

  7. NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

    Science.gov (United States)

    Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

    2012-05-01

    The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

  8. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-26

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

  9. Regulatory mechanisms of skeletal muscle protein turnover during exercise

    DEFF Research Database (Denmark)

    Rose, Adam John; Richter, Erik

    2009-01-01

    Skeletal muscle protein turnover is a relatively slow metabolic process that is altered by various physiological stimuli such as feeding/fasting and exercise. During exercise, catabolism of amino acids contributes very little to ATP turnover in working muscle. With regards to protein turnover......, there is now consistent data from tracer studies in rodents and humans showing that global protein synthesis is blunted in working skeletal muscle. Whether there is altered skeletal muscle protein breakdown during exercise remains unclear. The blunting of protein synthesis is believed to be mediated...... downstream of changes in intracellular Ca(2+) and energy turnover. In particular, a signaling cascade involving Ca(2+)-calmodulin-eEF2 kinase-eEF2 is implicated. The possible functional significance of altered protein turnover in working skeletal muscle during exercise is discussed. Further work...

  10. Protein complex prediction via dense subgraphs and false positive analysis.

    Directory of Open Access Journals (Sweden)

    Cecilia Hernandez

    Full Text Available Many proteins work together with others in groups called complexes in order to achieve a specific function. Discovering protein complexes is important for understanding biological processes and predict protein functions in living organisms. Large-scale and throughput techniques have made possible to compile protein-protein interaction networks (PPI networks, which have been used in several computational approaches for detecting protein complexes. Those predictions might guide future biologic experimental research. Some approaches are topology-based, where highly connected proteins are predicted to be complexes; some propose different clustering algorithms using partitioning, overlaps among clusters for networks modeled with unweighted or weighted graphs; and others use density of clusters and information based on protein functionality. However, some schemes still require much processing time or the quality of their results can be improved. Furthermore, most of the results obtained with computational tools are not accompanied by an analysis of false positives. We propose an effective and efficient mining algorithm for discovering highly connected subgraphs, which is our base for defining protein complexes. Our representation is based on transforming the PPI network into a directed acyclic graph that reduces the number of represented edges and the search space for discovering subgraphs. Our approach considers weighted and unweighted PPI networks. We compare our best alternative using PPI networks from Saccharomyces cerevisiae (yeast and Homo sapiens (human with state-of-the-art approaches in terms of clustering, biological metrics and execution times, as well as three gold standards for yeast and two for human. Furthermore, we analyze false positive predicted complexes searching the PDBe (Protein Data Bank in Europe database in order to identify matching protein complexes that have been purified and structurally characterized. Our analysis shows

  11. Global Stability of Complex-Valued Genetic Regulatory Networks with Delays on Time Scales

    Directory of Open Access Journals (Sweden)

    Wang Yajing

    2016-01-01

    Full Text Available In this paper, the global exponential stability of complex-valued genetic regulatory networks with delays is investigated. Besides presenting conditions guaranteeing the existence of a unique equilibrium pattern, its global exponential stability is discussed. Some numerical examples for different time scales.

  12. DNA-protein complexes induced by chromate and other carcinogens

    International Nuclear Information System (INIS)

    Costa, M.

    1991-01-01

    DNA-protein complexes induced in intact Chinese hamster ovary cells by chromate have been isolated, analyzed, and compared with those induced by cis-platinum, ultraviolet light, and formaldehyde. Actin has been identified as one of the major proteins complexed to DNA by chromate based upon its molecular weight, isoelectric point, positive reaction with an actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of similar molecular weight and isoelectric point, positive reaction with an actin polyclonal antibody, and proteolytic mapping. Chromate and cis-platinum both complex proteins of similar molecular weight and isoelectric points, and these complexes can be disrupted by chelating agents and sulfhydryl reducing agents, suggesting that the metal itself is participating in binding rather than having a catalytic or indirect role (i.e., oxygen radicals). In contrast, formaldehyde complexed histones to the DNA, and these complexes were not disrupted by chelating or reducing agents. An antiserum raised to chromate-induced DNA-protein complexes reacted primarily with 97,000 kDa protein that did not silver stain. Slot blots, as well as Western blots, were used to detect formation of p97 DNA crosslinks. This protein was complexed to the DNA by all four agents studied

  13. Recording information on protein complexes in an information management system.

    Science.gov (United States)

    Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

    2011-08-01

    The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Principles of assembly reveal a periodic table of protein complexes.

    Science.gov (United States)

    Ahnert, Sebastian E; Marsh, Joseph A; Hernández, Helena; Robinson, Carol V; Teichmann, Sarah A

    2015-12-11

    Structural insights into protein complexes have had a broad impact on our understanding of biological function and evolution. In this work, we sought a comprehensive understanding of the general principles underlying quaternary structure organization in protein complexes. We first examined the fundamental steps by which protein complexes can assemble, using experimental and structure-based characterization of assembly pathways. Most assembly transitions can be classified into three basic types, which can then be used to exhaustively enumerate a large set of possible quaternary structure topologies. These topologies, which include the vast majority of observed protein complex structures, enable a natural organization of protein complexes into a periodic table. On the basis of this table, we can accurately predict the expected frequencies of quaternary structure topologies, including those not yet observed. These results have important implications for quaternary structure prediction, modeling, and engineering. Copyright © 2015, American Association for the Advancement of Science.

  15. Trypanin, a component of the flagellar Dynein regulatory complex, is essential in bloodstream form African trypanosomes.

    Directory of Open Access Journals (Sweden)

    Katherine S Ralston

    2006-09-01

    Full Text Available The Trypanosoma brucei flagellum is a multifunctional organelle with critical roles in motility, cellular morphogenesis, and cell division. Although motility is thought to be important throughout the trypanosome lifecycle, most studies of flagellum structure and function have been restricted to the procyclic lifecycle stage, and our knowledge of the bloodstream form flagellum is limited. We have previously shown that trypanin functions as part of a flagellar dynein regulatory system that transmits regulatory signals from the central pair apparatus and radial spokes to axonemal dyneins. Here we investigate the requirement for this dynein regulatory system in bloodstream form trypanosomes. We demonstrate that trypanin is localized to the flagellum of bloodstream form trypanosomes, in a pattern identical to that seen in procyclic cells. Surprisingly, trypanin RNA interference is lethal in the bloodstream form. These knockdown mutants fail to initiate cytokinesis, but undergo multiple rounds of organelle replication, accumulating multiple flagella, nuclei, kinetoplasts, mitochondria, and flagellum attachment zone structures. These findings suggest that normal flagellar beat is essential in bloodstream form trypanosomes and underscore the emerging concept that there is a dichotomy between trypanosome lifecycle stages with respect to factors that contribute to cell division and cell morphogenesis. This is the first time that a defined dynein regulatory complex has been shown to be essential in any organism and implicates the dynein regulatory complex and other enzymatic regulators of flagellar motility as candidate drug targets for the treatment of African sleeping sickness.

  16. Protein complex prediction based on k-connected subgraphs in protein interaction network

    Directory of Open Access Journals (Sweden)

    Habibi Mahnaz

    2010-09-01

    Full Text Available Abstract Background Protein complexes play an important role in cellular mechanisms. Recently, several methods have been presented to predict protein complexes in a protein interaction network. In these methods, a protein complex is predicted as a dense subgraph of protein interactions. However, interactions data are incomplete and a protein complex does not have to be a complete or dense subgraph. Results We propose a more appropriate protein complex prediction method, CFA, that is based on connectivity number on subgraphs. We evaluate CFA using several protein interaction networks on reference protein complexes in two benchmark data sets (MIPS and Aloy, containing 1142 and 61 known complexes respectively. We compare CFA to some existing protein complex prediction methods (CMC, MCL, PCP and RNSC in terms of recall and precision. We show that CFA predicts more complexes correctly at a competitive level of precision. Conclusions Many real complexes with different connectivity level in protein interaction network can be predicted based on connectivity number. Our CFA program and results are freely available from http://www.bioinf.cs.ipm.ir/softwares/cfa/CFA.rar.

  17. The multifaceted activity of the VirF regulatory protein in the Shigella lifestyle

    Directory of Open Access Journals (Sweden)

    Maria Letizia Di Martino

    2016-09-01

    Full Text Available Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV. The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis.

  18. UO{sub 2}{sup 2+}/protein complexation sites screening

    Energy Technology Data Exchange (ETDEWEB)

    Guilbaud, P.; Pible, O

    2004-07-01

    Uranium(VI) is likely to make strong coordination with some proteins in the plasma and in targeted cells. In the frame of a nuclear toxicology program, a biochemical strategy has been developed to identify these targets in complex biological media. The present work focuses on an approach based on the screening of 3D protein structures in order to identify proteins able to bind UO{sub 2}{sup 2+} and the corresponding complexation sites in these proteins. Our preliminary results show that indeed a few proteins display a high affinity to uranyl salt. The site of interaction may be mapped using molecular modeling, providing coherent results with the biochemical data. (authors)

  19. Computational Modeling of Complex Protein Activity Networks

    NARCIS (Netherlands)

    Schivo, Stefano; Leijten, Jeroen; Karperien, Marcel; Post, Janine N.; Prignet, Claude

    2017-01-01

    Because of the numerous entities interacting, the complexity of the networks that regulate cell fate makes it impossible to analyze and understand them using the human brain alone. Computational modeling is a powerful method to unravel complex systems. We recently described the development of a

  20. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

    Directory of Open Access Journals (Sweden)

    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  1. RNA-Binding Proteins in Trichomonas vaginalis: Atypical Multifunctional Proteins Involved in a Posttranscriptional Iron Regulatory Mechanism

    Science.gov (United States)

    Figueroa-Angulo, Elisa E.; Calla-Choque, Jaeson S.; Mancilla-Olea, Maria Inocente; Arroyo, Rossana

    2015-01-01

    Iron homeostasis is highly regulated in vertebrates through a regulatory system mediated by RNA-protein interactions between the iron regulatory proteins (IRPs) that interact with an iron responsive element (IRE) located in certain mRNAs, dubbed the IRE-IRP regulatory system. Trichomonas vaginalis, the causal agent of trichomoniasis, presents high iron dependency to regulate its growth, metabolism, and virulence properties. Although T. vaginalis lacks IRPs or proteins with aconitase activity, possesses gene expression mechanisms of iron regulation at the transcriptional and posttranscriptional levels. However, only one gene with iron regulation at the transcriptional level has been described. Recently, our research group described an iron posttranscriptional regulatory mechanism in the T. vaginalis tvcp4 and tvcp12 cysteine proteinase mRNAs. The tvcp4 and tvcp12 mRNAs have a stem-loop structure in the 5'-coding region or in the 3'-UTR, respectively that interacts with T. vaginalis multifunctional proteins HSP70, α-Actinin, and Actin under iron starvation condition, causing translation inhibition or mRNA stabilization similar to the previously characterized IRE-IRP system in eukaryotes. Herein, we summarize recent progress and shed some light on atypical RNA-binding proteins that may participate in the iron posttranscriptional regulation in T. vaginalis. PMID:26703754

  2. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

    2015-01-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  3. Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

    KAUST Repository

    Kovács, Krisztián A.

    2015-11-01

    CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

  4. Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

    Science.gov (United States)

    Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

    2017-10-01

    Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

  5. Regulatory crosstalk by protein kinases on CFTR trafficking and activity

    Science.gov (United States)

    Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

    2016-01-01

    Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

  6. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells.

    OpenAIRE

    Jiang, M; Pandey, S; Tran, V T; Fong, H K

    1991-01-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein alpha subunits (G alpha) including Gs alpha, Gi-1 alpha, Gi-2 alpha, Gi-3 alpha, and Gz alpha (or Gx alpha), where Gs and Gi are proteins that stimulate or inhibit adenylyl cyclase, respectively, and Gz is a protein that may mediate pertussis toxin-insensi...

  7. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions

    DEFF Research Database (Denmark)

    Luo, Yonglun; Friis, Jenny Blechingberg; Fernandes, Ana Miguel

    2015-01-01

    at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. Conclusions The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes...... involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.......Background FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins...

  8. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly

    Science.gov (United States)

    Wells, Jonathan N.; Bergendahl, L. Therese; Marsh, Joseph A.

    2016-01-01

    Summary The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization. PMID:26804901

  9. Challenging the dogma: the hidden layer of non-protein-coding RNAs in complex organisms.

    Science.gov (United States)

    Mattick, John S

    2003-10-01

    The central dogma of biology holds that genetic information normally flows from DNA to RNA to protein. As a consequence it has been generally assumed that genes generally code for proteins, and that proteins fulfil not only most structural and catalytic but also most regulatory functions, in all cells, from microbes to mammals. However, the latter may not be the case in complex organisms. A number of startling observations about the extent of non-protein-coding RNA (ncRNA) transcription in the higher eukaryotes and the range of genetic and epigenetic phenomena that are RNA-directed suggests that the traditional view of the structure of genetic regulatory systems in animals and plants may be incorrect. ncRNA dominates the genomic output of the higher organisms and has been shown to control chromosome architecture, mRNA turnover and the developmental timing of protein expression, and may also regulate transcription and alternative splicing. This paper re-examines the available evidence and suggests a new framework for considering and understanding the genomic programming of biological complexity, autopoietic development and phenotypic variation. Copyright 2003 Wiley Periodicals, Inc.

  10. Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

    Directory of Open Access Journals (Sweden)

    Tin Nguyen

    Full Text Available MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

  11. Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

    Science.gov (United States)

    Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

    2012-01-01

    MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

  12. Co-suppression of sterol-regulatory element binding protein ...

    African Journals Online (AJOL)

    Administrator

    2011-06-22

    Jun 22, 2011 ... protein mediates etiolation in Arabidopsis thaliana .... (A) The scheme of At5g35220 gene and pEGAD-At5g35220; .... stem length of 42-day-old plants; root length of 5-day-old seedlings grown on MS medium; fresh weight of ...

  13. Patterning protein complexes on DNA nanostructures using a GFP nanobody.

    Science.gov (United States)

    Sommese, R F; Hariadi, R F; Kim, K; Liu, M; Tyska, M J; Sivaramakrishnan, S

    2016-11-01

    DNA nanostructures have become an important and powerful tool for studying protein function over the last 5 years. One of the challenges, though, has been the development of universal methods for patterning protein complexes on DNA nanostructures. Herein, we present a new approach for labeling DNA nanostructures by functionalizing them with a GFP nanobody. We demonstrate the ability to precisely control protein attachment via our nanobody linker using two enzymatic model systems, namely adenylyl cyclase activity and myosin motility. Finally, we test the power of this attachment method by patterning unpurified, endogenously expressed Arp2/3 protein complex from cell lysate. By bridging DNA nanostructures with a fluorescent protein ubiquitous throughout cell and developmental biology and protein biochemistry, this approach significantly streamlines the application of DNA nanostructures as a programmable scaffold in biological studies. © 2016 The Protein Society.

  14. Feast/famine regulatory proteins (FFRPs): Escherichia coli Lrp, AsnC and related archaeal transcription factors.

    Science.gov (United States)

    Yokoyama, Katsushi; Ishijima, Sanae A; Clowney, Lester; Koike, Hideaki; Aramaki, Hironori; Tanaka, Chikako; Makino, Kozo; Suzuki, Masashi

    2006-01-01

    Feast/famine regulatory proteins comprise a diverse family of transcription factors, which have been referred to in various individual identifications, including Escherichia coli leucine-responsive regulatory protein and asparagine synthase C gene product. A full length feast/famine regulatory protein consists of the N-terminal DNA-binding domain and the C-domain, which is involved in dimerization and further assembly, thereby producing, for example, a disc or a chromatin-like cylinder. Various ligands of the size of amino acids bind at the interface between feast/famine regulatory protein dimers, thereby altering their assembly forms. Also, the combination of feast/famine regulatory protein subunits forming the same assembly is altered. In this way, a small number of feast/famine regulatory proteins are able to regulate a large number of genes in response to various environmental changes. Because feast/famine regulatory proteins are shared by archaea and eubacteria, the genome-wide regulation by feast/famine regulatory proteins is traceable back to their common ancestor, being the prototype of highly differentiated transcription regulatory mechanisms found in organisms nowadays.

  15. Quantifying the energetics of cooperativity in a ternary protein complex

    DEFF Research Database (Denmark)

    Andersen, Peter S; Schuck, Peter; Sundberg, Eric J

    2002-01-01

    and mathematical modeling to describe the energetics of cooperativity in a trimolecular protein complex. As a model system for quantifying cooperativity, we studied the ternary complex formed by the simultaneous interaction of a superantigen with major histocompatibility complex and T cell receptor, for which...... a structural model is available. This system exhibits positive and negative cooperativity, as well as augmentation of the temperature dependence of binding kinetics upon the cooperative interaction of individual protein components in the complex. Our experimental and theoretical analysis may be applicable...... to other systems involving cooperativity....

  16. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  17. Prediction of heterodimeric protein complexes from weighted protein-protein interaction networks using novel features and kernel functions.

    Directory of Open Access Journals (Sweden)

    Peiying Ruan

    Full Text Available Since many proteins express their functional activity by interacting with other proteins and forming protein complexes, it is very useful to identify sets of proteins that form complexes. For that purpose, many prediction methods for protein complexes from protein-protein interactions have been developed such as MCL, MCODE, RNSC, PCP, RRW, and NWE. These methods have dealt with only complexes with size of more than three because the methods often are based on some density of subgraphs. However, heterodimeric protein complexes that consist of two distinct proteins occupy a large part according to several comprehensive databases of known complexes. In this paper, we propose several feature space mappings from protein-protein interaction data, in which each interaction is weighted based on reliability. Furthermore, we make use of prior knowledge on protein domains to develop feature space mappings, domain composition kernel and its combination kernel with our proposed features. We perform ten-fold cross-validation computational experiments. These results suggest that our proposed kernel considerably outperforms the naive Bayes-based method, which is the best existing method for predicting heterodimeric protein complexes.

  18. Linking structural features of protein complexes and biological function.

    Science.gov (United States)

    Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

    2015-09-01

    Protein-protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure-function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. © 2015 The Protein Society.

  19. Partitioning of genetic variation between regulatory and coding gene segments: the predominance of software variation in genes encoding introvert proteins.

    Science.gov (United States)

    Mitchison, A

    1997-01-01

    In considering genetic variation in eukaryotes, a fundamental distinction can be made between variation in regulatory (software) and coding (hardware) gene segments. For quantitative traits the bulk of variation, particularly that near the population mean, appears to reside in regulatory segments. The main exceptions to this rule concern proteins which handle extrinsic substances, here termed extrovert proteins. The immune system includes an unusually large proportion of this exceptional category, but even so its chief source of variation may well be polymorphism in regulatory gene segments. The main evidence for this view emerges from genome scanning for quantitative trait loci (QTL), which in the case of the immune system points to a major contribution of pro-inflammatory cytokine genes. Further support comes from sequencing of major histocompatibility complex (Mhc) class II promoters, where a high level of polymorphism has been detected. These Mhc promoters appear to act, in part at least, by gating the back-signal from T cells into antigen-presenting cells. Both these forms of polymorphism are likely to be sustained by the need for flexibility in the immune response. Future work on promoter polymorphism is likely to benefit from the input from genome informatics.

  20. Simplified Method for Predicting a Functional Class of Proteins in Transcription Factor Complexes

    KAUST Repository

    Piatek, Marek J.

    2013-07-12

    Background:Initiation of transcription is essential for most of the cellular responses to environmental conditions and for cell and tissue specificity. This process is regulated through numerous proteins, their ligands and mutual interactions, as well as interactions with DNA. The key such regulatory proteins are transcription factors (TFs) and transcription co-factors (TcoFs). TcoFs are important since they modulate the transcription initiation process through interaction with TFs. In eukaryotes, transcription requires that TFs form different protein complexes with various nuclear proteins. To better understand transcription regulation, it is important to know the functional class of proteins interacting with TFs during transcription initiation. Such information is not fully available, since not all proteins that act as TFs or TcoFs are yet annotated as such, due to generally partial functional annotation of proteins. In this study we have developed a method to predict, using only sequence composition of the interacting proteins, the functional class of human TF binding partners to be (i) TF, (ii) TcoF, or (iii) other nuclear protein. This allows for complementing the annotation of the currently known pool of nuclear proteins. Since only the knowledge of protein sequences is required in addition to protein interaction, the method should be easily applicable to many species.Results:Based on experimentally validated interactions between human TFs with different TFs, TcoFs and other nuclear proteins, our two classification systems (implemented as a web-based application) achieve high accuracies in distinguishing TFs and TcoFs from other nuclear proteins, and TFs from TcoFs respectively.Conclusion:As demonstrated, given the fact that two proteins are capable of forming direct physical interactions and using only information about their sequence composition, we have developed a completely new method for predicting a functional class of TF interacting protein partners

  1. Stabilization of Proteins and Noncovalent Protein Complexes during Electrospray Ionization by Amino Acid Additives.

    Science.gov (United States)

    Zhang, Hua; Lu, Haiyan; Chingin, Konstantin; Chen, Huanwen

    2015-07-21

    Ionization of proteins and noncovalent protein complexes with minimal disturbance to their native structure presents a great challenge for biological mass spectrometry (MS). In living organisms, the native structure of intracellular proteins is commonly stabilized by solute amino acids (AAs) accumulated in cells at very high concentrations. Inspired by nature, we hypothesized that AAs could also pose a stabilizing effect on the native structure of proteins and noncovalent protein complexes during ionization. To test this hypothesis, here we explored MS response for various protein complexes upon the addition of free AAs at mM concentrations into the electrospray ionization (ESI) solution. Thermal activation of ESI droplets in the MS inlet capillary was employed as a model destabilizing factor during ionization. Our results indicate that certain AAs, in particular proline (Pro), pose considerable positive effect on the stability of noncovalent protein complexes in ESI-MS without affecting the signal intensity of protein ions and original protein-ligand equilibrium, even when added at the 20 mM concentration. The data suggest that the degree of protein stabilization is primarily determined by the osmolytic and ampholytic characteristics of AA solutes. The highest stability and visibility of noncovalent protein complexes in ESI-MS are achieved using AA additives with neutral isoelectric point, moderate proton affinity, and unfavorable interaction with the native protein state. Overall, our results indicate that the simple addition of free amino acids into the working solution can notably improve the stability and accuracy of protein analysis by native ESI-MS.

  2. A complex regulatory network coordinating cell cycles during C. elegans development is revealed by a genome-wide RNAi screen.

    Science.gov (United States)

    Roy, Sarah H; Tobin, David V; Memar, Nadin; Beltz, Eleanor; Holmen, Jenna; Clayton, Joseph E; Chiu, Daniel J; Young, Laura D; Green, Travis H; Lubin, Isabella; Liu, Yuying; Conradt, Barbara; Saito, R Mako

    2014-02-28

    The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development. Copyright © 2014 Roy et al.

  3. Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

    Science.gov (United States)

    Xu, Jie; Zhang, Huimin; Srinivas, Swaminath

    2013-01-01

    The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of

  4. Drosophila protein interaction map (DPiM): a paradigm for metazoan protein complex interactions.

    Science.gov (United States)

    Guruharsha, K G; Obar, Robert A; Mintseris, Julian; Aishwarya, K; Krishnan, R T; Vijayraghavan, K; Artavanis-Tsakonas, Spyros

    2012-01-01

    Proteins perform essential cellular functions as part of protein complexes, often in conjunction with RNA, DNA, metabolites and other small molecules. The genome encodes thousands of proteins but not all of them are expressed in every cell type; and expressed proteins are not active at all times. Such diversity of protein expression and function accounts for the level of biological intricacy seen in nature. Defining protein-protein interactions in protein complexes, and establishing the when, what and where of potential interactions, is therefore crucial to understanding the cellular function of any protein-especially those that have not been well studied by traditional molecular genetic approaches. We generated a large-scale resource of affinity-tagged expression-ready clones and used co-affinity purification combined with tandem mass-spectrometry to identify protein partners of nearly 5,000 Drosophila melanogaster proteins. The resulting protein complex "map" provided a blueprint of metazoan protein complex organization. Here we describe how the map has provided valuable insights into protein function in addition to generating hundreds of testable hypotheses. We also discuss recent technological advancements that will be critical in addressing the next generation of questions arising from the map.

  5. Detecting protein complexes based on a combination of topological and biological properties in protein-protein interaction network

    Directory of Open Access Journals (Sweden)

    Pooja Sharma

    2018-06-01

    Full Text Available Protein complexes are known to play a major role in controlling cellular activity in a living being. Identifying complexes from raw protein protein interactions (PPIs is an important area of research. Earlier work has been limited mostly to yeast. Such protein complex identification methods, when applied to large human PPIs often give poor performance. We introduce a novel method called CSC to detect protein complexes. The method is evaluated in terms of positive predictive value, sensitivity and accuracy using the datasets of the model organism, yeast and humans. CSC outperforms several other competing algorithms for both organisms. Further, we present a framework to establish the usefulness of CSC in analyzing the influence of a given disease gene in a complex topologically as well as biologically considering eight major association factors. Keywords: Protein complex, Connectivity, Semantic similarity, Contribution

  6. Traveling-wave ion mobility mass spectrometry of protein complexes

    DEFF Research Database (Denmark)

    Salbo, Rune; Bush, Matthew F; Naver, Helle

    2012-01-01

    The collision cross-section (Ω) of a protein or protein complex ion can be measured using traveling-wave (T-wave) ion mobility (IM) mass spectrometry (MS) via calibration with compounds of known Ω. The T-wave Ω-values depend strongly on instrument parameters and calibrant selection. Optimization ...

  7. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

    Science.gov (United States)

    Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

    2013-12-17

    Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

  8. HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

    Science.gov (United States)

    De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

    2017-09-01

    Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Identification of Essential Proteins Based on a New Combination of Local Interaction Density and Protein Complexes.

    Directory of Open Access Journals (Sweden)

    Jiawei Luo

    Full Text Available Computational approaches aided by computer science have been used to predict essential proteins and are faster than expensive, time-consuming, laborious experimental approaches. However, the performance of such approaches is still poor, making practical applications of computational approaches difficult in some fields. Hence, the development of more suitable and efficient computing methods is necessary for identification of essential proteins.In this paper, we propose a new method for predicting essential proteins in a protein interaction network, local interaction density combined with protein complexes (LIDC, based on statistical analyses of essential proteins and protein complexes. First, we introduce a new local topological centrality, local interaction density (LID, of the yeast PPI network; second, we discuss a new integration strategy for multiple bioinformatics. The LIDC method was then developed through a combination of LID and protein complex information based on our new integration strategy. The purpose of LIDC is discovery of important features of essential proteins with their neighbors in real protein complexes, thereby improving the efficiency of identification.Experimental results based on three different PPI(protein-protein interaction networks of Saccharomyces cerevisiae and Escherichia coli showed that LIDC outperformed classical topological centrality measures and some recent combinational methods. Moreover, when predicting MIPS datasets, the better improvement of performance obtained by LIDC is over all nine reference methods (i.e., DC, BC, NC, LID, PeC, CoEWC, WDC, ION, and UC.LIDC is more effective for the prediction of essential proteins than other recently developed methods.

  10. Protein Complex Production from the Drug Discovery Standpoint.

    Science.gov (United States)

    Moarefi, Ismail

    2016-01-01

    Small molecule drug discovery critically depends on the availability of meaningful in vitro assays to guide medicinal chemistry programs that are aimed at optimizing drug potency and selectivity. As it becomes increasingly evident, most disease relevant drug targets do not act as a single protein. In the body, they are instead generally found in complex with protein cofactors that are highly relevant for their correct function and regulation. This review highlights selected examples of the increasing trend to use biologically relevant protein complexes for rational drug discovery to reduce costly late phase attritions due to lack of efficacy or toxicity.

  11. EWS and FUS bind a subset of transcribed genes encoding proteins enriched in RNA regulatory functions.

    Science.gov (United States)

    Luo, Yonglun; Blechingberg, Jenny; Fernandes, Ana Miguel; Li, Shengting; Fryland, Tue; Børglum, Anders D; Bolund, Lars; Nielsen, Anders Lade

    2015-11-14

    FUS (TLS) and EWS (EWSR1) belong to the FET-protein family of RNA and DNA binding proteins. FUS and EWS are structurally and functionally related and participate in transcriptional regulation and RNA processing. FUS and EWS are identified in translocation generated cancer fusion proteins and involved in the human neurological diseases amyotrophic lateral sclerosis and fronto-temporal lobar degeneration. To determine the gene regulatory functions of FUS and EWS at the level of chromatin, we have performed chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Our results show that FUS and EWS bind to a subset of actively transcribed genes, that binding often is downstream the poly(A)-signal, and that binding overlaps with RNA polymerase II. Functional examinations of selected target genes identified that FUS and EWS can regulate gene expression at different levels. Gene Ontology analyses showed that FUS and EWS target genes preferentially encode proteins involved in regulatory processes at the RNA level. The presented results yield new insights into gene interactions of EWS and FUS and have identified a set of FUS and EWS target genes involved in pathways at the RNA regulatory level with potential to mediate normal and disease-associated functions of the FUS and EWS proteins.

  12. Emerging functions of multi-protein complex Mediator with special emphasis on plants.

    Science.gov (United States)

    Malik, Naveen; Agarwal, Pinky; Tyagi, Akhilesh

    2017-10-01

    Mediator is a multi-subunit protein complex which is involved in transcriptional regulation in yeast and other eukaryotes. As a co-activator, it connects information from transcriptional activators/repressors to transcriptional machinery including RNA polymerase II and general transcription factors. It is not only involved in transcription initiation but also has important roles to play in transcription elongation and termination. Functional attributes of different Mediator subunits have been largely defined in yeast and mammalian systems earlier, while such studies in plants have gained momentum recently. Mediator regulates various processes related to plant development and is also involved in biotic and abiotic stress response. Thus, plant Mediator, like yeast and mammalian Mediator complex, is indispensable for plant growth and survival. Interaction of its multiple subunits with other regulatory proteins and their ectopic expression or knockdown in model plant like Arabidopsis and certain crop plants are paving the way to biochemical analysis and unravel molecular mechanisms of action of Mediator in plants.

  13. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  14. Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

    Directory of Open Access Journals (Sweden)

    Jun Ren

    2014-01-01

    Full Text Available Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes.

  15. Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites

    Science.gov (United States)

    Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas

    2017-01-01

    The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly. PMID:28949297

  16. Comprehensive inventory of protein complexes in the Protein Data Bank from consistent classification of interfaces

    Directory of Open Access Journals (Sweden)

    Gorin Andrey A

    2008-05-01

    Full Text Available Abstract Background Protein-protein interactions are ubiquitous and essential for all cellular processes. High-resolution X-ray crystallographic structures of protein complexes can reveal the details of their function and provide a basis for many computational and experimental approaches. Differentiation between biological and non-biological contacts and reconstruction of the intact complex is a challenging computational problem. A successful solution can provide additional insights into the fundamental principles of biological recognition and reduce errors in many algorithms and databases utilizing interaction information extracted from the Protein Data Bank (PDB. Results We have developed a method for identifying protein complexes in the PDB X-ray structures by a four step procedure: (1 comprehensively collecting all protein-protein interfaces; (2 clustering similar protein-protein interfaces together; (3 estimating the probability that each cluster is relevant based on a diverse set of properties; and (4 combining these scores for each PDB entry in order to predict the complex structure. The resulting clusters of biologically relevant interfaces provide a reliable catalog of evolutionary conserved protein-protein interactions. These interfaces, as well as the predicted protein complexes, are available from the Protein Interface Server (PInS website (see Availability and requirements section. Conclusion Our method demonstrates an almost two-fold reduction of the annotation error rate as evaluated on a large benchmark set of complexes validated from the literature. We also estimate relative contributions of each interface property to the accurate discrimination of biologically relevant interfaces and discuss possible directions for further improving the prediction method.

  17. Auto-Regulatory RNA Editing Fine-Tunes mRNA Re-Coding and Complex Behaviour in Drosophila

    Science.gov (United States)

    Savva, Yiannis A.; Jepson, James E.C; Sahin, Asli; Sugden, Arthur U.; Dorsky, Jacquelyn S.; Alpert, Lauren; Lawrence, Charles; Reenan, Robert A.

    2014-01-01

    Auto-regulatory feedback loops are a common molecular strategy used to optimize protein function. In Drosophila many mRNAs involved in neuro-transmission are re-coded at the RNA level by the RNA editing enzyme dADAR, leading to the incorporation of amino acids that are not directly encoded by the genome. dADAR also re-codes its own transcript, but the consequences of this auto-regulation in vivo are unclear. Here we show that hard-wiring or abolishing endogenous dADAR auto-regulation dramatically remodels the landscape of re-coding events in a site-specific manner. These molecular phenotypes correlate with altered localization of dADAR within the nuclear compartment. Furthermore, auto-editing exhibits sexually dimorphic patterns of spatial regulation and can be modified by abiotic environmental factors. Finally, we demonstrate that modifying dAdar auto-editing affects adaptive complex behaviors. Our results reveal the in vivo relevance of auto-regulatory control over post-transcriptional mRNA re-coding events in fine-tuning brain function and organismal behavior. PMID:22531175

  18. Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

    Science.gov (United States)

    Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

    2017-06-02

    Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

  19. Decomposition of overlapping protein complexes: A graph theoretical method for analyzing static and dynamic protein associations

    Directory of Open Access Journals (Sweden)

    Guimarães Katia S

    2006-04-01

    Full Text Available Abstract Background Most cellular processes are carried out by multi-protein complexes, groups of proteins that bind together to perform a specific task. Some proteins form stable complexes, while other proteins form transient associations and are part of several complexes at different stages of a cellular process. A better understanding of this higher-order organization of proteins into overlapping complexes is an important step towards unveiling functional and evolutionary mechanisms behind biological networks. Results We propose a new method for identifying and representing overlapping protein complexes (or larger units called functional groups within a protein interaction network. We develop a graph-theoretical framework that enables automatic construction of such representation. We illustrate the effectiveness of our method by applying it to TNFα/NF-κB and pheromone signaling pathways. Conclusion The proposed representation helps in understanding the transitions between functional groups and allows for tracking a protein's path through a cascade of functional groups. Therefore, depending on the nature of the network, our representation is capable of elucidating temporal relations between functional groups. Our results show that the proposed method opens a new avenue for the analysis of protein interaction networks.

  20. Regulatory effects of intrinsic IL-10 in IgG immune complex-induced lung injury

    DEFF Research Database (Denmark)

    Shanley, T P; Schmal, H; Friedl, H P

    1995-01-01

    IL-10 has regulatory effects in vitro on cytokine production by activated macrophages. In the IgG immune complex model of lung injury, exogenously administered IL-10 has been shown to suppress in vivo formation of TNF-alpha, up-regulation of vascular ICAM-1, neutrophil recruitment, and ensuing lung....... Blocking of IL-10 by Ab resulted in a 52% increase in lung vascular permeability, a 56% increase in TNF-alpha activity in bronchoalveolar lavage fluids, and a 47 to 48% increase in bronchoalveolar lavage neutrophils and lung myeloperoxidase content. These findings suggest that IL-10 is an important natural...

  1. Structural studies of bacterial transcriptional regulatory proteins by multidimensional heteronuclear NMR

    Energy Technology Data Exchange (ETDEWEB)

    Volkman, Brian Finley [Univ. of California, Berkeley, CA (United States)

    1995-02-01

    Nuclear magnetic resonance spectroscopy was used to elucidate detailed structural information for peptide and protein molecules. A small peptide was designed and synthesized, and its three-dimensional structure was calculated using distance information derived from two-dimensional NMR measurements. The peptide was used to induce antibodies in mice, and the cross-reactivity of the antibodies with a related protein was analyzed with enzyme-linked immunosorbent assays. Two proteins which are involved in regulation of transcription in bacteria were also studied. The ferric uptake regulation (Fur) protein is a metal-dependent repressor which controls iron uptake in bacteria. Two- and three-dimensional NMR techniques, coupled with uniform and selective isotope labeling allowed the nearly complete assignment of the resonances of the metal-binding domain of the Fur protein. NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a "receiver domain" in the family of "two-component" regulatory systems. Phosphorylation of the N-terminal domain of NTRC activates the initiation of transcription of aeries encoding proteins involved in nitrogen regulation. Three- and four-dimensional NMR spectroscopy methods have been used to complete the resonance assignments and determine the solution structure of the N-terminal receiver domain of the NTRC protein. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY reveals a very similar fold, with the only significant difference being the position of helix 4 relative to the rest of the protein. The determination of the structure of the NTRC receiver domain is the first step toward understanding a mechanism of signal transduction which is common to many bacterial regulatory systems.

  2. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma.

    Science.gov (United States)

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-09-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted from primary cell culture of osteosarcoma (n=7) and osteoblasts of cancellous bone (n=7) were studied. Using 2-DE based proteomics and LC-MS/MS analysis, we successfully determined seven differentially expressed protein spots. Four upregulated proteins and three downregulated proteins were observed in this study in which KH-type splicing regulatory protein (KSRP) was selected for further exploration. KSRP was significantly upregulated in osteosarcoma cells compared to osteoblasts using western blot assay. In addition, immunohistochemistry demonstrated that KSRP was also highly expressed in osteosarcoma tissue of independent cases from the experimental group. More importantly, KSRP silencing of osteosarcoma cell lines significantly decreased cell proliferation, migration ability, as well as implantation and growth ability in chick chorioallantoic membrane assay. Taken together, these findings demonstrate, that KSRP plays important roles in regulatory controls of osteosarcoma pathogenesis and serves as a potentially therapeutic target of osteosarcoma.

  3. Predicting protein complexes from weighted protein-protein interaction graphs with a novel unsupervised methodology: Evolutionary enhanced Markov clustering.

    Science.gov (United States)

    Theofilatos, Konstantinos; Pavlopoulou, Niki; Papasavvas, Christoforos; Likothanassis, Spiros; Dimitrakopoulos, Christos; Georgopoulos, Efstratios; Moschopoulos, Charalampos; Mavroudi, Seferina

    2015-03-01

    Proteins are considered to be the most important individual components of biological systems and they combine to form physical protein complexes which are responsible for certain molecular functions. Despite the large availability of protein-protein interaction (PPI) information, not much information is available about protein complexes. Experimental methods are limited in terms of time, efficiency, cost and performance constraints. Existing computational methods have provided encouraging preliminary results, but they phase certain disadvantages as they require parameter tuning, some of them cannot handle weighted PPI data and others do not allow a protein to participate in more than one protein complex. In the present paper, we propose a new fully unsupervised methodology for predicting protein complexes from weighted PPI graphs. The proposed methodology is called evolutionary enhanced Markov clustering (EE-MC) and it is a hybrid combination of an adaptive evolutionary algorithm and a state-of-the-art clustering algorithm named enhanced Markov clustering. EE-MC was compared with state-of-the-art methodologies when applied to datasets from the human and the yeast Saccharomyces cerevisiae organisms. Using public available datasets, EE-MC outperformed existing methodologies (in some datasets the separation metric was increased by 10-20%). Moreover, when applied to new human datasets its performance was encouraging in the prediction of protein complexes which consist of proteins with high functional similarity. In specific, 5737 protein complexes were predicted and 72.58% of them are enriched for at least one gene ontology (GO) function term. EE-MC is by design able to overcome intrinsic limitations of existing methodologies such as their inability to handle weighted PPI networks, their constraint to assign every protein in exactly one cluster and the difficulties they face concerning the parameter tuning. This fact was experimentally validated and moreover, new

  4. Detecting coordinated regulation of multi-protein complexes using logic analysis of gene expression

    Directory of Open Access Journals (Sweden)

    Yeates Todd O

    2009-12-01

    Full Text Available Abstract Background Many of the functional units in cells are multi-protein complexes such as RNA polymerase, the ribosome, and the proteasome. For such units to work together, one might expect a high level of regulation to enable co-appearance or repression of sets of complexes at the required time. However, this type of coordinated regulation between whole complexes is difficult to detect by existing methods for analyzing mRNA co-expression. We propose a new methodology that is able to detect such higher order relationships. Results We detect coordinated regulation of multiple protein complexes using logic analysis of gene expression data. Specifically, we identify gene triplets composed of genes whose expression profiles are found to be related by various types of logic functions. In order to focus on complexes, we associate the members of a gene triplet with the distinct protein complexes to which they belong. In this way, we identify complexes related by specific kinds of regulatory relationships. For example, we may find that the transcription of complex C is increased only if the transcription of both complex A AND complex B is repressed. We identify hundreds of examples of coordinated regulation among complexes under various stress conditions. Many of these examples involve the ribosome. Some of our examples have been previously identified in the literature, while others are novel. One notable example is the relationship between the transcription of the ribosome, RNA polymerase and mannosyltransferase II, which is involved in N-linked glycan processing in the Golgi. Conclusions The analysis proposed here focuses on relationships among triplets of genes that are not evident when genes are examined in a pairwise fashion as in typical clustering methods. By grouping gene triplets, we are able to decipher coordinated regulation among sets of three complexes. Moreover, using all triplets that involve coordinated regulation with the ribosome

  5. The Contribution of Serine 194 Phosphorylation to Steroidogenic Acute Regulatory Protein Function

    OpenAIRE

    Sasaki, Goro; Zubair, Mohamad; Ishii, Tomohiro; Mitsui, Toshikatsu; Hasegawa, Tomonobu; Auchus, Richard J.

    2014-01-01

    The steroidogenic acute regulatory protein (StAR) facilitates the delivery of cholesterol to the inner mitochondrial membrane, where the cholesterol side-chain cleavage enzyme catalyzes the initial step of steroid hormone biosynthesis. StAR was initially identified in adrenocortical cells as a phosphoprotein, the expression and phosphorylation of which were stimulated by corticotropin. A number of in vitro studies have implicated cAMP-dependent phosphorylation at serine 194 (S194, S195 in hum...

  6. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    International Nuclear Information System (INIS)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha; Jamal, Shazia; Levi, Edi; Rishi, Arun K.; Datta, Nabanita S.

    2013-01-01

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  7. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Sonali; Mahalingam, Chandrika D.; Das, Varsha [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Jamal, Shazia [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Levi, Edi [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Department of Pathology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Rishi, Arun K. [Department of Oncology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); VA Medical Center, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Datta, Nabanita S., E-mail: ndatta@med.wayne.edu [Department of Internal Medicine/Endocrinology, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States); Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48201 (United States)

    2013-07-12

    Highlights: •CARP-1 is identified for the first time in bone cells. •PTH downregulates CARP-1 expression in differentiated osteoblasts. •PTH displaces CARP-1 from nucleus to the cytoplasm in differentiated osteoblasts. •Downregulation of CARP-1 by PTH involves PKA, PKC and P-p38 MAPK pathways. -- Abstract: Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30 min to 5 h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1

  8. Uncovering packaging features of co-regulated modules based on human protein interaction and transcriptional regulatory networks

    Directory of Open Access Journals (Sweden)

    He Weiming

    2010-07-01

    Full Text Available Abstract Background Network co-regulated modules are believed to have the functionality of packaging multiple biological entities, and can thus be assumed to coordinate many biological functions in their network neighbouring regions. Results Here, we weighted edges of a human protein interaction network and a transcriptional regulatory network to construct an integrated network, and introduce a probabilistic model and a bipartite graph framework to exploit human co-regulated modules and uncover their specific features in packaging different biological entities (genes, protein complexes or metabolic pathways. Finally, we identified 96 human co-regulated modules based on this method, and evaluate its effectiveness by comparing it with four other methods. Conclusions Dysfunctions in co-regulated interactions often occur in the development of cancer. Therefore, we focussed on an example co-regulated module and found that it could integrate a number of cancer-related genes. This was extended to causal dysfunctions of some complexes maintained by several physically interacting proteins, thus coordinating several metabolic pathways that directly underlie cancer.

  9. Effects of sterol regulatory element-binding protein (SREBP in chickens

    Directory of Open Access Journals (Sweden)

    Alipour Fahimeh

    2012-02-01

    Full Text Available Abstract Sterol regulatory element binding protein- 1 and -2 (SREBP-1 and -2 are key transcription factors involved in the biosynthesis of cholesterol and fatty acids. The SREBP have mostly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, though, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. Since a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we review Size and Tissue expression Pattern of SREBP and role of this protein in chickens.

  10. The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear.

    Science.gov (United States)

    Colantonio, Jessica R; Vermot, Julien; Wu, David; Langenbacher, Adam D; Fraser, Scott; Chen, Jau-Nian; Hill, Kent L

    2009-01-08

    In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized 'ear stones' (or otoliths). Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

  11. Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

    Science.gov (United States)

    Belov, Arseniy M.; Viner, Rosa; Santos, Marcia R.; Horn, David M.; Bern, Marshall; Karger, Barry L.; Ivanov, Alexander R.

    2017-12-01

    Native mass spectrometry (MS) is a rapidly advancing field in the analysis of proteins, protein complexes, and macromolecular species of various types. The majority of native MS experiments reported to-date has been conducted using direct infusion of purified analytes into a mass spectrometer. In this study, capillary zone electrophoresis (CZE) was coupled online to Orbitrap mass spectrometers using a commercial sheathless interface to enable high-performance separation, identification, and structural characterization of limited amounts of purified proteins and protein complexes, the latter with preserved non-covalent associations under native conditions. The performance of both bare-fused silica and polyacrylamide-coated capillaries was assessed using mixtures of protein standards known to form non-covalent protein-protein and protein-ligand complexes. High-efficiency separation of native complexes is demonstrated using both capillary types, while the polyacrylamide neutral-coated capillary showed better reproducibility and higher efficiency for more complex samples. The platform was then evaluated for the determination of monoclonal antibody aggregation and for analysis of proteomes of limited complexity using a ribosomal isolate from E. coli. Native CZE-MS, using accurate single stage and tandem-MS measurements, enabled identification of proteoforms and non-covalent complexes at femtomole levels. This study demonstrates that native CZE-MS can serve as an orthogonal and complementary technique to conventional native MS methodologies with the advantages of low sample consumption, minimal sample processing and losses, and high throughput and sensitivity. This study presents a novel platform for analysis of ribosomes and other macromolecular complexes and organelles, with the potential for discovery of novel structural features defining cellular phenotypes (e.g., specialized ribosomes). [Figure not available: see fulltext.

  12. Effects of ionizing radiations on DNA-protein complexes

    International Nuclear Information System (INIS)

    Gillard, N.

    2005-11-01

    The radio-induced destruction of DNA-protein complexes may have serious consequences for systems implicated in important cellular functions. The first system which has been studied is the lactose operon system, that regulates gene expression in Escherichia coli. First of all, the repressor-operator complex is destroyed after irradiation of the complex or of the protein alone. The damaging of the domain of repressor binding to DNA (headpiece) has been demonstrated and studied from the point of view of peptide chain integrity, conformation and amino acids damages. Secondly, dysfunctions of the in vitro induction of an irradiated repressor-unirradiated DNA complex have been observed. These perturbations, due to a decrease of the number of inducer binding sites, are correlated to the damaging of tryptophan residues. Moreover, the inducer protects the repressor when they are irradiated together, both by acting as a scavenger in the bulk, and by the masking of its binding site on the protein. The second studied system is formed by Fpg (for Formamido pyrimidine glycosylase), a DNA repair protein and a DNA with an oxidative lesion. The results show that irradiation disturbs the repair both by decreasing its efficiency of DNA lesion recognition and binding, and by altering its enzymatic activity. (author)

  13. Colorful packages : fluorescent proteins in complex coacervate core micelles

    NARCIS (Netherlands)

    Nolles, Antsje

    2018-01-01

    This thesis explores the encapsulation of fluorescent proteins (FPs) into complex coacervate core micelles (C3Ms) and features the impact of this encapsulation on the biophysical properties of the FPs. In total eight different FPs were investigated originating from two different classes

  14. Comparative Study of Elastic Network Model and Protein Contact Network for Protein Complexes: The Hemoglobin Case

    Directory of Open Access Journals (Sweden)

    Guang Hu

    2017-01-01

    Full Text Available The overall topology and interfacial interactions play key roles in understanding structural and functional principles of protein complexes. Elastic Network Model (ENM and Protein Contact Network (PCN are two widely used methods for high throughput investigation of structures and interactions within protein complexes. In this work, the comparative analysis of ENM and PCN relative to hemoglobin (Hb was taken as case study. We examine four types of structural and dynamical paradigms, namely, conformational change between different states of Hbs, modular analysis, allosteric mechanisms studies, and interface characterization of an Hb. The comparative study shows that ENM has an advantage in studying dynamical properties and protein-protein interfaces, while PCN is better for describing protein structures quantitatively both from local and from global levels. We suggest that the integration of ENM and PCN would give a potential but powerful tool in structural systems biology.

  15. New insights into the regulatory function of CYFIP1 in the context of WAVE- and FMRP-containing complexes

    Directory of Open Access Journals (Sweden)

    Sabiha Abekhoukh

    2017-04-01

    Full Text Available Cytoplasmic FMRP interacting protein 1 (CYFIP1 is a candidate gene for intellectual disability (ID, autism, schizophrenia and epilepsy. It is a member of a family of proteins that is highly conserved during evolution, sharing high homology with its Drosophila homolog, dCYFIP. CYFIP1 interacts with the Fragile X mental retardation protein (FMRP, encoded by the FMR1 gene, whose absence causes Fragile X syndrome, and with the translation initiation factor eIF4E. It is a member of the WAVE regulatory complex (WRC, thus representing a link between translational regulation and the actin cytoskeleton. Here, we present data showing a correlation between mRNA levels of CYFIP1 and other members of the WRC. This suggests a tight regulation of the levels of the WRC members, not only by post-translational mechanisms, as previously hypothesized. Moreover, we studied the impact of loss of function of both CYFIP1 and FMRP on neuronal growth and differentiation in two animal models – fly and mouse. We show that these two proteins antagonize each other's function not only during neuromuscular junction growth in the fly but also during new neuronal differentiation in the olfactory bulb of adult mice. Mechanistically, FMRP and CYFIP1 modulate mTor signaling in an antagonistic manner, likely via independent pathways, supporting the results obtained in mouse as well as in fly at the morphological level. Collectively, our results illustrate a new model to explain the cellular roles of FMRP and CYFIP1 and the molecular significance of their interaction.

  16. Which clustering algorithm is better for predicting protein complexes?

    Directory of Open Access Journals (Sweden)

    Moschopoulos Charalampos N

    2011-12-01

    Full Text Available Abstract Background Protein-Protein interactions (PPI play a key role in determining the outcome of most cellular processes. The correct identification and characterization of protein interactions and the networks, which they comprise, is critical for understanding the molecular mechanisms within the cell. Large-scale techniques such as pull down assays and tandem affinity purification are used in order to detect protein interactions in an organism. Today, relatively new high-throughput methods like yeast two hybrid, mass spectrometry, microarrays, and phage display are also used to reveal protein interaction networks. Results In this paper we evaluated four different clustering algorithms using six different interaction datasets. We parameterized the MCL, Spectral, RNSC and Affinity Propagation algorithms and applied them to six PPI datasets produced experimentally by Yeast 2 Hybrid (Y2H and Tandem Affinity Purification (TAP methods. The predicted clusters, so called protein complexes, were then compared and benchmarked with already known complexes stored in published databases. Conclusions While results may differ upon parameterization, the MCL and RNSC algorithms seem to be more promising and more accurate at predicting PPI complexes. Moreover, they predict more complexes than other reviewed algorithms in absolute numbers. On the other hand the spectral clustering algorithm achieves the highest valid prediction rate in our experiments. However, it is nearly always outperformed by both RNSC and MCL in terms of the geometrical accuracy while it generates the fewest valid clusters than any other reviewed algorithm. This article demonstrates various metrics to evaluate the accuracy of such predictions as they are presented in the text below. Supplementary material can be found at: http://www.bioacademy.gr/bioinformatics/projects/ppireview.htm

  17. Protein complexes associated with the Kaposi's sarcoma-associated herpesvirus-encoded LANA

    International Nuclear Information System (INIS)

    Kaul, Rajeev; Verma, Subhash C.; Robertson, Erle S.

    2007-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the major biological cofactor contributing to development of Kaposi's sarcoma. KSHV establishes a latent infection in human B cells expressing the latency-associated nuclear antigen (LANA), a critical factor in the regulation of viral latency. LANA is known to modulate viral and cellular gene expression. We report here on some initial proteomic studies to identify cellular proteins associated with the amino and carboxy-terminal domains of LANA. The results of these studies show an association of known cellular proteins which support LANA functions and have identified additional LANA-associated proteins. These results provide new evidence for complexes involving LANA with a number of previously unreported functional classes of proteins including DNA polymerase, RNA helicase and cell cycle control proteins. The results also indicate that the amino terminus of LANA can interact with its carboxy-terminal domain. This interaction is potentially important for facilitating associations with other cell cycle regulatory proteins which include CENP-F identified in association with both the amino and carboxy-termini. These novel associations add to the diversity of LANA functions in relation to the maintenance of latency and subsequent transformation of KSHV infected cells

  18. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Directory of Open Access Journals (Sweden)

    Hongtao Hu

    of a second round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

  19. The Complexity of Posttranscriptional Small RNA Regulatory Networks Revealed by In Silico Analysis of Gossypium arboreum L. Leaf, Flower and Boll Small Regulatory RNAs.

    Science.gov (United States)

    Hu, Hongtao; Rashotte, Aaron M; Singh, Narendra K; Weaver, David B; Goertzen, Leslie R; Singh, Shree R; Locy, Robert D

    2015-01-01

    round of both cis- and trans-cleavage of additional siRNAs, leading to the formation of complex sRNA regulatory networks mediating posttranscriptional gene silencing. Results from this study extended our knowledge on G. arboreum sRNAs and their biological importance, which would facilitate future studies on regulatory mechanism of tissue development in cotton and other plant species.

  20. Extreme disorder in an ultrahigh-affinity protein complex

    Science.gov (United States)

    Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

    2018-03-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

  1. Extreme disorder in an ultrahigh-affinity protein complex

    DEFF Research Database (Denmark)

    Borgia, Alessandro; Borgia, Madeleine B; Bugge, Katrine

    2018-01-01

    Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions...... with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring...... or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex...

  2. From nonspecific DNA-protein encounter complexes to the prediction of DNA-protein interactions.

    Directory of Open Access Journals (Sweden)

    Mu Gao

    2009-03-01

    Full Text Available DNA-protein interactions are involved in many essential biological activities. Because there is no simple mapping code between DNA base pairs and protein amino acids, the prediction of DNA-protein interactions is a challenging problem. Here, we present a novel computational approach for predicting DNA-binding protein residues and DNA-protein interaction modes without knowing its specific DNA target sequence. Given the structure of a DNA-binding protein, the method first generates an ensemble of complex structures obtained by rigid-body docking with a nonspecific canonical B-DNA. Representative models are subsequently selected through clustering and ranking by their DNA-protein interfacial energy. Analysis of these encounter complex models suggests that the recognition sites for specific DNA binding are usually favorable interaction sites for the nonspecific DNA probe and that nonspecific DNA-protein interaction modes exhibit some similarity to specific DNA-protein binding modes. Although the method requires as input the knowledge that the protein binds DNA, in benchmark tests, it achieves better performance in identifying DNA-binding sites than three previously established methods, which are based on sophisticated machine-learning techniques. We further apply our method to protein structures predicted through modeling and demonstrate that our method performs satisfactorily on protein models whose root-mean-square Calpha deviation from native is up to 5 A from their native structures. This study provides valuable structural insights into how a specific DNA-binding protein interacts with a nonspecific DNA sequence. The similarity between the specific DNA-protein interaction mode and nonspecific interaction modes may reflect an important sampling step in search of its specific DNA targets by a DNA-binding protein.

  3. Dual functions of a small regulatory subunit in the mitochondrial calcium uniporter complex.

    Science.gov (United States)

    Tsai, Ming-Feng; Phillips, Charles B; Ranaghan, Matthew; Tsai, Chen-Wei; Wu, Yujiao; Willliams, Carole; Miller, Christopher

    2016-04-21

    Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.

  4. Evolution of DNA replication protein complexes in eukaryotes and Archaea.

    Directory of Open Access Journals (Sweden)

    Nicholas Chia

    Full Text Available BACKGROUND: The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA, replication factor C (RFC, and the minichromosome maintenance (MCM complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best. METHODOLOGY/PRINCIPAL FINDINGS: While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex-all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea. CONCLUSION/SIGNIFICANCE: This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.

  5. Encapsulation of Protein-Polysaccharide HIP Complex in Polymeric Nanoparticles

    Directory of Open Access Journals (Sweden)

    Ripal Gaudana

    2011-01-01

    Full Text Available The objective of the present study is to formulate and characterize a nanoparticulate-based formulation of a macromolecule in a hydrophobic ion pairing (HIP complex form. So far, HIP complexation approach has been studied only for proteins with molecular weight of 10–20 kDa. Hence, we have selected bovine serum albumin (BSA having higher molecular weight (66.3 kDa as a model protein and dextran sulphate (DS as a complexing polymer to generate HIP complex. We have prepared and optimized the HIP complex formation process of BSA with DS. Ionic interactions between basic amino acids of BSA with sulphate groups of DS were confirmed by FTIR analysis. Further, nanoparticles were prepared and characterized with respect to size and surface morphology. We observed significant entrapment of BSA in nanoparticles prepared with minimal amounts of PLGA polymer. Finally, results of circular dichroism and intrinsic fluorescence assay have clearly indicated that HIP complexation and method of nanoparticle preparation did not alter the secondary and tertiary structures of BSA.

  6. Effects of ionizing radiations on DNA-protein complexes; Effets des radiations ionisantes sur des complexes ADN-proteine

    Energy Technology Data Exchange (ETDEWEB)

    Gillard, N

    2005-11-15

    The radio-induced destruction of DNA-protein complexes may have serious consequences for systems implicated in important cellular functions. The first system which has been studied is the lactose operon system, that regulates gene expression in Escherichia coli. First of all, the repressor-operator complex is destroyed after irradiation of the complex or of the protein alone. The damaging of the domain of repressor binding to DNA (headpiece) has been demonstrated and studied from the point of view of peptide chain integrity, conformation and amino acids damages. Secondly, dysfunctions of the in vitro induction of an irradiated repressor-unirradiated DNA complex have been observed. These perturbations, due to a decrease of the number of inducer binding sites, are correlated to the damaging of tryptophan residues. Moreover, the inducer protects the repressor when they are irradiated together, both by acting as a scavenger in the bulk, and by the masking of its binding site on the protein. The second studied system is formed by Fpg (for Formamido pyrimidine glycosylase), a DNA repair protein and a DNA with an oxidative lesion. The results show that irradiation disturbs the repair both by decreasing its efficiency of DNA lesion recognition and binding, and by altering its enzymatic activity. (author)

  7. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

    Directory of Open Access Journals (Sweden)

    Wu Yihe

    2012-04-01

    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

  8. Distinct configurations of protein complexes and biochemical pathways revealed by epistatic interaction network motifs

    LENUS (Irish Health Repository)

    Casey, Fergal

    2011-08-22

    Abstract Background Gene and protein interactions are commonly represented as networks, with the genes or proteins comprising the nodes and the relationship between them as edges. Motifs, or small local configurations of edges and nodes that arise repeatedly, can be used to simplify the interpretation of networks. Results We examined triplet motifs in a network of quantitative epistatic genetic relationships, and found a non-random distribution of particular motif classes. Individual motif classes were found to be associated with different functional properties, suggestive of an underlying biological significance. These associations were apparent not only for motif classes, but for individual positions within the motifs. As expected, NNN (all negative) motifs were strongly associated with previously reported genetic (i.e. synthetic lethal) interactions, while PPP (all positive) motifs were associated with protein complexes. The two other motif classes (NNP: a positive interaction spanned by two negative interactions, and NPP: a negative spanned by two positives) showed very distinct functional associations, with physical interactions dominating for the former but alternative enrichments, typical of biochemical pathways, dominating for the latter. Conclusion We present a model showing how NNP motifs can be used to recognize supportive relationships between protein complexes, while NPP motifs often identify opposing or regulatory behaviour between a gene and an associated pathway. The ability to use motifs to point toward underlying biological organizational themes is likely to be increasingly important as more extensive epistasis mapping projects in higher organisms begin.

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

    Directory of Open Access Journals (Sweden)

    Cheng-Hsien Wu

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

  10. A Bacillus megaterium System for the Production of Recombinant Proteins and Protein Complexes.

    Science.gov (United States)

    Biedendieck, Rebekka

    2016-01-01

    For many years the Gram-positive bacterium Bacillus megaterium has been used for the production and secretion of recombinant proteins. For this purpose it was systematically optimized. Plasmids with different inducible promoter systems, with different compatible origins, with small tags for protein purification and with various specific signals for protein secretion were combined with genetically improved host strains. Finally, the development of appropriate cultivation conditions for the production strains established this organism as a bacterial cell factory even for large proteins. Along with the overproduction of individual proteins the organism is now also used for the simultaneous coproduction of up to 14 recombinant proteins, multiple subsequently interacting or forming protein complexes. Some of these recombinant strains are successfully used for bioconversion or the biosynthesis of valuable components including vitamins. The titers in the g per liter scale for the intra- and extracellular recombinant protein production prove the high potential of B. megaterium for industrial applications. It is currently further enhanced for the production of recombinant proteins and multi-subunit protein complexes using directed genetic engineering approaches based on transcriptome, proteome, metabolome and fluxome data.

  11. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank

    2005-01-01

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  12. Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

    OpenAIRE

    Sanda, Takaomi; Lawton, Lee N.; Barrasa, M. Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A.; Jamieson, Catriona H.M.; Staudt, Louis M.; Young, Richard A.; Look, A. Thomas

    2012-01-01

    The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3 and RUNX1. We show that TAL1 forms a positive interconnected auto-regulatory loop with GATA3 and RUNX1, and that the TAL1 complex directly activates the MY...

  13. A plant virus movement protein forms ringlike complexes with the major nucleolar protein, fibrillarin, in vitro.

    Science.gov (United States)

    Canetta, Elisabetta; Kim, Sang Hyon; Kalinina, Natalia O; Shaw, Jane; Adya, Ashok K; Gillespie, Trudi; Brown, John W S; Taliansky, Michael

    2008-02-29

    Fibrillarin, one of the major proteins of the nucleolus, has methyltransferase activity directing 2'-O-ribose methylation of rRNA and snRNAs and is required for rRNA processing. The ability of the plant umbravirus, groundnut rosette virus, to move long distances through the phloem, the specialized plant vascular system, has been shown to strictly depend on the interaction of one of its proteins, the ORF3 protein (protein encoded by open reading frame 3), with fibrillarin. This interaction is essential for several stages in the groundnut rosette virus life cycle such as nucleolar import of the ORF3 protein via Cajal bodies, relocalization of some fibrillarin from the nucleolus to cytoplasm, and assembly of cytoplasmic umbraviral ribonucleoprotein particles that are themselves required for the long-distance spread of the virus and systemic infection. Here, using atomic force microscopy, we determine the architecture of these complexes as single-layered ringlike structures with a diameter of 18-22 nm and a height of 2.0+/-0.4 nm, which consist of several (n=6-8) distinct protein granules. We also estimate the molar ratio of fibrillarin to ORF3 protein in the complexes as approximately 1:1. Based on these data, we propose a model of the structural organization of fibrillarin-ORF3 protein complexes and discuss potential mechanistic and functional implications that may also apply to other viruses.

  14. Complex regulatory network encompassing the Csr, c-di-GMP and motility systems of Salmonella Typhimurium.

    Science.gov (United States)

    Jonas, Kristina; Edwards, Adrianne N; Ahmad, Irfan; Romeo, Tony; Römling, Ute; Melefors, Ojar

    2010-02-01

    Bacterial survival depends on the ability to switch between sessile and motile lifestyles in response to changing environmental conditions. In many species, this switch is governed by (3'-5')-cyclic-diguanosine monophosphate (c-di-GMP), a signalling molecule, which is metabolized by proteins containing GGDEF and/or EAL domains. Salmonella Typhimurium contains 20 such proteins. Here, we show that the RNA-binding protein CsrA regulates the expression of eight genes encoding GGDEF, GGDEF-EAL and EAL domain proteins. CsrA bound directly to the mRNA leaders of five of these genes, suggesting that it may regulate these genes post-transcriptionally. The c-di-GMP-specific phosphodiesterase STM3611, which reciprocally controls flagella function and production of biofilm matrix components, was regulated by CsrA binding to the mRNA, but was also indirectly regulated by CsrA through the FlhDC/FliA flagella cascade and STM1344. STM1344 is an unconventional (c-di-GMP-inactive) EAL domain protein, recently identified as a negative regulator of flagella gene expression. Here, we demonstrate that CsrA directly downregulates expression of STM1344, which in turn regulates STM3611 through fliA and thus reciprocally controls motility and biofilm factors. Altogether, our data reveal that the concerted and complex regulation of several genes encoding GGDEF/EAL domain proteins allows CsrA to control the motility-sessility switch in S. Typhimurium at multiple levels.

  15. Improving the extraction of complex regulatory events from scientific text by using ontology-based inference.

    Science.gov (United States)

    Kim, Jung-Jae; Rebholz-Schuhmann, Dietrich

    2011-10-06

    The extraction of complex events from biomedical text is a challenging task and requires in-depth semantic analysis. Previous approaches associate lexical and syntactic resources with ontologies for the semantic analysis, but fall short in testing the benefits from the use of domain knowledge. We developed a system that deduces implicit events from explicitly expressed events by using inference rules that encode domain knowledge. We evaluated the system with the inference module on three tasks: First, when tested against a corpus with manually annotated events, the inference module of our system contributes 53.2% of correct extractions, but does not cause any incorrect results. Second, the system overall reproduces 33.1% of the transcription regulatory events contained in RegulonDB (up to 85.0% precision) and the inference module is required for 93.8% of the reproduced events. Third, we applied the system with minimum adaptations to the identification of cell activity regulation events, confirming that the inference improves the performance of the system also on this task. Our research shows that the inference based on domain knowledge plays a significant role in extracting complex events from text. This approach has great potential in recognizing the complex concepts of such biomedical ontologies as Gene Ontology in the literature.

  16. Improving the extraction of complex regulatory events from scientific text by using ontology-based inference

    Directory of Open Access Journals (Sweden)

    Kim Jung-jae

    2011-10-01

    Full Text Available Abstract Background The extraction of complex events from biomedical text is a challenging task and requires in-depth semantic analysis. Previous approaches associate lexical and syntactic resources with ontologies for the semantic analysis, but fall short in testing the benefits from the use of domain knowledge. Results We developed a system that deduces implicit events from explicitly expressed events by using inference rules that encode domain knowledge. We evaluated the system with the inference module on three tasks: First, when tested against a corpus with manually annotated events, the inference module of our system contributes 53.2% of correct extractions, but does not cause any incorrect results. Second, the system overall reproduces 33.1% of the transcription regulatory events contained in RegulonDB (up to 85.0% precision and the inference module is required for 93.8% of the reproduced events. Third, we applied the system with minimum adaptations to the identification of cell activity regulation events, confirming that the inference improves the performance of the system also on this task. Conclusions Our research shows that the inference based on domain knowledge plays a significant role in extracting complex events from text. This approach has great potential in recognizing the complex concepts of such biomedical ontologies as Gene Ontology in the literature.

  17. WAVE regulatory complex activation by cooperating GTPases Arf and Rac1

    DEFF Research Database (Denmark)

    Koronakis, Vassilis; Hume, Peter J; Humphreys, Daniel

    2011-01-01

    The WAVE regulatory complex (WRC) is a critical element in the control of actin polymerization at the eukaryotic cell membrane, but how WRC is activated remains uncertain. While Rho GTPase Rac1 can bind and activate WRC in vitro, this interaction is of low affinity, suggesting other factors may...... be important. By reconstituting WAVE-dependent actin assembly on membrane-coated beads in mammalian cell extracts, we found that Rac1 was not sufficient to engender bead motility, and we uncovered a key requirement for Arf GTPases. In vitro, Rac1 and Arf1 were individually able to bind weakly to recombinant...... be central components in WAVE signalling, acting directly, alongside Rac1....

  18. Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

    Science.gov (United States)

    Rim, Jong S; Kozak, Leslie P

    2002-09-13

    Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

  19. Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes

    Directory of Open Access Journals (Sweden)

    Bunai Christine L

    2009-02-01

    Full Text Available Abstract Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr, an alkaline solution (180 mM Na2CO3, pH 11.3 and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14. Separation of proteins by 2D gel electrophoresis was followed by identification of more than 600 gene products by MS. Data from differential 2D gel display experiments, comparing protein abundances in cytoplasmic, periplasmic and all three membrane fractions, were used to assign proteins found in the membrane fractions to three protein categories: (i integral membrane proteins and peripheral membrane proteins with low solubility in aqueous solutions (220 entries; (ii peripheral membrane proteins with moderate to high solubility in aqueous solutions (127 entries; (iii cytoplasmic or ribosomal membrane-contaminating proteins (80 entries. Thirty-one proteins were experimentally associated with the outer membrane (OM. Circa 50 proteins thought to be part of membrane-localized, multi-subunit complexes were identified in high Mr fractions of membrane extracts via size exclusion chromatography. This data supported biologically meaningful assignments of many proteins to the membrane periphery. Since only 32 inner membrane (IM proteins with two or more predicted transmembrane domains (TMDs were profiled in 2D gels, we resorted to a proteomic analysis by 2D-LC-MS/MS. Ninety-four additional IM proteins with two or more TMDs were identified. The total number of proteins associated with Y. pestis membranes increased to 456 and included representatives of all six β-barrel OM protein families and 25 distinct IM transporter families.

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

    Directory of Open Access Journals (Sweden)

    Lisa M. Tuttle

    2018-03-01

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

  1. Immersion freezing of ice nucleation active protein complexes

    Directory of Open Access Journals (Sweden)

    S. Hartmann

    2013-06-01

    Full Text Available Utilising the Leipzig Aerosol Cloud Interaction Simulator (LACIS, the immersion freezing behaviour of droplet ensembles containing monodisperse particles, generated from a Snomax™ solution/suspension, was investigated. Thereto ice fractions were measured in the temperature range between −5 °C to −38 °C. Snomax™ is an industrial product applied for artificial snow production and contains Pseudomonas syringae} bacteria which have long been used as model organism for atmospheric relevant ice nucleation active (INA bacteria. The ice nucleation activity of such bacteria is controlled by INA protein complexes in their outer membrane. In our experiments, ice fractions increased steeply in the temperature range from about −6 °C to about −10 °C and then levelled off at ice fractions smaller than one. The plateau implies that not all examined droplets contained an INA protein complex. Assuming the INA protein complexes to be Poisson distributed over the investigated droplet populations, we developed the CHESS model (stoCHastic modEl of similar and poiSSon distributed ice nuclei which allows for the calculation of ice fractions as function of temperature and time for a given nucleation rate. Matching calculated and measured ice fractions, we determined and parameterised the nucleation rate of INA protein complexes exhibiting class III ice nucleation behaviour. Utilising the CHESS model, together with the determined nucleation rate, we compared predictions from the model to experimental data from the literature and found good agreement. We found that (a the heterogeneous ice nucleation rate expression quantifying the ice nucleation behaviour of the INA protein complex is capable of describing the ice nucleation behaviour observed in various experiments for both, Snomax™ and P. syringae bacteria, (b the ice nucleation rate, and its temperature dependence, seem to be very similar regardless of whether the INA protein complexes inducing ice

  2. The complex between SOS3 and SOS2 regulatory domain from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Barrena, María José; Moreno-Pérez, Sandra; Angulo, Iván; Martínez-Ripoll, Martín; Albert, Armando, E-mail: xalbert@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química Física ‘Rocasolano’, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid (Spain)

    2007-07-01

    Recombinant SOS3 and SOS2 regulatory domain from A. thaliana have been coexpressed in E. coli, purified and crystallized by the hanging-drop vapour-diffusion method. An X-ray data set has been collected at 2.0 Å resolution. The salt-tolerance genes SOS3 (salt overly sensitive 3) and SOS2 (salt overly sensitive 2) regulatory domain of Arabidopsis thaliana were cloned into a polycistronic plasmid and the protein complex was expressed in Escherichia coli, allowing purification to homogeneity in three chromatographic steps. Crystals were grown using vapour-diffusion techniques. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 44.14, b = 57.39, c = 141.90 Å.

  3. Supercharging Protein Complexes from Aqueous Solution Disrupts their Native Conformations

    Science.gov (United States)

    Sterling, Harry J.; Kintzer, Alexander F.; Feld, Geoffrey K.; Cassou, Catherine A.; Krantz, Bryan A.; Williams, Evan R.

    2012-02-01

    The effects of aqueous solution supercharging on the solution- and gas-phase structures of two protein complexes were investigated using traveling-wave ion mobility-mass spectrometry (TWIMS-MS). Low initial concentrations of m-nitrobenzyl alcohol ( m-NBA) in the electrospray ionization (ESI) solution can effectively increase the charge of concanavalin A dimers and tetramers, but at higher m-NBA concentrations, the increases in charge are accompanied by solution-phase dissociation of the dimers and up to a ~22% increase in the collision cross section (CCS) of the tetramers. With just 0.8% m-NBA added to the ESI solution of a ~630 kDa anthrax toxin octamer complex, the average charge is increased by only ~4% compared with the "native" complex, but it is sufficiently destabilized so that extensive gas-phase fragmentation occurs in the relatively high pressure regions of the TWIMS device. Anthrax toxin complexes exist in either a prechannel or a transmembrane channel state. With m-NBA, the prechannel state of the complex has the same CCS/charge ratio in the gas phase as the transmembrane channel state of the same complex formed without m-NBA, yet undergoes extensive dissociation, indicating that destabilization from supercharging occurs in the ESI droplet prior to ion formation and is not a result of Coulombic destabilization in the gas phase as a result of higher charging. These results demonstrate that the supercharging of large protein complexes is the result of conformational changes induced by the reagents in the ESI droplets, where enrichment of the supercharging reagent during droplet evaporation occurs.

  4. Crystallization of bi-functional ligand protein complexes.

    Science.gov (United States)

    Antoni, Claudia; Vera, Laura; Devel, Laurent; Catalani, Maria Pia; Czarny, Bertrand; Cassar-Lajeunesse, Evelyn; Nuti, Elisa; Rossello, Armando; Dive, Vincent; Stura, Enrico Adriano

    2013-06-01

    Homodimerization is important in signal transduction and can play a crucial role in many other biological systems. To obtaining structural information for the design of molecules able to control the signalization pathways, the proteins involved will have to be crystallized in complex with ligands that induce dimerization. Bi-functional drugs have been generated by linking two ligands together chemically and the relative crystallizability of complexes with mono-functional and bi-functional ligands has been evaluated. There are problems associated with crystallization with such ligands, but overall, the advantages appear to be greater than the drawbacks. The study involves two matrix metalloproteinases, MMP-12 and MMP-9. Using flexible and rigid linkers we show that it is possible to control the crystal packing and that by changing the ligand-enzyme stoichiometric ratio, one can toggle between having one bi-functional ligand binding to two enzymes and having the same ligand bound to each enzyme. The nature of linker and its point of attachment on the ligand can be varied to aid crystallization, and such variations can also provide valuable structural information about the interactions made by the linker with the protein. We report here the crystallization and structure determination of seven ligand-dimerized complexes. These results suggest that the use of bi-functional drugs can be extended beyond the realm of protein dimerization to include all drug design projects. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Characterization of known protein complexes using k-connectivity and other topological measures

    Science.gov (United States)

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  6. Radiation damage to DNA in DNA-protein complexes.

    Science.gov (United States)

    Spotheim-Maurizot, M; Davídková, M

    2011-06-03

    The most aggressive product of water radiolysis, the hydroxyl (OH) radical, is responsible for the indirect effect of ionizing radiations on DNA in solution and aerobic conditions. According to radiolytic footprinting experiments, the resulting strand breaks and base modifications are inhomogeneously distributed along the DNA molecule irradiated free or bound to ligands (polyamines, thiols, proteins). A Monte-Carlo based model of simulation of the reaction of OH radicals with the macromolecules, called RADACK, allows calculating the relative probability of damage of each nucleotide of DNA irradiated alone or in complexes with proteins. RADACK calculations require the knowledge of the three dimensional structure of DNA and its complexes (determined by X-ray crystallography, NMR spectroscopy or molecular modeling). The confrontation of the calculated values with the results of the radiolytic footprinting experiments together with molecular modeling calculations show that: (1) the extent and location of the lesions are strongly dependent on the structure of DNA, which in turns is modulated by the base sequence and by the binding of proteins and (2) the regions in contact with the protein can be protected against the attack by the hydroxyl radicals via masking of the binding site and by scavenging of the radicals. 2011 Elsevier B.V. All rights reserved.

  7. CISAPS: Complex Informational Spectrum for the Analysis of Protein Sequences

    Directory of Open Access Journals (Sweden)

    Charalambos Chrysostomou

    2015-01-01

    Full Text Available Complex informational spectrum analysis for protein sequences (CISAPS and its web-based server are developed and presented. As recent studies show, only the use of the absolute spectrum in the analysis of protein sequences using the informational spectrum analysis is proven to be insufficient. Therefore, CISAPS is developed to consider and provide results in three forms including absolute, real, and imaginary spectrum. Biologically related features to the analysis of influenza A subtypes as presented as a case study in this study can also appear individually either in the real or imaginary spectrum. As the results presented, protein classes can present similarities or differences according to the features extracted from CISAPS web server. These associations are probable to be related with the protein feature that the specific amino acid index represents. In addition, various technical issues such as zero-padding and windowing that may affect the analysis are also addressed. CISAPS uses an expanded list of 611 unique amino acid indices where each one represents a different property to perform the analysis. This web-based server enables researchers with little knowledge of signal processing methods to apply and include complex informational spectrum analysis to their work.

  8. Interaction of the anaphase-promoting complex/cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins

    DEFF Research Database (Denmark)

    Seeger, Michael; Hartmann-Petersen, Rasmus; Wilkinson, Caroline R M

    2003-01-01

    Fission yeast Rhp23 and Pus1 represent two families of multiubiquitin chain-binding proteins that associate with the proteasome. We show that both proteins bind to different regions of the proteasome subunit Mts4. The binding site for Pus1 was mapped to a cluster of repetitive sequences also found...... in the proteasome subunit SpRpn2 and the anaphase-promoting complex/cyclosome (APC/C) subunit Cut4. The putative role of Pus1 as a factor involved in allocation of ubiquitinylated substrates for the proteasome is discussed....

  9. Taking advantage of local structure descriptors to analyze interresidue contacts in protein structures and protein complexes.

    Science.gov (United States)

    Martin, Juliette; Regad, Leslie; Etchebest, Catherine; Camproux, Anne-Claude

    2008-11-15

    Interresidue protein contacts in proteins structures and at protein-protein interface are classically described by the amino acid types of interacting residues and the local structural context of the contact, if any, is described using secondary structures. In this study, we present an alternate analysis of interresidue contact using local structures defined by the structural alphabet introduced by Camproux et al. This structural alphabet allows to describe a 3D structure as a sequence of prototype fragments called structural letters, of 27 different types. Each residue can then be assigned to a particular local structure, even in loop regions. The analysis of interresidue contacts within protein structures defined using Voronoï tessellations reveals that pairwise contact specificity is greater in terms of structural letters than amino acids. Using a simple heuristic based on specificity score comparison, we find that 74% of the long-range contacts within protein structures are better described using structural letters than amino acid types. The investigation is extended to a set of protein-protein complexes, showing that the similar global rules apply as for intraprotein contacts, with 64% of the interprotein contacts best described by local structures. We then present an evaluation of pairing functions integrating structural letters to decoy scoring and show that some complexes could benefit from the use of structural letter-based pairing functions.

  10. Radioprotection by polyethylene glycol-protein complexes in mice

    International Nuclear Information System (INIS)

    Gray, B.H.; Stull, R.W.

    1983-01-01

    Polyethylene glycol of about 5000 D was activated with cyanuric chloride, and the activated compound was complexed to each of three proteins. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase were each radioprotectants when administered prophylactically to female B6CBF1 mice before irradiation. The dose reduction factor for these mice was 1.2 when 5000 units of polyethylene glycol-catalase was administered before 60 Co irradiation. Female B6CBF1 mice administered prophylactic intravenous injections of catalase, polyethylene glycol-albumin, or heat-denatured polyethylene glycol-catalase had survival rates similar to phosphate-buffered saline-injected control mice following 60 Co irradiation. Polyethylene glycol-superoxide dismutase and polyethylene glycol-catalase have radioprotective activity in B6CBF1 mice, which appears to depend in part on enzymatic activities of the complex. However, no radioprotective effect was observed in male C57BL/6 mice injected with each polyethylene glycol-protein complex at either 3 or 24 hr before irradiation. The mechanism for radioprotection by these complexes may depend in part on other factors

  11. Protein chimerism: novel source of protein diversity in humans adds complexity to bottom-up proteomics.

    Science.gov (United States)

    Casado-Vela, Juan; Lacal, Juan Carlos; Elortza, Felix

    2013-01-01

    Three main molecular mechanisms are considered to contribute expanding the repertoire and diversity of proteins present in living organisms: first, at DNA level (gene polymorphisms and single nucleotide polymorphisms); second, at messenger RNA (pre-mRNA and mRNA) level including alternative splicing (also termed differential splicing or cis-splicing); finally, at the protein level mainly driven through PTM and specific proteolytic cleavages. Chimeric mRNAs constitute an alternative source of protein diversity, which can be generated either by chromosomal translocations or by trans-splicing events. The occurrence of chimeric mRNAs and proteins is a frequent event in cells from the immune system and cancer cells, mainly as a consequence of gene rearrangements. Recent reports support that chimeric proteins may also be expressed at low levels under normal physiological circumstances, thus, representing a novel source of protein diversity. Notably, recent publications demonstrate that chimeric protein products can be successfully identified through bottom-up proteomic analyses. Several questions remain unsolved, such as the physiological role and impact of such chimeric proteins or the potential occurrence of chimeric proteins in higher eukaryotic organisms different from humans. The occurrence of chimeric proteins certainly seems to be another unforeseen source of complexity for the proteome. It may be a process to take in mind not only when performing bottom-up proteomic analyses in cancer studies but also in general bottom-up proteomics experiments. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. iTRAQ-Based Quantitative Proteomics Identifies Potential Regulatory Proteins Involved in Chicken Eggshell Brownness.

    Directory of Open Access Journals (Sweden)

    Guangqi Li

    Full Text Available Brown eggs are popular in many countries and consumers regard eggshell brownness as an important indicator of egg quality. However, the potential regulatory proteins and detailed molecular mechanisms regulating eggshell brownness have yet to be clearly defined. In the present study, we performed quantitative proteomics analysis with iTRAQ technology in the shell gland epithelium of hens laying dark and light brown eggs to investigate the candidate proteins and molecular mechanisms underlying variation in chicken eggshell brownness. The results indicated 147 differentially expressed proteins between these two groups, among which 65 and 82 proteins were significantly up-regulated in the light and dark groups, respectively. Functional analysis indicated that in the light group, the down-regulated iron-sulfur cluster assembly protein (Iba57 would decrease the synthesis of protoporphyrin IX; furthermore, the up-regulated protein solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator, member 5 (SLC25A5 and down-regulated translocator protein (TSPO would lead to increased amounts of protoporphyrin IX transported into the mitochondria matrix to form heme with iron, which is supplied by ovotransferrin protein (TF. In other words, chickens from the light group produce less protoporphyrin IX, which is mainly used for heme synthesis. Therefore, the exported protoporphyrin IX available for eggshell deposition and brownness is reduced in the light group. The current study provides valuable information to elucidate variation of chicken eggshell brownness, and demonstrates the feasibility and sensitivity of iTRAQ-based quantitative proteomics analysis in providing useful insights into the molecular mechanisms underlying brown eggshell pigmentation.

  13. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  14. Core regulatory network motif underlies the ocellar complex patterning in Drosophila melanogaster

    Science.gov (United States)

    Aguilar-Hidalgo, D.; Lemos, M. C.; Córdoba, A.

    2015-03-01

    During organogenesis, developmental programs governed by Gene Regulatory Networks (GRN) define the functionality, size and shape of the different constituents of living organisms. Robustness, thus, is an essential characteristic that GRNs need to fulfill in order to maintain viability and reproducibility in a species. In the present work we analyze the robustness of the patterning for the ocellar complex formation in Drosophila melanogaster fly. We have systematically pruned the GRN that drives the development of this visual system to obtain the minimum pathway able to satisfy this pattern. We found that the mechanism underlying the patterning obeys to the dynamics of a 3-nodes network motif with a double negative feedback loop fed by a morphogenetic gradient that triggers the inhibition in a French flag problem fashion. A Boolean modeling of the GRN confirms robustness in the patterning mechanism showing the same result for different network complexity levels. Interestingly, the network provides a steady state solution in the interocellar part of the patterning and an oscillatory regime in the ocelli. This theoretical result predicts that the ocellar pattern may underlie oscillatory dynamics in its genetic regulation.

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

    Science.gov (United States)

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

    2018-03-20

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

  16. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the α subunit of G i and other G proteins in solution. However, the occurrence of the phosphorylation of G 1 within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the α subunits of G i undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with [γ 32 P]ATP and [ 32 P]H 3 PO 4 , respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G iα -despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G zα , or antibodies for both G zα and G iα , precipitated a 40-kDa phosphoprotein

  17. Improved functional overview of protein complexes using inferred epistatic relationships

    LENUS (Irish Health Repository)

    Ryan, Colm

    2011-05-23

    Abstract Background Epistatic Miniarray Profiling(E-MAP) quantifies the net effect on growth rate of disrupting pairs of genes, often producing phenotypes that may be more (negative epistasis) or less (positive epistasis) severe than the phenotype predicted based on single gene disruptions. Epistatic interactions are important for understanding cell biology because they define relationships between individual genes, and between sets of genes involved in biochemical pathways and protein complexes. Each E-MAP screen quantifies the interactions between a logically selected subset of genes (e.g. genes whose products share a common function). Interactions that occur between genes involved in different cellular processes are not as frequently measured, yet these interactions are important for providing an overview of cellular organization. Results We introduce a method for combining overlapping E-MAP screens and inferring new interactions between them. We use this method to infer with high confidence 2,240 new strongly epistatic interactions and 34,469 weakly epistatic or neutral interactions. We show that accuracy of the predicted interactions approaches that of replicate experiments and that, like measured interactions, they are enriched for features such as shared biochemical pathways and knockout phenotypes. We constructed an expanded epistasis map for yeast cell protein complexes and show that our new interactions increase the evidence for previously proposed inter-complex connections, and predict many new links. We validated a number of these in the laboratory, including new interactions linking the SWR-C chromatin modifying complex and the nuclear transport apparatus. Conclusion Overall, our data support a modular model of yeast cell protein network organization and show how prediction methods can considerably extend the information that can be extracted from overlapping E-MAP screens.

  18. Biodegradation of the chitin-protein complex in crustacean cuticle

    Science.gov (United States)

    Artur, Stankiewicz B.; Mastalerz, Maria; Hof, C.H.J.; Bierstedt, A.; Flannery, M.B.; Briggs, D.E.G.; Evershed, R.P.

    1998-01-01

    Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative analysis of amino acids (by HPLC) and chitin showed that the major loss of proteins and chitin occurred between weeks 1 and 2. After 8 weeks tyrosine, tryptophan and valine are the most prominent amino acid moieties, showing their resistance to degradation. The presence of cyclic ketones in the pyrolysates indicates that mucopolysaccharides or other bound non-chitinous carbohydrates are also resistant to decay. There is no evidence of structural degradation of chitin prior to 8 weeks when FTIR revealed a reduction in chitin-specific bands. The chemical changes are paralleled by structural changes in the cuticle, which becomes an increasingly open structure consisting of loose chitinous fibres. The rapid rate of decay in the experiments suggests that where chitin and protein are preserved in fossil cuticles degradation must have been inhibited.Arthropod cuticles consist predominantly of chitin cross-linked with proteins. While there is some experimental evidence that this chitin-protein complex may resist decay, the chemical changes that occur during degradation have not been investigated in detail. The stomatopod crustacean Neogonodactylus oerstedii was decayed in the laboratory under anoxic conditions. A combination of pyrolysis-gas chromatography/mass spectrometry and FTIR revealed extensive chemical changes after just 2 weeks that resulted in a cuticle composition dominated by chitin. Quantitative

  19. Nuclear pore complex protein mediated nuclear localization of dicer protein in human cells.

    Directory of Open Access Journals (Sweden)

    Yoshinari Ando

    Full Text Available Human DICER1 protein cleaves double-stranded RNA into small sizes, a crucial step in production of single-stranded RNAs which are mediating factors of cytoplasmic RNA interference. Here, we clearly demonstrate that human DICER1 protein localizes not only to the cytoplasm but also to the nucleoplasm. We also find that human DICER1 protein associates with the NUP153 protein, one component of the nuclear pore complex. This association is detected predominantly in the cytoplasm but is also clearly distinguishable at the nuclear periphery. Additional characterization of the NUP153-DICER1 association suggests NUP153 plays a crucial role in the nuclear localization of the DICER1 protein.

  20. Model of a DNA-protein complex of the architectural monomeric protein MC1 from Euryarchaea.

    Directory of Open Access Journals (Sweden)

    Françoise Paquet

    Full Text Available In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1 from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.

  1. Phenotypic complexity of T regulatory subsets in patients with B-chronic lymphocytic leukemia.

    Science.gov (United States)

    Biancotto, Angélique; Dagur, Pradeep K; Fuchs, John C; Wiestner, Adrian; Bagwell, C Bruce; McCoy, J Philip

    2012-02-01

    Increased numbers of T regulatory (T(reg)) cells are found in B-chronic lymphocytic leukemia, but the nature and function of these T(regs) remains unclear. Detailed characterization of the T(regs) in chronic lymphocytic leukemia has not been performed and the degree of heterogeneity of among these cells has not been studied to date. Using 15-color flow cytometry we show that T(reg) cells, defined using CD4, CD25, and forkhead box P3 (FOXP3), can be divided into multiple complex subsets based on markers used for naïve, memory, and effector delineation as well as markers of T(reg) activation. Furthermore FOXP3(+) cells can be identified among CD4(+)CD25(-) as well as CD8(+)CD4(-) populations in increased proportions in patients with chronic lymphocytic leukemia compared with healthy donors. Significantly different frequencies of naïve and effector T(regs) populations are found in healthy donor controls compared with donors with chronic lymphocytic leukemia. A population of CCR7(+)CD39(+) T(regs) was significantly associated with chronic lymphocytic leukemia. This population demonstrated slightly reduced suppressive activity compared with total T(regs) or T(regs) of healthy donors. These data suggest that FOXP3-expressing cells, particularly in patients with chronic lymphocytic leukemia are much more complex for T(reg) sub-populations and transitions than previously reported. These findings demonstrate the complexity of regulation of T-cell responses in chronic lymphocytic leukemia and illustrate the use of high-dimensional analysis of cellular phenotypes in facilitating understanding of the intricacies of cellular immune responses and their dysregulation in cancer.

  2. High-resolution diffraction from crystals of a membrane-protein complex: bacterial outer membrane protein OmpC complexed with the antibacterial eukaryotic protein lactoferrin

    International Nuclear Information System (INIS)

    Sundara Baalaji, N.; Acharya, K. Ravi; Singh, T. P.; Krishnaswamy, S.

    2005-01-01

    Crystals of the complex formed between the bacterial membrane protein OmpC and the antibacterial protein lactoferrin suitable for high-resolution structure determination have been obtained. The crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å. Crystals of the complex formed between the outer membrane protein OmpC from Escherichia coli and the eukaryotic antibacterial protein lactoferrin from Camelus dromedarius (camel) have been obtained using a detergent environment. Initial data processing suggests that the crystals belong to the hexagonal space group P6, with unit-cell parameters a = b = 116.3, c = 152.4 Å, α = β = 90, γ = 120°. This indicated a Matthews coefficient (V M ) of 3.3 Å 3 Da −1 , corresponding to a possible molecular complex involving four molecules of lactoferrin and two porin trimers in the unit cell (4832 amino acids; 533.8 kDa) with 63% solvent content. A complete set of diffraction data was collected to 3 Å resolution at 100 K. Structure determination by molecular replacement is in progress. Structural study of this first surface-exposed membrane-protein complex with an antibacterial protein will provide insights into the mechanism of action of OmpC as well as lactoferrin

  3. Learning by Preparing to Teach: Fostering Self-Regulatory Processes and Achievement during Complex Mathematics Problem Solving

    Science.gov (United States)

    Muis, Krista R.; Psaradellis, Cynthia; Chevrier, Marianne; Di Leo, Ivana; Lajoie, Susanne P.

    2016-01-01

    We developed an intervention based on the learning by teaching paradigm to foster self-regulatory processes and better learning outcomes during complex mathematics problem solving in a technology-rich learning environment. Seventy-eight elementary students were randomly assigned to 1 of 2 conditions: learning by preparing to teach, or learning for…

  4. Nuclear Magnetic Resonance structural studies of peptides and proteins from the vaso-regulatory System

    International Nuclear Information System (INIS)

    Sizun, Philippe

    1991-01-01

    The aim of the present work is to show how Nuclear Magnetic Resonance (NMR) allows to determine the 3D structure of peptides and proteins in solution. A comparative study of peptides involved in the vaso-regulatory System (form small hormonal peptide to the 65 amido-acid protein hirudin) has allowed to design most efficient NMR 1D and 2D strategies. It rapidly appeared that the size of the peptide plays a key role in the structuration of the molecule, smallest peptides being weakly structured owing to the lack of cooperative effects. As the molecular size increases or if conformational locks are present (disulfide bridges) the probability of stable secondary structure increases. For the protein hirudin, a combination of ail available NMR parameters deduced form dedicated experiments (chemical shifts, coupling constants, overhauser effects, accessibility of amide protons) and molecular modelling under constraints allows a clear 3D structure to be proposed for this protein in solution. Finally, a comparative study of the experimental structures and of those deduced form prediction rules has shed light on the concept of structural predisposition, the latter being of high value for a better understanding of structure-activity relationships. (author) [fr

  5. Structural basis for regulation of rhizobial nodulation and symbiosis gene expression by the regulatory protein NolR.

    Science.gov (United States)

    Lee, Soon Goo; Krishnan, Hari B; Jez, Joseph M

    2014-04-29

    The symbiosis between rhizobial microbes and host plants involves the coordinated expression of multiple genes, which leads to nodule formation and nitrogen fixation. As part of the transcriptional machinery for nodulation and symbiosis across a range of Rhizobium, NolR serves as a global regulatory protein. Here, we present the X-ray crystal structures of NolR in the unliganded form and complexed with two different 22-base pair (bp) double-stranded operator sequences (oligos AT and AA). Structural and biochemical analysis of NolR reveals protein-DNA interactions with an asymmetric operator site and defines a mechanism for conformational switching of a key residue (Gln56) to accommodate variation in target DNA sequences from diverse rhizobial genes for nodulation and symbiosis. This conformational switching alters the energetic contributions to DNA binding without changes in affinity for the target sequence. Two possible models for the role of NolR in the regulation of different nodulation and symbiosis genes are proposed. To our knowledge, these studies provide the first structural insight on the regulation of genes involved in the agriculturally and ecologically important symbiosis of microbes and plants that leads to nodule formation and nitrogen fixation.

  6. Defects in tor regulatory complexes retard aging and carbonyl/oxidative stress development in yeast Sассharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    B. V. Homza

    2014-02-01

    Full Text Available TOR signaling pathway first described in yeast S. сerevisiae is the highly conserved regulator of eukaryotic cell growth, aging and stress resistance. The effect of nitrogen sources, in particular amino acids, on the activity of TOR signaling pathway is well studied, however its relation to carbohydrates is poor understood. The aim of the present study is expanding of our understanding of potential role of TOR regulatory complexes in development of carbonyl/oxidative stress that can result from yeast cultivation on glucose and fructose. It has been shown that the level of α-dicarbonyl compounds and protein carbonyl groups increased with time of yeast cultivation and was higher in cells grown on fructose that demonstrated their accelerated aging and carbonyl/oxidative stress development as compared with cells grown on glucose. The strains defective in TOR proteins cultivated in the presence of glucose as well as fructose demonstrated lower markers of the stress and aging than parental strain. Thus these data confirmed the previous conclusion on fructose more potent ability to cause carbonyl/oxidative stress and accelerated aging in S. cerevisiae as compared with glucose. However, defects in TOR regulatory complexes retard aging and development of the stress in yeast independent on the type of carbohydrate in the cultivation medium.

  7. Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

    Science.gov (United States)

    Sudhir, Putty-Reddy; Chen, Chung-Hsuan

    2016-03-22

    A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.

  8. Protein dynamics during presynaptic complex assembly on individual ssDNA molecules

    Science.gov (United States)

    Gibb, Bryan; Ye, Ling F.; Kwon, YoungHo; Niu, Hengyao; Sung, Patrick; Greene, Eric C.

    2014-01-01

    Homologous recombination is a conserved pathway for repairing double–stranded breaks, which are processed to yield single–stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single–molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Together, our work illustrates the spatial and temporal progression of RPA and Rad52 association with the presynaptic complex, and reveals a novel RPA–Rad52–Rad51–ssDNA intermediate, which has implications for understanding how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages recombination. PMID:25195049

  9. Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort.

    Directory of Open Access Journals (Sweden)

    Jane E Salmon

    2011-03-01

    Full Text Available Pregnancy in women with systemic lupus erythematosus (SLE or antiphospholipid antibodies (APL Ab--autoimmune conditions characterized by complement-mediated injury--is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins--membrane cofactor protein (MCP, complement factor I (CFI, and complement factor H (CFH--in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%. Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.ClinicalTrials.gov NCT00198068.

  10. Protein: FBA2 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA2 19S regulatory particles(RP) Rpn11 yip5 26S proteasome non-ATPase regulatory subunit 14 26S pr...oteasome regulatory complex subunit p37B, 26S proteasome regulatory subunit rpn11, Yippee-interacting protein 5 7227 Drosophila melanogaster Q9V3H2 Q9V3H2 19075009 ...

  11. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    International Nuclear Information System (INIS)

    Nielsen, Anders Lade

    2009-01-01

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  12. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, Anders Lade, E-mail: aln@humgen.au.dk [Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C (Denmark)

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  13. WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System.

    Science.gov (United States)

    Herrou, Julien; Czyż, Daniel M; Willett, Jonathan W; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean

    2016-04-01

    The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family. Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes

  14. DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

    OpenAIRE

    Kristie, T M; Roizman, B

    1986-01-01

    Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

  15. Understanding Epistatic Interactions between Genes Targeted by Non-coding Regulatory Elements in Complex Diseases

    Directory of Open Access Journals (Sweden)

    Min Kyung Sung

    2014-12-01

    Full Text Available Genome-wide association studies have proven the highly polygenic architecture of complex diseases or traits; therefore, single-locus-based methods are usually unable to detect all involved loci, especially when individual loci exert small effects. Moreover, the majority of associated single-nucleotide polymorphisms resides in non-coding regions, making it difficult to understand their phenotypic contribution. In this work, we studied epistatic interactions associated with three common diseases using Korea Association Resource (KARE data: type 2 diabetes mellitus (DM, hypertension (HT, and coronary artery disease (CAD. We showed that epistatic single-nucleotide polymorphisms (SNPs were enriched in enhancers, as well as in DNase I footprints (the Encyclopedia of DNA Elements [ENCODE] Project Consortium 2012, which suggested that the disruption of the regulatory regions where transcription factors bind may be involved in the disease mechanism. Accordingly, to identify the genes affected by the SNPs, we employed whole-genome multiple-cell-type enhancer data which discovered using DNase I profiles and Cap Analysis Gene Expression (CAGE. Assigned genes were significantly enriched in known disease associated gene sets, which were explored based on the literature, suggesting that this approach is useful for detecting relevant affected genes. In our knowledge-based epistatic network, the three diseases share many associated genes and are also closely related with each other through many epistatic interactions. These findings elucidate the genetic basis of the close relationship between DM, HT, and CAD.

  16. Modulation of Correlated Segment Fluctuations in IDPs upon Complex Formation as an Allosteric Regulatory Mechanism.

    Science.gov (United States)

    Beier, Andreas; Schwarz, Thomas C; Kurzbach, Dennis; Platzer, Gerald; Tribuzio, Francesca; Konrat, Robert

    2018-05-05

    Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of "fuzzy complexes." Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of "energetic frustration" and provides an explanation for the puzzling observation of disordered allostery in IDPs. Copyright © 2018. Published by Elsevier Ltd.

  17. Design principles for cancer therapy guided by changes in complexity of protein-protein interaction networks.

    Science.gov (United States)

    Benzekry, Sebastian; Tuszynski, Jack A; Rietman, Edward A; Lakka Klement, Giannoula

    2015-05-28

    The ever-increasing expanse of online bioinformatics data is enabling new ways to, not only explore the visualization of these data, but also to apply novel mathematical methods to extract meaningful information for clinically relevant analysis of pathways and treatment decisions. One of the methods used for computing topological characteristics of a space at different spatial resolutions is persistent homology. This concept can also be applied to network theory, and more specifically to protein-protein interaction networks, where the number of rings in an individual cancer network represents a measure of complexity. We observed a linear correlation of R = -0.55 between persistent homology and 5-year survival of patients with a variety of cancers. This relationship was used to predict the proteins within a protein-protein interaction network with the most impact on cancer progression. By re-computing the persistent homology after computationally removing an individual node (protein) from the protein-protein interaction network, we were able to evaluate whether such an inhibition would lead to improvement in patient survival. The power of this approach lied in its ability to identify the effects of inhibition of multiple proteins and in the ability to expose whether the effect of a single inhibition may be amplified by inhibition of other proteins. More importantly, we illustrate specific examples of persistent homology calculations, which correctly predict the survival benefit observed effects in clinical trials using inhibitors of the identified molecular target. We propose that computational approaches such as persistent homology may be used in the future for selection of molecular therapies in clinic. The technique uses a mathematical algorithm to evaluate the node (protein) whose inhibition has the highest potential to reduce network complexity. The greater the drop in persistent homology, the greater reduction in network complexity, and thus a larger

  18. Simulating evolution of protein complexes through gene duplication and co-option.

    Science.gov (United States)

    Haarsma, Loren; Nelesen, Serita; VanAndel, Ethan; Lamine, James; VandeHaar, Peter

    2016-06-21

    We present a model of the evolution of protein complexes with novel functions through gene duplication, mutation, and co-option. Under a wide variety of input parameters, digital organisms evolve complexes of 2-5 bound proteins which have novel functions but whose component proteins are not independently functional. Evolution of complexes with novel functions happens more quickly as gene duplication rates increase, point mutation rates increase, protein complex functional probability increases, protein complex functional strength increases, and protein family size decreases. Evolution of complexity is inhibited when the metabolic costs of making proteins exceeds the fitness gain of having functional proteins, or when point mutation rates get so large the functional proteins undergo deleterious mutations faster than new functional complexes can evolve. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

    Directory of Open Access Journals (Sweden)

    Grassmann Ralph

    2005-09-01

    Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

  20. A complex regulatory network controls aerobic ethanol oxidation in Pseudomonas aeruginosa: indication of four levels of sensor kinases and response regulators.

    Science.gov (United States)

    Mern, Demissew S; Ha, Seung-Wook; Khodaverdi, Viola; Gliese, Nicole; Görisch, Helmut

    2010-05-01

    In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS', and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS' do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS', resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.

  1. DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-08-19

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

  2. DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

    Science.gov (United States)

    Keyamura, Kenji; Katayama, Tsutomu

    2011-01-01

    Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

  3. Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA.

    Science.gov (United States)

    Baxter, Jamie C; Sutton, Mark D

    2012-08-01

    The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable. © 2012 Blackwell Publishing Ltd.

  4. Systematic comparison of the response properties of protein and RNA mediated gene regulatory motifs.

    Science.gov (United States)

    Iyengar, Bharat Ravi; Pillai, Beena; Venkatesh, K V; Gadgil, Chetan J

    2017-05-30

    We present a framework enabling the dissection of the effects of motif structure (feedback or feedforward), the nature of the controller (RNA or protein), and the regulation mode (transcriptional, post-transcriptional or translational) on the response to a step change in the input. We have used a common model framework for gene expression where both motif structures have an activating input and repressing regulator, with the same set of parameters, to enable a comparison of the responses. We studied the global sensitivity of the system properties, such as steady-state gain, overshoot, peak time, and peak duration, to parameters. We find that, in all motifs, overshoot correlated negatively whereas peak duration varied concavely with peak time. Differences in the other system properties were found to be mainly dependent on the nature of the controller rather than the motif structure. Protein mediated motifs showed a higher degree of adaptation i.e. a tendency to return to baseline levels; in particular, feedforward motifs exhibited perfect adaptation. RNA mediated motifs had a mild regulatory effect; they also exhibited a lower peaking tendency and mean overshoot. Protein mediated feedforward motifs showed higher overshoot and lower peak time compared to the corresponding feedback motifs.

  5. Expression of the cytoskeleton regulatory protein Mena in human gastric carcinoma and its prognostic significance.

    Science.gov (United States)

    Xu, Lihua; Tan, Huo; Liu, Ruiming; Huang, Qungai; Zhang, Nana; Li, Xi; Wang, Jiani

    2017-11-01

    The cytoskeleton regulatory protein Mena is reportedly overexpressed in breast cancer; however, data regarding its expression level and clinical significance in gastric carcinoma (GC) is limited. The aim of the present study was to investigate Mena expression levels and prognostic significance in GC. Mena mRNA expression level was determined by reverse transcription-quantitative polymerase chain reaction in 10 paired GC and adjacent normal tissues. The Mena protein expression level was analyzed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. Statistical analyses were also performed to evaluate the clinicopathological significance of Mena. The results revealed that the mRNA expression level of Mena was significantly higher in G Ct issues compared with in adjacent normal tissues from10 paired samples. In the paraffin-embedded tissue samples, the protein expression level of Mena was higher in G Ct issues compared with in adjacent normal tissues. Compared with adjacent normal tissues, Mena overexpression was observed in 52.83% (56/106) of patients. The overexpression of Mena was significantly associated with the T stage (P=0.033), tumor-node-metastasis (TNM) stage (PMena expression level was an independent prognostic factor for overall survival time. In conclusion, Mena wasoverexpressed in G C tissues and significantly associated with the T stage, TNM stage and overall survival time. Mena may therefore be suitable as a prognostic indicator for patients with GC.

  6. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    Science.gov (United States)

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

  7. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  8. Protein associations in DnaA-ATP hydrolysis mediated by the Hda-replicase clamp complex.

    Science.gov (United States)

    Su'etsugu, Masayuki; Shimuta, Toh-Ru; Ishida, Takuma; Kawakami, Hironori; Katayama, Tsutomu

    2005-02-25

    In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner. The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta-sliding clamp, a subunit of the replicase holoenzyme. A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule. Purified mutant Hda proteins were used in a staged in vitro RIDA system followed by a pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif. Arg(168) in the AAA(+) Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis, but not in clamp binding. Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex. Single cells contain approximately 50 Hda dimers, consistent with the results of in vitro experiments. These findings and the features of AAA(+) proteins, including DnaA, suggest the following model. DnaA-ATP is hydrolyzed at a binding interface between the AAA(+) domains of DnaA and Hda; the DnaA N-terminal domain supports this interaction; and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

  9. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli

    DEFF Research Database (Denmark)

    Seo, Sang Woo; Kim, Donghyuk; Latif, Haythem

    2014-01-01

    The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism. However, the full regulatory potential of Fur remains undefined. Here we comprehensively reconstruct the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response...

  10. Properties of Sequence Conservation in Upstream Regulatory and Protein Coding Sequences among Paralogs in Arabidopsis thaliana

    Science.gov (United States)

    Richardson, Dale N.; Wiehe, Thomas

    Whole genome duplication (WGD) has catalyzed the formation of new species, genes with novel functions, altered expression patterns, complexified signaling pathways and has provided organisms a level of genetic robustness. We studied the long-term evolution and interrelationships of 5’ upstream regulatory sequences (URSs), protein coding sequences (CDSs) and expression correlations (EC) of duplicated gene pairs in Arabidopsis. Three distinct methods revealed significant evolutionary conservation between paralogous URSs and were highly correlated with microarray-based expression correlation of the respective gene pairs. Positional information on exact matches between sequences unveiled the contribution of micro-chromosomal rearrangements on expression divergence. A three-way rank analysis of URS similarity, CDS divergence and EC uncovered specific gene functional biases. Transcription factor activity was associated with gene pairs exhibiting conserved URSs and divergent CDSs, whereas a broad array of metabolic enzymes was found to be associated with gene pairs showing diverged URSs but conserved CDSs.

  11. Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

    Science.gov (United States)

    Ribeiro, Daniel A; Nascimento, Fabio D; Fracalossi, Ana Carolina C; Gomes, Thiago S; Oshima, Celina T F; Franco, Marcello F

    2010-01-01

    The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

  12. Core transcriptional regulatory circuit controlled by the TAL1 complex in human T cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Sanda, Takaomi; Lawton, Lee N; Barrasa, M Inmaculada; Fan, Zi Peng; Kohlhammer, Holger; Gutierrez, Alejandro; Ma, Wenxue; Tatarek, Jessica; Ahn, Yebin; Kelliher, Michelle A; Jamieson, Catriona H M; Staudt, Louis M; Young, Richard A; Look, A Thomas

    2012-08-14

    The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Molecular recognition in complexes of TRF proteins with telomeric DNA.

    Directory of Open Access Journals (Sweden)

    Miłosz Wieczór

    Full Text Available Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.

  14. Regime complexity and expertise in transnational governance: Strategizing in the face of regulatory uncertainty

    Directory of Open Access Journals (Sweden)

    Sigrid Quack

    2013-01-01

    Full Text Available The rise and spread of transnational governance arrangements has added to the legal indeterminacy of existing regime complexes. The combined regulatory uncertainty resulting from international regime complexes and transnational polycentric governance heightens the role of expertise in managing this institutional complexity. The rising importance of knowledgeable actors with claims to policy-relevant expertise, according to many scholars, is expected further to advantage well-resourced and powerful actors. However, attention to recent developments in accounting and copyright, as two transnational governance fields that have been dominated by a small group of powerful actors for more than three decades, sheds doubt on the generalizability of such arguments. Although representing least likely cases for change, the empirical evidence presented in this paper shows how apparently weak or marginalized actors – whether they are part of public bureaucracies or civil society – developed expertise-based strategies to claim greater involvement and influence in rule and standard-setting. Their strategizing on regime complexity opened up previously shielded policy spaces to broader audiences, thereby transforming actor constellations, preferences and problem definitions in the two policy fields. These findings suggest that under conditions of complexity, indeterminacy and uncertainty, claims to expertise-based rule are becoming increasingly contested – even in transnational governance fields that have a long-established trajectory of rule-setting and rule-implementation monopolized by small groups of professionals, industrialists or technical diplomats. El surgimiento y la difusión de las disposiciones de gobierno transnacional ha contribuido a la indeterminación jurídica de los complejos regímenes existentes. La incertidumbre regulatoria resultante de los complejos regímenes internacionales y del gobierno policéntrico transnacional realza el

  15. PROXiMATE: a database of mutant protein-protein complex thermodynamics and kinetics.

    Science.gov (United States)

    Jemimah, Sherlyn; Yugandhar, K; Michael Gromiha, M

    2017-09-01

    We have developed PROXiMATE, a database of thermodynamic data for more than 6000 missense mutations in 174 heterodimeric protein-protein complexes, supplemented with interaction network data from STRING database, solvent accessibility, sequence, structural and functional information, experimental conditions and literature information. Additional features include complex structure visualization, search and display options, download options and a provision for users to upload their data. The database is freely available at http://www.iitm.ac.in/bioinfo/PROXiMATE/ . The website is implemented in Python, and supports recent versions of major browsers such as IE10, Firefox, Chrome and Opera. gromiha@iitm.ac.in. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  16. pH-Regulatory Proteins as Potential Targets in Breast Cancer

    DEFF Research Database (Denmark)

    Andersen, Anne Poder

    tissues. The focus of the present PhD study is on understanding the mechanisms through which pH-regulatory transporters are regulated by the breast tumor microenvironment, and how these transporters in turn favor cancer progression. In Paper I, we summarized the recent knowledge on the dynamic...... exhibit distinct spatial organization during 3D growth of MCF-7 and MDA-MB-231 breast cancer cells. By pharmacological inhibition and stable shRNA-mediated knockdown, we addressed the specific contributions of the transporters to spheroid growth and show that the specific transporters contribute to breast...... development in the more complex in vivo setting. In Paper III, we found that chemical induction of breast cancer in NBCn1 knockout (KO) mice is delayed and tumor growth rates reduced, compared to those in wildtype (WT) mice, demonstrating a causal link between NBCn1 and breast cancer development. In Paper IV...

  17. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

  18. Mannan-binding protein forms complexes with alpha-2-macroglobulin. A protein model for the interaction

    DEFF Research Database (Denmark)

    Storgaard, P; Holm Nielsen, E; Skriver, E

    1995-01-01

    We report that alpha-2-macroglobulin (alpha 2M) can form complexes with a high molecular weight porcine mannan-binding protein (pMBP-28). The alpha 2M/pMBP-28 complexes was isolated by PEG-precipitation and affinity chromatography on mannan-Sepharose, protein A-Sepharose and anti-IgM Sepharose......-PAGE, which reacted with antibodies against alpha 2M and pMBP-28, respectively, in Western blotting. Furthermore, alpha 2M/pMBP-28 complexes were demonstrated by electron microscopy. Fractionation of pMBP-containing D-mannose eluate from mannan-Sepharose on Superose 6 showed two protein peaks which reacted...... with anti-C1 s antibodies in ELISA, one of about 650-800 kDa, which in addition contained pMBP-28 and anti-alpha 2M reactive material, the other with an M(r) of 100-150 kDa. The latter peak revealed rhomboid molecules (7 x 15 nm) in the electron microscope and a 67 kDa band in SDS-PAGE under reducing...

  19. Discovering functional interdependence relationship in PPI networks for protein complex identification.

    Science.gov (United States)

    Lam, Winnie W M; Chan, Keith C C

    2012-04-01

    Protein molecules interact with each other in protein complexes to perform many vital functions, and different computational techniques have been developed to identify protein complexes in protein-protein interaction (PPI) networks. These techniques are developed to search for subgraphs of high connectivity in PPI networks under the assumption that the proteins in a protein complex are highly interconnected. While these techniques have been shown to be quite effective, it is also possible that the matching rate between the protein complexes they discover and those that are previously determined experimentally be relatively low and the "false-alarm" rate can be relatively high. This is especially the case when the assumption of proteins in protein complexes being more highly interconnected be relatively invalid. To increase the matching rate and reduce the false-alarm rate, we have developed a technique that can work effectively without having to make this assumption. The name of the technique called protein complex identification by discovering functional interdependence (PCIFI) searches for protein complexes in PPI networks by taking into consideration both the functional interdependence relationship between protein molecules and the network topology of the network. The PCIFI works in several steps. The first step is to construct a multiple-function protein network graph by labeling each vertex with one or more of the molecular functions it performs. The second step is to filter out protein interactions between protein pairs that are not functionally interdependent of each other in the statistical sense. The third step is to make use of an information-theoretic measure to determine the strength of the functional interdependence between all remaining interacting protein pairs. Finally, the last step is to try to form protein complexes based on the measure of the strength of functional interdependence and the connectivity between proteins. For performance evaluation

  20. Behaviour of intrinsically disordered proteins in protein-protein complexes with an emphasis on fuzziness

    DEFF Research Database (Denmark)

    Olsen, Johan Gotthardt; Teilum, Kaare; Kragelund, Birthe Brandt

    2017-01-01

    in their malleability, which enables them to bind several different partners with high specificity. In addition, their interactions with other macromolecules can be regulated by a variable amount of chemically diverse post-translational modifications. Four kinetically and energetically different types of complexes...

  1. Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

    Science.gov (United States)

    Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

    2017-05-01

    Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression was significantly positively correlated with triglyceride in both polycystic ovary syndrome and endometrial cancer (p = 0.028 and p = 0.027, respectively). Quantitative serum sterol regulatory element

  2. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  3. The Search Engine for Multi-Proteoform Complexes: An Online Tool for the Identification and Stoichiometry Determination of Protein Complexes.

    Science.gov (United States)

    Skinner, Owen S; Schachner, Luis F; Kelleher, Neil L

    2016-12-08

    Recent advances in top-down mass spectrometry using native electrospray now enable the analysis of intact protein complexes with relatively small sample amounts in an untargeted mode. Here, we describe how to characterize both homo- and heteropolymeric complexes with high molecular specificity using input data produced by tandem mass spectrometry of whole protein assemblies. The tool described is a "search engine for multi-proteoform complexes," (SEMPC) and is available for free online. The output is a list of candidate multi-proteoform complexes and scoring metrics, which are used to define a distinct set of one or more unique protein subunits, their overall stoichiometry in the intact complex, and their pre- and post-translational modifications. Thus, we present an approach for the identification and characterization of intact protein complexes from native mass spectrometry data. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  4. Distinct forms of the β subunit of GTP-binding regulatory proteins identified by molecular cloning

    International Nuclear Information System (INIS)

    Fong, H.K.W.; Amatruda, T.T. III; Birren, B.W.; Simon, M.I.

    1987-01-01

    Two distinct β subunits of guanine nucleotide-binding regulatory proteins have been identified by cDNA cloning and are referred to as β 1 and β 1 subunits. The bovine transducin β subunit (β 1 ) has been cloned previously. The author now isolated and analyzed cDNA clones that encode the β 2 subunit from bovine adrenal, bovine brain, and a human myeloid leukemia cell line, HL-60. The 340-residue M/sub r/ 37,329 Β 2 protein is 90% identical with β 1 in predicted amino acid sequence, and it is also organized as a series of repetitive homologous segments. The major mRNA that encodes the bovine β 2 subunit is 1.7 kilobases in length. It is expressed at lower levels than β 1 subunit mRNA in all tissues examined. The β 1 and β 2 messages are expressed in cloned human cell lines. Hybridization of cDNA probes to bovine DNA showed that β 1 and β 2 are encoded by separate genes. The amino acid sequences for the bovine and human β 2 subunit are identical, as are the amino acid sequences for the bovine and human β 1 subunit. This evolutionary conservation suggests that the two β subunits have different roles in the signal transduction process

  5. KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

    Science.gov (United States)

    Fujita, Yuji; Masuda, Kiyoshi; Hamada, Junichi; Shoda, Katsutoshi; Naruto, Takuya; Hamada, Satoshi; Miyakami, Yuko; Kohmoto, Tomohiro; Watanabe, Miki; Takahashi, Rizu; Tange, Shoichiro; Saito, Masako; Kudo, Yasusei; Fujiwara, Hitoshi; Ichikawa, Daisuke; Tangoku, Akira; Otsuji, Eigo; Imoto, Issei

    2017-11-24

    KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

  6. Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

    Science.gov (United States)

    Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

    2014-01-31

    Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

  7. Translation initiation mediated by nuclear cap-binding protein complex.

    Science.gov (United States)

    Ryu, Incheol; Kim, Yoon Ki

    2017-04-01

    In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

  8. Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A.

    Directory of Open Access Journals (Sweden)

    Margarita Zayas

    2016-01-01

    Full Text Available Hepatitis C virus (HCV nonstructural protein (NS5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI and two intrinsically disordered domains (DII and DIII interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2. We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core-RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i SC-dependent recruitment of replication complexes to core protein and (ii BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.

  9. COMK ENCODES THE COMPETENCE TRANSCRIPTION FACTOR, THE KEY REGULATORY PROTEIN FOR COMPETENCE DEVELOPMENT IN BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    VANSINDEREN, D; LUTTINGER, A; KONG, LY; DUBNAU, D; VENEMA, G; HAMOEN, L

    comK is a positive autoregulatory gene occupying a central position in the com petence-signal-transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late

  10. Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus

    NARCIS (Netherlands)

    Sedelnikova, S.E.; Smits, S.H.J.; Leonard, P.M.; Brinkman, A.B.; Oost, van der J.; Rafferty, J.B.

    2001-01-01

    The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in

  11. The regulatory beta-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis

    DEFF Research Database (Denmark)

    Yde, C W; Olsen, B B; Meek, D

    2008-01-01

    25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory beta-subunit of protein kinase CK2 by RNA...

  12. Epitopes of human immunodeficiency virus regulatory proteins tat, nef, and rev are expressed in normal human tissue

    NARCIS (Netherlands)

    Parmentier, H. K.; van Wichen, D. F.; Meyling, F. H.; Goudsmit, J.; Schuurman, H. J.

    1992-01-01

    The expression of regulatory proteins tat, rev, and nef of human immunodeficiency virus type-1 (HIV-1) and tat of HIV-2 was studied in frozen sections of lymph nodes from HIV-1-infected individuals, and various tissues from uninfected persons. In HIV-1-positive lymph nodes, monoclonal antibodies to

  13. Solving structures of protein complexes by molecular replacement with Phaser

    International Nuclear Information System (INIS)

    McCoy, Airlie J.

    2006-01-01

    Four case studies in using maximum-likelihood molecular replacement, as implemented in the program Phaser, to solve structures of protein complexes are described. Molecular replacement (MR) generally becomes more difficult as the number of components in the asymmetric unit requiring separate MR models (i.e. the dimensionality of the search) increases. When the proportion of the total scattering contributed by each search component is small, the signal in the search for each component in isolation is weak or non-existent. Maximum-likelihood MR functions enable complex asymmetric units to be built up from individual components with a ‘tree search with pruning’ approach. This method, as implemented in the automated search procedure of the program Phaser, has been very successful in solving many previously intractable MR problems. However, there are a number of cases in which the automated search procedure of Phaser is suboptimal or encounters difficulties. These include cases where there are a large number of copies of the same component in the asymmetric unit or where the components of the asymmetric unit have greatly varying B factors. Two case studies are presented to illustrate how Phaser can be used to best advantage in the standard ‘automated MR’ mode and two case studies are used to show how to modify the automated search strategy for problematic cases

  14. Protein Loop Dynamics Are Complex and Depend on the Motions of the Whole Protein

    Directory of Open Access Journals (Sweden)

    Michael T. Zimmermann

    2012-04-01

    Full Text Available We investigate the relationship between the motions of the same peptide loop segment incorporated within a protein structure and motions of free or end-constrained peptides. As a reference point we also compare against alanine chains having the same length as the loop. Both the analysis of atomic molecular dynamics trajectories and structure-based elastic network models, reveal no general dependence on loop length or on the number of solvent exposed residues. Rather, the whole structure affects the motions in complex ways that depend strongly and specifically on the tertiary structure of the whole protein. Both the Elastic Network Models and Molecular Dynamics confirm the differences in loop dynamics between the free and structured contexts; there is strong agreement between the behaviors observed from molecular dynamics and the elastic network models. There is no apparent simple relationship between loop mobility and its size, exposure, or position within a loop. Free peptides do not behave the same as the loops in the proteins. Surface loops do not behave as if they were random coils, and the tertiary structure has a critical influence upon the apparent motions. This strongly implies that entropy evaluation of protein loops requires knowledge of the motions of the entire protein structure.

  15. Nitric oxide-mediated modulation of iron regulatory proteins: implication for cellular iron homeostasis.

    Science.gov (United States)

    Kim, Sangwon; Ponka, Prem

    2002-01-01

    Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) that are located in the 3' untranslated region (UTR) and the 5' UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO(.), a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels and a decrease in ferritin synthesis. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO(+) (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels and a dramatic increase in ferritin synthesis. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels and an increase in ferritin synthesis in LPS/IFN-gamma-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO(+)-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

  16. Changes in regulatory molecules for lymphangiogenesis in intestinal lymphangiectasia with enteric protein loss.

    Science.gov (United States)

    Hokari, Ryota; Kitagawa, Noritake; Watanabe, Chikako; Komoto, Shunsuke; Kurihara, Chie; Okada, Yoshikiyo; Kawaguchi, Atsushi; Nagao, Shigeaki; Hibi, Toshifumi; Miura, Soichiro

    2008-07-01

    Vascular endothelial growth factor receptor 3 (VEGFR3) and LYVE-1 are specifically expressed in the endothelium of the lymphatic systems. VEGF-C, D, FOXC2, Prox 1, and SOX18 are known to play central roles in lymphatic development. We investigated the expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa of idiopathic intestinal lymphangiectasia. Biopsy samples were obtained from duodenal biopsies in patients with intestinal lymphangiectasia complicated with protein-losing from white spot lesions in which lymphangiectasia was histologically confirmed. Immunohistochemical analysis for VEGFR3 and LYVE-1 was performed. mRNA expression of VEGF-C, VEGF-D, VEGFR3, and transcription factors was determined by the quantitative reverse transcription-polymerase chain reaction method. In the control mucosa, VEGFR3 was weakly expressed on the central lymphatic vessels in the lamina propria and LYVE-1 was expressed mainly on the lymphatic vessels in the submucosa. In intestinal lymphangiectasia, VEGFR3 and LYVE-1 expression levels were increased on the mucosal surface corresponding to widely dilated lymphatic vessels, while they were decreased in the deeper mucosa. mRNA expression study showed a significant increase in the expression level of VEGFR3 in lymphangiectasia, but the expression of VEGF-C and -D mRNA was significantly suppressed compared with that in controls despite the presence of lymphangiectasia. The mRNA expression levels of FOXC2 and SOX18 were also decreased, whereas Prox 1 was not altered. There is an altered expression of regulatory molecules for lymphangiogenesis in the duodenal mucosa in these patients.

  17. Overproduction of lactimidomycin by cross-overexpression of genes encoding Streptomyces antibiotic regulatory proteins.

    Science.gov (United States)

    Zhang, Bo; Yang, Dong; Yan, Yijun; Pan, Guohui; Xiang, Wensheng; Shen, Ben

    2016-03-01

    The glutarimide-containing polyketides represent a fascinating class of natural products that exhibit a multitude of biological activities. We have recently cloned and sequenced the biosynthetic gene clusters for three members of the glutarimide-containing polyketides-iso-migrastatin (iso-MGS) from Streptomyces platensis NRRL 18993, lactimidomycin (LTM) from Streptomyces amphibiosporus ATCC 53964, and cycloheximide (CHX) from Streptomyces sp. YIM56141. Comparative analysis of the three clusters identified mgsA and chxA, from the mgs and chx gene clusters, respectively, that were predicted to encode the PimR-like Streptomyces antibiotic regulatory proteins (SARPs) but failed to reveal any regulatory gene from the ltm gene cluster. Overexpression of mgsA or chxA in S. platensis NRRL 18993, Streptomyces sp. YIM56141 or SB11024, and a recombinant strain of Streptomyces coelicolor M145 carrying the intact mgs gene cluster has no significant effect on iso-MGS or CHX production, suggesting that MgsA or ChxA regulation may not be rate-limiting for iso-MGS and CHX production in these producers. In contrast, overexpression of mgsA or chxA in S. amphibiosporus ATCC 53964 resulted in a significant increase in LTM production, with LTM titer reaching 106 mg/L, which is five-fold higher than that of the wild-type strain. These results support MgsA and ChxA as members of the SARP family of positive regulators for the iso-MGS and CHX biosynthetic machinery and demonstrate the feasibility to improve glutarimide-containing polyketide production in Streptomyces strains by exploiting common regulators.

  18. 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.

  19. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1–AMPK complex

    International Nuclear Information System (INIS)

    Nakagawa, Koji; Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie; Asaka, Masahiro; Takeda, Hiroshi; Kobayashi, Masanobu

    2012-01-01

    Highlights: ► The nuclear protein Artemis physically interacts with AMPKα2. ► Artemis co-localizes with AMPKα2 in the nucleus. ► Artemis promotes phosphorylation and activation of AMPK. ► The interaction between AMPKα2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1–AMPK complex.

  20. The nuclear protein Artemis promotes AMPK activation by stabilizing the LKB1-AMPK complex

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, Koji, E-mail: k_nakagawa@pharm.hokudai.ac.jp [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Uehata, Yasuko; Natsuizaka, Mitsuteru; Kohara, Toshihisa; Darmanin, Stephanie [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Asaka, Masahiro [Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Takeda, Hiroshi [Department of Pathophysiology and Therapeutics, Division of Pharmascience, Faculty of Pharmaceutical Sciences, Hokkaido University, N12 W6, Kita-ku, Sapporo, Hokkaido 060-0812 (Japan); Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); Kobayashi, Masanobu [Department of Cancer Preventive Medicine, Graduate School of Medicine, Hokkaido University, N15 W7, Kita-ku, Sapporo, Hokkaido 060-8638 (Japan); School of Nursing and Social Services, Health Sciences University of Hokkaido, Ishikari-Toubetsu, Hokkaido 061-0293 (Japan)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer The nuclear protein Artemis physically interacts with AMPK{alpha}2. Black-Right-Pointing-Pointer Artemis co-localizes with AMPK{alpha}2 in the nucleus. Black-Right-Pointing-Pointer Artemis promotes phosphorylation and activation of AMPK. Black-Right-Pointing-Pointer The interaction between AMPK{alpha}2 and LKB1 is stabilized by Artemis. -- Abstract: AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic {alpha} subunit and regulatory {beta} and {gamma} subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the {alpha}-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPK{alpha}2-binding protein. Artemis was found to co-immunoprecipitate with AMPK{alpha}2, and the co-localization of Artemis with AMPK{alpha}2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPK{alpha}2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPK{alpha}2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex.

  1. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinsil; Ha, Hye-Jeong [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kim, Sujin [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Ah-Reum [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Sook-Jeong [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Hoe, Kwang-Lae, E-mail: kwanghoe@cnu.ac.kr [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Kim, Dong-Uk, E-mail: kimdongu@kribb.re.kr [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

  2. Stargazin regulates AMPA receptor trafficking through adaptor protein complexes during long-term depression

    Science.gov (United States)

    Matsuda, Shinji; Kakegawa, Wataru; Budisantoso, Timotheus; Nomura, Toshihiro; Kohda, Kazuhisa; Yuzaki, Michisuke

    2013-11-01

    Long-term depression (LTD) underlies learning and memory in various brain regions. Although postsynaptic AMPA receptor trafficking mediates LTD, its underlying molecular mechanisms remain largely unclear. Here we show that stargazin, a transmembrane AMPA receptor regulatory protein, forms a ternary complex with adaptor proteins AP-2 and AP-3A in hippocampal neurons, depending on its phosphorylation state. Inhibiting the stargazin-AP-2 interaction disrupts NMDA-induced AMPA receptor endocytosis, and inhibiting that of stargazin-AP-3A abrogates the late endosomal/lysosomal trafficking of AMPA receptors, thereby upregulating receptor recycling to the cell surface. Similarly, stargazin’s interaction with AP-2 or AP-3A is necessary for low-frequency stimulus-evoked LTD in CA1 hippocampal neurons. Thus, stargazin has a crucial role in NMDA-dependent LTD by regulating two trafficking pathways of AMPA receptors—transport from the cell surface to early endosomes and from early endosomes to late endosomes/lysosomes—through its sequential binding to AP-2 and AP-3A.

  3. Moonlighting microtubule-associated proteins: regulatory functions by day and pathological functions at night.

    Science.gov (United States)

    Oláh, J; Tőkési, N; Lehotzky, A; Orosz, F; Ovádi, J

    2013-11-01

    The sensing, integrating, and coordinating features of the eukaryotic cells are achieved by the complex ultrastructural arrays and multifarious functions of the cytoskeletal network. Cytoskeleton comprises fibrous protein networks of microtubules, actin, and intermediate filaments. These filamentous polymer structures are highly dynamic and undergo constant and rapid reorganization during cellular processes. The microtubular system plays a crucial role in the brain, as it is involved in an enormous number of cellular events including cell differentiation and pathological inclusion formation. These multifarious functions of microtubules can be achieved by their decoration with proteins/enzymes that exert specific effects on the dynamics and organization of the cytoskeleton and mediate distinct functions due to their moonlighting features. This mini-review focuses on two aspects of the microtubule cytoskeleton. On the one hand, we describe the heteroassociation of tubulin/microtubules with metabolic enzymes, which in addition to their catalytic activities stabilize microtubule structures via their cross-linking functions. On the other hand, we focus on the recently identified moonlighting tubulin polymerization promoting protein, TPPP/p25. TPPP/p25 is a microtubule-associated protein and it displays distinct physiological or pathological (aberrant) functions; thus it is a prototype of Neomorphic Moonlighting Proteins. The expression of TPPP/p25 is finely controlled in the human brain; this protein is indispensable for the development of projections of oligodendrocytes that are responsible for the ensheathment of axons. The nonphysiological, higher or lower TPPP/p25 level leads to distinct CNS diseases. Mechanisms contributing to the control of microtubule stability and dynamics by metabolic enzymes and TPPP/p25 will be discussed. Copyright © 2013 Wiley Periodicals, Inc.

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

    Directory of Open Access Journals (Sweden)

    Madlen eNietzsche

    2014-02-01

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

  5. Recovering protein-protein and domain-domain interactions from aggregation of IP-MS proteomics of coregulator complexes.

    Directory of Open Access Journals (Sweden)

    Amin R Mazloom

    2011-12-01

    Full Text Available Coregulator proteins (CoRegs are part of multi-protein complexes that transiently assemble with transcription factors and chromatin modifiers to regulate gene expression. In this study we analyzed data from 3,290 immuno-precipitations (IP followed by mass spectrometry (MS applied to human cell lines aimed at identifying CoRegs complexes. Using the semi-quantitative spectral counts, we scored binary protein-protein and domain-domain associations with several equations. Unlike previous applications, our methods scored prey-prey protein-protein interactions regardless of the baits used. We also predicted domain-domain interactions underlying predicted protein-protein interactions. The quality of predicted protein-protein and domain-domain interactions was evaluated using known binary interactions from the literature, whereas one protein-protein interaction, between STRN and CTTNBP2NL, was validated experimentally; and one domain-domain interaction, between the HEAT domain of PPP2R1A and the Pkinase domain of STK25, was validated using molecular docking simulations. The scoring schemes presented here recovered known, and predicted many new, complexes, protein-protein, and domain-domain interactions. The networks that resulted from the predictions are provided as a web-based interactive application at http://maayanlab.net/HT-IP-MS-2-PPI-DDI/.

  6. INFLUENCE OF NATURAL ADDITIVES ON PROTEIN COMPLEX OF BREAD

    Directory of Open Access Journals (Sweden)

    Dana Urminská

    2010-11-01

    Full Text Available The study focuses on researching the influence of natural additives on certain technological characteristics of mixtures used for bread production, more particularly the influence of N substances in used raw material on selected qualitative parameters of bread. The blends for bread production to be analysed were prepared by mixing wheat flour with an addition of oat, buckwheat, lentil and chickpea wholegrain flour in different portions (10, 20, 30, 40 and 50 %. The experiment showed that the addition of natural additives worsened the protein complex of the blends used in bread production (worsening also qualitative parameters known as product volume. The loaves prepared with an addition of buckwheat, oat, lentil and chickpea were evaluated to be of a lesser quality from a technological viewpoint when compared with pure wheat loaves. The lower content of gluten forming proteins and the generally changed protein composition of blends due to additives caused a lower percentage of wet gluten content, its lower extensibility and swelling capacity. The sedimentation value (Zeleny index decreased proportionally with the increase of addition until the level was unsatisfactory for raw material intended for bakery purposes. The N content in experimental loaves was higher than in the reference loaves and it increased according to the selected additive and its portion in the blend (more with the addition of lentil and chickpea, less in case of buckwheat and oat which is considered as positive from a nutritional point of view. But from the technological point of view the additives did not show any positive influence and caused a lower loaf bread volume. The most significant decrease of the loaf bread volume was found with the addition of 50 % of buckwheat (- 45.6 %. Better results were obtained with a lower portion of the additive: loaf with an addition of 30 % of chickpea (volume decreased by 12.8 % > loaf with an addition of 30 % of lentil (volume

  7. Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Andrew Loyd [Univ. of California, Berkeley, CA (United States). Dept. of Chemistry

    1996-05-01

    Solution NMR was primarily used to characterize structure and dynamics in two different eukaryotic protein systems: the δ-Al-ε activation domain from c-jun and the Drosophila RNA-binding protein Sex-lethal. The second system is the Drosophila Sex-lethal (Sxl) protein, an RNA-binding protein which is the ``master switch`` in sex determination. Sxl contains two adjacent RNA-binding domains (RBDs) of the RNP consensus-type. The NMR spectrum of the second RBD (Sxl-RBD2) was assigned using multidimensional heteronuclear NMR, and an intermediate-resolution family of structures was calculated from primarily NOE distance restraints. The overall fold was determined to be similar to other RBDs: a βαβ-βαβ pattern of secondary structure, with the two helices packed against a 4-stranded anti-parallel β-sheet. In addition 15N T1, T2, and 15N/1H NOE relaxation measurements were carried out to characterize the backbone dynamics of Sxl-RBD2 in solution. RNA corresponding to the polypyrimidine tract of transformer pre-mRNA was generated and titrated into 3 different Sxl-RBD protein constructs. Combining Sxl-RBD1+2 (bht RBDs) with this RNA formed a specific, high affinity protein/RNA complex that is amenable to further NMR characterization. The backbone 1H, 13C, and 15N resonances of Sxl-RBD1+2 were assigned using a triple-resonance approach, and 15N relaxation experiments were carried out to characterize the backbone dynamics of this complex. The changes in chemical shift in Sxl-RBD1+2 upon binding RNA are observed using Sxl-RBD2 as a substitute for unbound Sxl-RBD1+2. This allowed the binding interface to be qualitatively mapped for the second domain.

  8. G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells*

    OpenAIRE

    Vellano, Christopher P.; Maher, Ellen M.; Hepler, John R.; Blumer, Joe B.

    2011-01-01

    Background: Regulator of G protein signaling 14 (RGS14) is a G protein regulatory (GPR) protein that participates in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs).

  9. The physiological functions of iron regulatory proteins in iron homeostasis - an update

    Directory of Open Access Journals (Sweden)

    De-Liang eZhang

    2014-06-01

    Full Text Available Iron regulatory proteins (IRPs regulate the expression of genes involved in iron metabolism by binding to RNA stem-loop structures known as iron responsive elements (IREs in target mRNAs. IRP binding inhibits the translation of mRNAs that contain an IRE in the 5’untranslated region of the transcripts, and increases the stability of mRNAs that contain IREs in the 3'untranslated region of transcripts. By these mechanisms, IRPs increase cellular iron absorption and decrease storage and export of iron to maintain an optimal intracellular iron balance. There are two members of the mammalian IRP protein family, IRP1 and IRP2, and they have redundant functions as evidenced by the embryonic lethality of the mice that completely lack IRP expression (Irp1-/-/Irp2-/- mice, which contrasts with the fact that Irp1-/- and Irp2-/- mice are viable. In addition, Irp2-/- mice also display neurodegenerative symptoms and microcytic hypochromic anemia, suggesting that IRP2 function predominates in the nervous system and erythropoietic homeostasis. Though the physiological significance of IRP1 had been unclear since Irp1-/- animals were first assessed in the early 1990’s, recent studies indicate that IRP1 plays an essential function in orchestrating the balance between erythropoiesis and bodily iron homeostasis. Additionally, Irp1-/- mice develop pulmonary hypertension, and they experience sudden death when maintained on an iron-deficient diet, indicating that IRP1 has a critical role in the pulmonary and cardiovascular systems. This review summarizes recent progress that has been made in understanding the physiological roles of IRP1 and IRP2, and further discusses the implications for clinical research on patients with idiopathic polycythemia, pulmonary hypertension and neurodegeneration.

  10. Espins are multifunctional actin cytoskeletal regulatory proteins in the microvilli of chemosensory and mechanosensory cells

    Science.gov (United States)

    Sekerková, Gabriella; Zheng, Lili; Loomis, Patricia A.; Changyaleket, Benjarat; Whitlon, Donna S.; Mugnaini, Enrico; Bartles, James R.

    2010-01-01

    Espins are associated with the parallel actin bundles of hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction in mice and humans. Here, we report that espins are also concentrated in the microvilli of a number of other sensory cells: vomeronasal organ sensory neurons, solitary chemoreceptor cells, taste cells and Merkel cells. Moreover, we show that hair cells and these other sensory cells contain novel espin isoforms that arise from a different transcriptional start site and differ significantly from other espin isoforms in their complement of ligand-binding activities and their effects on actin polymerization. The novel espin isoforms of sensory cells bundled actin filaments with high affinity in a Ca2+-resistant fashion, bound actin monomer via a WASP homology 2 domain, bound profilin via a single proline-rich peptide, and caused a dramatic elongation of microvillus-type parallel actin bundles in transfected epithelial cells. In addition, the novel espin isoforms of sensory cells differed from other espin isoforms in that they potently inhibited actin polymerization in vitro, did not bind the Src homology 3 domain of the adapter protein insulin receptor substrate p53 and did not bind the acidic, signaling phospholipid phosphatidylinositol 4,5- bisphosphate. Thus, the espins constitute a family of multifunctional actin cytoskeletal regulatory proteins with the potential to differentially influence the organization, dimensions, dynamics and signaling capabilities of the actin filament-rich, microvillus-type specializations that mediate sensory transduction in a variety of mechanosensory and chemosensory cells. PMID:15190118

  11. Human Sterol Regulatory Element-Binding Protein 1a Contributes Significantly to Hepatic Lipogenic Gene Expression

    Directory of Open Access Journals (Sweden)

    Andreas Bitter

    2015-01-01

    Full Text Available Background/Aims: Sterol regulatory element-binding protein (SREBP 1, the master regulator of lipogenesis, was shown to be associated with non-alcoholic fatty liver disease, which is attributed to its major isoform SREBP1c. Based on studies in mice, the minor isoform SREBP1a is regarded as negligible for hepatic lipogenesis. This study aims to elucidate the expression and functional role of SREBP1a in human liver. Methods: mRNA expression of both isoforms was quantified in cohorts of human livers and primary human hepatocytes. Hepatocytes were treated with PF-429242 to inhibit the proteolytic activation of SREBP precursor protein. SREBP1a-specifc and pan-SREBP1 knock-down were performed by transfection of respective siRNAs. Lipogenic SREBP-target gene expression was analyzed by real-time RT-PCR. Results: In human liver, SREBP1a accounts for up to half of the total SREBP1 pool. Treatment with PF-429242 indicated SREBP-dependent auto-regulation of SREBP1a, which however was much weaker than of SREBP1c. SREBP1a-specifc knock-down also reduced significantly the expression of SREBP1c and of SREBP-target genes. Regarding most SREBP-target genes, simultaneous knock-down of both isoforms resulted in effects of only similar extent as SREBP1a-specific knock-down. Conclusion: We here showed that SREBP1a is significantly contributing to the human hepatic SREBP1 pool and has a share in human hepatic lipogenic gene expression.

  12. A novel protein-protein interaction in the RES (REtention and Splicing) complex.

    Science.gov (United States)

    Tripsianes, Konstantinos; Friberg, Anders; Barrandon, Charlotte; Brooks, Mark; van Tilbeurgh, Herman; Seraphin, Bertrand; Sattler, Michael

    2014-10-10

    The retention and splicing (RES) complex is a conserved spliceosome-associated module that was shown to enhance splicing of a subset of transcripts and promote the nuclear retention of unspliced pre-mRNAs in yeast. The heterotrimeric RES complex is organized around the Snu17p protein that binds to both the Bud13p and Pml1p subunits. Snu17p exhibits an RRM domain that resembles a U2AF homology motif (UHM) and Bud13p harbors a Trp residue reminiscent of an UHM-ligand motif (ULM). It has therefore been proposed that the interaction between Snu17p and Bud13p resembles canonical UHM-ULM complexes. Here, we have used biochemical and NMR structural analysis to characterize the structure of the yeast Snu17p-Bud13p complex. Unlike known UHMs that sequester the Trp residue of the ULM ligand in a hydrophobic pocket, Snu17p and Bud13p utilize a large interaction surface formed around the two helices of the Snu17p domain. In total 18 residues of the Bud13p ligand wrap around the Snu17p helical surface in an U-turn-like arrangement. The invariant Trp(232) in Bud13p is located in the center of the turn, and contacts surface residues of Snu17p. The structural data are supported by mutational analysis and indicate that Snu17p provides an extended binding surface with Bud13p that is notably distinct from canonical UHM-ULM interactions. Our data highlight structural diversity in RRM-protein interactions, analogous to the one seen for nucleic acid interactions. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

    International Nuclear Information System (INIS)

    Wang Haidong; Niu, Catherine Hui; Yang Qiaoqin; Badea, Ildiko

    2011-01-01

    In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.

  14. Study on protein conformation and adsorption behaviors in nanodiamond particle-protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Wang Haidong [Department of Biomedical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Niu, Catherine Hui [Department of Chemical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Yang Qiaoqin [Department of Mechanical Engineering, University of Saskatchewan, Saskatoon, S7N 5A9 (Canada); Badea, Ildiko, E-mail: catherine.niu@usask.ca [Drug Design and Discovery Research Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, S7N 5C9 (Canada)

    2011-04-08

    In the present research, the conformation of bovine serum albumin (BSA) in the nanodiamond particle (ND)-BSA complex was studied by Fourier transform infrared spectroscopy, fluorescence spectroscopy, UV-vis spectroscopy, and circular dichroism spectroscopy. The spectroscopic study revealed that most BSA structural features could be preserved in the complex though the BSA underwent conformational changes in the complex due to ND-BSA interaction. In addition, BSA adsorption isotherms and zeta-potential measurements were employed to investigate the pH dependence of the ND-BSA interaction. The changes in surface charge of the ND-BSA complex with pH variations indicated that the binding of BSA to ND might lead to not only the adsorption of BSA onto the ND surface but also the partial breakup of ND aggregates into relatively small ND-BSA aggregates because of the strong binding force between ND and BSA. The results show that ND is an excellent platform for protein immobilization with high affinity and holds great potential to be used for biosensor applications.

  15. Characterization of the regulatory subunit from brain cyclic AMP-dependent protein kinase II

    International Nuclear Information System (INIS)

    Stein, J.C.

    1985-01-01

    Tryptic peptides derived from the regulatory subunits of brain and heart cAMP-dependent protein kinase II were mapped by reverse phase HPLC. At 280 nm, 15 unique peptides were found only in the heart RII digest, while 5 other peptides were obtained only from brain RII. At 210 nm, 13 brain-RII specific and 15 heart-RII specific tryptic peptides were identified and resolved. Two-dimensional mapping analyses revealed that several 37 P-labeled tryptic fragments derived from the autophosphorylation and the photoaffinity labeled cAMP-binding sites of brain RII were separate and distinct from the 32 P-peptides isolated from similarly treated heart RII. The tryptic phosphopeptide containing the autophosphorylation site in brain RII was purified. The sequence and phosphorylation site is: Arg-Ala-Ser(P)-Val-Cys-Ala-Glu-Ala-Tyr-Asn-Pro-Asp-Glu-Glu-Glu-Asp-Asp-Ala-Glu. Astrocytes and neurons exhibit high levels of the brain RII enzyme, while oligodendrocytes contain the heart RII enzyme. Monoclonal antibodies to bovine cerebral cortex RII were made and characterized. The antibodies elucidated a subtle difference between membrane-associated and cytosolic RII from cerebral cortex

  16. Prognostic value of cell cycle regulatory proteins in muscle-infiltrating bladder cancer.

    Science.gov (United States)

    Galmozzi, Fabia; Rubagotti, Alessandra; Romagnoli, Andrea; Carmignani, Giorgio; Perdelli, Luisa; Gatteschi, Beatrice; Boccardo, Francesco

    2006-12-01

    The aims of this study were to investigate the expression levels of proteins involved in cell cycle regulation in specimens of bladder cancer and to correlate them with the clinicopathological characteristics, proliferative activity and survival. Eighty-two specimens obtained from patients affected by muscle-invasive bladder cancer were evaluated immunohistochemically for p53, p21 and cyclin D1 expression, as well as for the tumour proliferation index, Ki-67. The statistical analysis included Kaplan-Meier curves with log-rank test and Cox proportional hazards models. In univariate analyses, low Ki-67 proliferation index (P = 0.045) and negative p21 immunoreactivity (P = 0.04) were associated to patient's overall survival (OS), but in multivariate models p21 did not reach statistical significance. When the combinations of the variables were assessed in two separate multivariate models that included tumour stage, grading, lymph node status, vascular invasion and perineural invasion, the combined variables p21/Ki-67 or p21/cyclin D1 expression were independent predictors for OS; in particular, patients with positive p21/high Ki-67 (P = 0.015) or positive p21/negative cyclin D1 (P = 0.04) showed the worst survival outcome. Important alterations in the cell cycle regulatory pathways occur in muscle-invasive bladder cancer and the combined use of cell cycle regulators appears to provide significant prognostic information that could be used to select the patients most suitable for multimodal therapeutic approaches.

  17. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  18. Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

    Science.gov (United States)

    Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

    2017-03-31

    The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Fas/CD95 regulatory protein Faim2 is neuroprotective after transient brain ischemia.

    Science.gov (United States)

    Reich, Arno; Spering, Christopher; Gertz, Karen; Harms, Christoph; Gerhardt, Ellen; Kronenberg, Golo; Nave, Klaus A; Schwab, Markus; Tauber, Simone C; Drinkut, Anja; Harms, Kristian; Beier, Chrstioph P; Voigt, Aaron; Göbbels, Sandra; Endres, Matthias; Schulz, Jörg B

    2011-01-05

    Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen-glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.

  20. [Glucokinase and glucokinase regulatory proteins as molecular targets for novel antidiabetic drugs].

    Science.gov (United States)

    Rubtsov, P M; Igudin, E L; Tiulpakov, A N

    2015-01-01

    The impairment of glucose homeostasis leads to hyperglycemia and type-2 diabetes mellitus. Glucokinase (GK), an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and intestine enterocytes, is a key regulator of glucose homeostasis. In hepatocytes, GK controls the glucose uptake and glycogen synthesis and inhibits the glucose synthesis via the gluconeogenesis pathway. Glucokinase regulatory protein (GKRP) synthesized in hepatocytes acts as an endogenous GK inhibitor. During fasting, GKRP binds GK, inactivates it, and transports it into the cell nucleus, thus isolating it from the hepatocyte carbohydrate metabolism. In the beginning of the 2000s, the research was mainly focused on the development and trials of the small molecule GK activators as potential antidiabetic glucose-lowering drugs. However, the use of such substances increased the risk of hypoglycemia, and clinical studies of most synthetic GK activators are currently discontinued. Allosteric inhibitors of the GK-GKRP interaction are coming as alternative agents increasing the GK activity that can substitute GKA. In this review, we discuss the recent advances and the current state of art in the development of potential antidiabetic drugs targeted to GK as a key regulator of glucose homeostasis.

  1. HAMLET - A protein-lipid complex with broad tumoricidal activity.

    Science.gov (United States)

    Ho, James C S; Nadeem, Aftab; Svanborg, Catharina

    2017-01-15

    HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma.

    Science.gov (United States)

    ten Kate, J; van den Ingh, H F; Khan, P M; Bosman, F T

    1986-04-15

    Immunoreactive adenosine deaminase complexing protein (ADCP) was studied in 91 human colorectal adenocarcinomas. The expression of ADCP was correlated with that of secretory component (SC) and carcinoembryonic antigen (CEA), with the histological grade and the Dukes' stage of the carcinomas. The histological grade was scored semi-quantitatively according to 5 structural and 4 cytological variables. ADCP expression was observed in 3 different staining patterns, namely: (1) diffuse cytoplasmic (77% of the carcinomas); (2) granular cytoplasmic (13%); and (3) membrane-associated (66%). These patterns were observed alone or in combination. Eleven percent of the carcinomas exhibited no ADCP immunoreactivity. Linear regression analysis showed that the expression of ADCP correlates with that of SC and CEA. However, no significant correlation emerged between the histological parameters or the Dukes' stage and any of the immunohistological parameters. Comparison of the histological characteristics of carcinomas exhibiting little or no ADCP immunoreactivity with those showing extensive immunoreactivity, showed that membranous ADCP immunoreactivity occurs more frequently in well-differentiated carcinomas. Structural parameters showed a better correlation with membranous ADCP expression than the cytological variables. It is concluded that membranous expression of ADCP and CEA are indicators of a high level of differentiation as reflected primarily in the structural characteristics of the tumor.

  3. A complex process - transforming scientific research into regulatory rules for environmental protection

    International Nuclear Information System (INIS)

    Yan, J.J.; Goss, D.; Huffman, A.

    2002-01-01

    The protection of isolated wetlands from consumptive use withdrawals has been a policy in the South Florida Water Management District (SFWMD) for over 15 years. A guideline for protecting isolated wetlands was established in the mid-1980's for the consumptive water use permitting program administered by the SFWMD. The guideline specifies groundwater drawdown criteria associated with well field pumpage. In 1994, the SFWMD convened a panel of wetland scientists to review the existing groundwater drawdown criteria. The panel concluded there was insufficient information to determine if the criteria were either too restrictive or insufficient in protecting wetlands. The panel recommended that the SFWMD conduct research to answer related questions. Since that time, staff at the SFWMD have developed a research plan, selected 38 isolated wetland monitoring sites in seven study areas, collected over four years of data, and developed an integrated surface water and groundwater simulation model. However, the staff at the SFWMD has had difficulties in transforming the research results into regulatory rules. The nature of an isolated wetland is quite complicated. Its setting changes significantly from time to time depending on the variation of rainfall, hydro-geological conditions, and human activities. A regulatory rule requires simple and more easily measurable criteria. The regulatory staff need simple tools to evaluate many permit applications within a limited time frame. The tools used in the research process are often complicated and time consuming. This paper describes the wetland research, and the difficulties of transforming research results into regulatory rules. (author)

  4. Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells

    OpenAIRE

    Wang, Xiaomei; Su, Jin; Sherman, Alexandra; Rogers, Geoffrey L.; Liao, Gongxian; Hoffman, Brad E.; Leong, Kam W.; Terhorst, Cox; Daniell, Henry; Herzog, Roland W.

    2015-01-01

    Coadministering FIX orally and systemically induces tolerance via complex immune regulation, involving tolerogenic dendritic and T-cell subsets.Induced CD4+CD25−LAP+ regulatory T cells with increased IL-10 and TGF-β expression and CD4+CD25+ regulatory T cells suppress antibody formation against FIX.

  5. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

    Energy Technology Data Exchange (ETDEWEB)

    Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami, E-mail: nagahama@my-pharm.ac.jp

    2015-11-20

    Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.

  6. AAA-ATPase NVL2 acts on MTR4-exosome complex to dissociate the nucleolar protein WDR74

    International Nuclear Information System (INIS)

    Hiraishi, Nobuhiro; Ishida, Yo-ichi; Nagahama, Masami

    2015-01-01

    Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis. - Highlights: • WDR74 accumulates in MTR4-exosome complex upon expression of dominant-negative NVL2. • WDR74 is co-localized with NVL2 in the nucleolus. • WDR74, along with NVL2, is involved in the synthesis of 60S ribosomal subunits.

  7. MicroProtein-mediated recruitment of CONSTANS into a TOPLESS trimeric complex represses flowering in Arabidopsis

    DEFF Research Database (Denmark)

    Graeff, Moritz; Straub, Daniel; Eguen, Tenai E.

    2016-01-01

    MicroProteins are short, single domain proteins that act by sequestering larger, multi-domain proteins into non-functional complexes. MicroProteins have been identified in plants and animals, where they are mostly involved in the regulation of developmental processes. Here we show that two...

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

    Science.gov (United States)

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

    2009-06-07

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

  9. Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome

    Science.gov (United States)

    Vorobyeva, Nadezhda E.; Mazina, Marina U.; Golovnin, Anton K.; Kopytova, Daria V.; Gurskiy, Dmitriy Y.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Georgiev, Pavel G.; Krasnov, Aleksey N.

    2013-01-01

    Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein. PMID:23609538

  10. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  11. The Prognostic Impact of Some Cell Cycle Regulatory Proteins in Egyptian Breast Cancer Patients

    International Nuclear Information System (INIS)

    KAMEL, A.; Mokhtar, N.; Elshaknkiry, N.; Yassin, D.; Elnahass, Y.; Zakarya, O.; Elbasmy, A.; Elmetenawy, W.

    2006-01-01

    Purpose: The particular goal of this work is to study some cell cycle regulatory proteins and their potential impact on prognosis of breast cancer; p53, cyclin D 1 and p27 are potential effectors being the major contributors to the control of the restriction (R) check point of the cell cycle. We also aimed to evaluate different techniques used to detect these cell cycle proteins. Material and Methods: Forty five breast cancer patients as well as 10 controls with non malignant pathology were assessed for cell cycle regulators each by 2 different techniques; p53 was assessed by enzyme immunoassay (EJA) and immunohistochemistry (lHC), cyclin D1 by Western Blotting (WB) and IHC and p27 by WB and me. The cut-off was calculated as the mean of the normal controls +2 SD. Patients were followed up for 4 years and their laboratory data were correlated with different clinical parameters and with other studied regulators. Results: Eighty seven percent of cases (39/45) were positive for p53 by EIA with a range from 20 to 4300, and a mean of 464±97 I pg/mg protein. By mc, 80% (24/30) of the cases showed varying degrees of positivity. Using WB, cyclin D 1 showed high expression levels above cut off values in 69% of patients (31/45) and in 67% (20/30) by me. The corresponding positive figures for p27 were 82% (37/45) and 73% (22/30) using the two techniques, respectively. No significant association was found between p53, cyclin 01 and p27 on one side and different clinical parameters as lymph node status, tumor size or presence of distant metastases on the other side. Survival was poor in patients with high p53 expression. Cyclin D1 positive cases showed comparable survival with negative cases, whereas high p27 levels favored a longer disease free survival. Conclusions: Techniques more suitable for assessment of each of these markers in our consideration were EIA for p53, WB for cyclin D1 and IHC for p27. Moreover, this study demonstrated that these markers were relevant to the

  12. Protein complex finding and ranking: An application to Alzheimer's ...

    Indian Academy of Sciences (India)

    Pooja Sharma

    2017-07-07

    Jul 7, 2017 ... and a few other model organisms. .... form proteins) affect the protein formation process. Muta- ..... We implemented the ComFiR method in MATLAB run- ning on ..... Van Dongen SM 2001 Graph clustering by flow simulation.

  13. MIPCE: An MI-based protein complex extraction technique

    Indian Academy of Sciences (India)

    2015-09-28

    Sep 28, 2015 ... In this work, a method is proposed, referred to as. MIPCE, to find protein ... Because of its importance in the studies of protein interaction network, there are ..... three publicly available benchmark real-life datasets. The method was ... protein interaction networks with improved balance and scal- ability; in ...

  14. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-05-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms--including humans--are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)(1) with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method.

  15. Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*

    Science.gov (United States)

    Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

    2013-01-01

    High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated β-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

  16. Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.

    Science.gov (United States)

    Wang, Xiaomei; Su, Jin; Sherman, Alexandra; Rogers, Geoffrey L; Liao, Gongxian; Hoffman, Brad E; Leong, Kam W; Terhorst, Cox; Daniell, Henry; Herzog, Roland W

    2015-04-09

    Coagulation factor replacement therapy for the X-linked bleeding disorder hemophilia is severely complicated by antibody ("inhibitor") formation. We previously found that oral delivery to hemophilic mice of cholera toxin B subunit-coagulation factor fusion proteins expressed in chloroplasts of transgenic plants suppressed inhibitor formation directed against factors VIII and IX and anaphylaxis against factor IX (FIX). This observation and the relatively high concentration of antigen in the chloroplasts prompted us to evaluate the underlying tolerance mechanisms. The combination of oral delivery of bioencapsulated FIX and intravenous replacement therapy induced a complex, interleukin-10 (IL-10)-dependent, antigen-specific systemic immune suppression of pathogenic antibody formation (immunoglobulin [Ig] 1/inhibitors, IgE) in hemophilia B mice. Tolerance induction was also successful in preimmune mice but required prolonged oral delivery once replacement therapy was resumed. Orally delivered antigen, initially targeted to epithelial cells, was taken up by dendritic cells throughout the small intestine and additionally by F4/80(+) cells in the duodenum. Consistent with the immunomodulatory responses, frequencies of tolerogenic CD103(+) and plasmacytoid dendritic cells were increased. Ultimately, latency-associated peptide expressing CD4(+) regulatory T cells (CD4(+)CD25(-)LAP(+) cells with upregulated IL-10 and transforming growth factor-β (TGF-β) expression) as well as conventional CD4(+)CD25(+) regulatory T cells systemically suppressed anti-FIX responses. © 2015 by The American Society of Hematology.

  17. Lysosomal-associated Transmembrane Protein 4B (LAPTM4B) Decreases Transforming Growth Factor β1 (TGF-β1) Production in Human Regulatory T Cells.

    Science.gov (United States)

    Huygens, Caroline; Liénart, Stéphanie; Dedobbeleer, Olivier; Stockis, Julie; Gauthy, Emilie; Coulie, Pierre G; Lucas, Sophie

    2015-08-14

    Production of active TGF-β1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-β1, favors its cleavage into latent inactive TGF-β1, induces the secretion and surface presentation of GARP·latent TGF-β1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-β1 complexes regulate TGF-β1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-β1, secretion of soluble latent TGF-β1, and surface presentation of GARP·TGF-β1 complexes by Tregs but does not contribute to TGF-β1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-β1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

    Science.gov (United States)

    Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

    2017-09-08

    Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

  19. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

    1999-03-05

    Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

  20. Radiation-induced dissociation of stable DNA-protein complexes in Erlich ascites carcinoma cells

    International Nuclear Information System (INIS)

    Juhasz, P.P.; Sirota, N.P.; Gaziev, A.I.

    1982-01-01

    DNA of Ehrlich ascites carcinoma cells prepared under conditions that were highly denaturing for proteins but not for DNA, contained a group of nonhistone residual proteins. The amount of these proteins increased during DNA replication. The DNA-protein complex observed was sensitive to proteolytic enzymes and/or SH-reagents. γ-irradiation cells with moderate doses leads to a decrease in the amount of DNA-protein complexes. High-dose gamma-irradiation produces enhanced linking of chromosomal proteins with DNA. (author)

  1. A Type-2 fuzzy data fusion approach for building reliable weighted protein interaction networks with application in protein complex detection.

    Science.gov (United States)

    Mehranfar, Adele; Ghadiri, Nasser; Kouhsar, Morteza; Golshani, Ashkan

    2017-09-01

    Detecting the protein complexes is an important task in analyzing the protein interaction networks. Although many algorithms predict protein complexes in different ways, surveys on the interaction networks indicate that about 50% of detected interactions are false positives. Consequently, the accuracy of existing methods needs to be improved. In this paper we propose a novel algorithm to detect the protein complexes in 'noisy' protein interaction data. First, we integrate several biological data sources to determine the reliability of each interaction and determine more accurate weights for the interactions. A data fusion component is used for this step, based on the interval type-2 fuzzy voter that provides an efficient combination of the information sources. This fusion component detects the errors and diminishes their effect on the detection protein complexes. So in the first step, the reliability scores have been assigned for every interaction in the network. In the second step, we have proposed a general protein complex detection algorithm by exploiting and adopting the strong points of other algorithms and existing hypotheses regarding real complexes. Finally, the proposed method has been applied for the yeast interaction datasets for predicting the interactions. The results show that our framework has a better performance regarding precision and F-measure than the existing approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. ComplexContact: a web server for inter-protein contact prediction using deep learning

    KAUST Repository

    Zeng, Hong; Wang, Sheng; Zhou, Tianming; Zhao, Feifeng; Li, Xiufeng; Wu, Qing; Xu, Jinbo

    2018-01-01

    ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

  3. ComplexContact: a web server for inter-protein contact prediction using deep learning

    KAUST Repository

    Zeng, Hong

    2018-05-20

    ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

  4. ComplexContact: a web server for inter-protein contact prediction using deep learning.

    Science.gov (United States)

    Zeng, Hong; Wang, Sheng; Zhou, Tianming; Zhao, Feifeng; Li, Xiufeng; Wu, Qing; Xu, Jinbo

    2018-05-22

    ComplexContact (http://raptorx2.uchicago.edu/ComplexContact/) is a web server for sequence-based interfacial residue-residue contact prediction of a putative protein complex. Interfacial residue-residue contacts are critical for understanding how proteins form complex and interact at residue level. When receiving a pair of protein sequences, ComplexContact first searches for their sequence homologs and builds two paired multiple sequence alignments (MSA), then it applies co-evolution analysis and a CASP-winning deep learning (DL) method to predict interfacial contacts from paired MSAs and visualizes the prediction as an image. The DL method was originally developed for intra-protein contact prediction and performed the best in CASP12. Our large-scale experimental test further shows that ComplexContact greatly outperforms pure co-evolution methods for inter-protein contact prediction, regardless of the species.

  5. Identification of Protein Complexes Using Weighted PageRank-Nibble Algorithm and Core-Attachment Structure.

    Science.gov (United States)

    Peng, Wei; Wang, Jianxin; Zhao, Bihai; Wang, Lusheng

    2015-01-01

    Protein complexes play a significant role in understanding the underlying mechanism of most cellular functions. Recently, many researchers have explored computational methods to identify protein complexes from protein-protein interaction (PPI) networks. One group of researchers focus on detecting local dense subgraphs which correspond to protein complexes by considering local neighbors. The drawback of this kind of approach is that the global information of the networks is ignored. Some methods such as Markov Clustering algorithm (MCL), PageRank-Nibble are proposed to find protein complexes based on random walk technique which can exploit the global structure of networks. However, these methods ignore the inherent core-attachment structure of protein complexes and treat adjacent node equally. In this paper, we design a weighted PageRank-Nibble algorithm which assigns each adjacent node with different probability, and propose a novel method named WPNCA to detect protein complex from PPI networks by using weighted PageRank-Nibble algorithm and core-attachment structure. Firstly, WPNCA partitions the PPI networks into multiple dense clusters by using weighted PageRank-Nibble algorithm. Then the cores of these clusters are detected and the rest of proteins in the clusters will be selected as attachments to form the final predicted protein complexes. The experiments on yeast data show that WPNCA outperforms the existing methods in terms of both accuracy and p-value. The software for WPNCA is available at "http://netlab.csu.edu.cn/bioinfomatics/weipeng/WPNCA/download.html".

  6. Analysis of protein-protein docking decoys using interaction fingerprints: application to the reconstruction of CaM-ligand complexes

    Directory of Open Access Journals (Sweden)

    Uchikoga Nobuyuki

    2010-05-01

    Full Text Available Abstract Background Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles. Results To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG, CaM kinase kinase (CaMKK and the plasma membrane Ca2+ ATPase pump (PMCA, and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK. Conclusions The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases.

  7. Involvement of the iron regulatory protein from Eisenia andrei earthworms in the regulation of cellular iron homeostasis.

    Directory of Open Access Journals (Sweden)

    Petra Procházková

    Full Text Available Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs of the 5'- or 3'-untranslated regions (UTR of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP. The earthworm IRE site in 5'-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.

  8. Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

    Science.gov (United States)

    Procházková, Petra; Škanta, František; Roubalová, Radka; Šilerová, Marcela; Dvořák, Jiří; Bilej, Martin

    2014-01-01

    Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant. PMID:25279857

  9. A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth.

    Science.gov (United States)

    Viloria, Katrina; Munasinghe, Amanda; Asher, Sharan; Bogyere, Roberto; Jones, Lucy; Hill, Natasha J

    2016-11-25

    SPARC is a matricellular protein that is involved in both pancreatic cancer and diabetes. It belongs to a wider family of proteins that share structural and functional similarities. Relatively little is known about this extended family, but evidence of regulatory interactions suggests the importance of a holistic approach to their study. We show that Hevin, SPOCKs, and SMOCs are strongly expressed within islets, ducts, and blood vessels, suggesting important roles for these proteins in the normal pancreas, while FSTL-1 expression is localised to the stromal compartment reminiscent of SPARC. In direct contrast to SPARC, however, FSTL-1 expression is reduced in pancreatic cancer. Consistent with this, FSTL-1 inhibited pancreatic cancer cell proliferation. The complexity of SPARC family proteins is further revealed by the detection of multiple cell-type specific isoforms that arise due to a combination of post-translational modification and alternative splicing. Identification of splice variants lacking a signal peptide suggests the existence of novel intracellular isoforms. This study underlines the importance of addressing the complexity of the SPARC family and provides a new framework to explain their controversial and contradictory effects. We also demonstrate for the first time that FSTL-1 suppresses pancreatic cancer cell growth.

  10. WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

    Energy Technology Data Exchange (ETDEWEB)

    Herrou, Julien; Czyż, Daniel M.; Willett, Jonathan W.; Kim, Hye-Sook; Chhor, Gekleng; Babnigg, Gyorgy; Kim, Youngchang; Crosson, Sean; Stock, A. M.

    2016-02-08

    ABSTRACT

    The general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpAwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite

  11. Supervised maximum-likelihood weighting of composite protein networks for complex prediction

    Directory of Open Access Journals (Sweden)

    Yong Chern Han

    2012-12-01

    Full Text Available Abstract Background Protein complexes participate in many important cellular functions, so finding the set of existent complexes is essential for understanding the organization and regulation of processes in the cell. With the availability of large amounts of high-throughput protein-protein interaction (PPI data, many algorithms have been proposed to discover protein complexes from PPI networks. However, such approaches are hindered by the high rate of noise in high-throughput PPI data, including spurious and missing interactions. Furthermore, many transient interactions are detected between proteins that are not from the same complex, while not all proteins from the same complex may actually interact. As a result, predicted complexes often do not match true complexes well, and many true complexes go undetected. Results We address these challenges by integrating PPI data with other heterogeneous data sources to construct a composite protein network, and using a supervised maximum-likelihood approach to weight each edge based on its posterior probability of belonging to a complex. We then use six different clustering algorithms, and an aggregative clustering strategy, to discover complexes in the weighted network. We test our method on Saccharomyces cerevisiae and Homo sapiens, and show that complex discovery is improved: compared to previously proposed supervised and unsupervised weighting approaches, our method recalls more known complexes, achieves higher precision at all recall levels, and generates novel complexes of greater functional similarity. Furthermore, our maximum-likelihood approach allows learned parameters to be used to visualize and evaluate the evidence of novel predictions, aiding human judgment of their credibility. Conclusions Our approach integrates multiple data sources with supervised learning to create a weighted composite protein network, and uses six clustering algorithms with an aggregative clustering strategy to

  12. Inhibitors Alter the Stochasticity of Regulatory Proteins to Force Cells to Switch to the Other State in the Bistable System.

    Science.gov (United States)

    Jhang, Wun-Sin; Lo, Shih-Chiang; Yeh, Chen-Chao; Shu, Che-Chi

    2017-06-30

    The cellular behaviors under the control of genetic circuits are subject to stochastic fluctuations, or noise. The stochasticity in gene regulation, far from a nuisance, has been gradually appreciated for its unusual function in cellular activities. In this work, with Chemical Master Equation (CME), we discovered that the addition of inhibitors altered the stochasticity of regulatory proteins. For a bistable system of a mutually inhibitory network, such a change of noise led to the migration of cells in the bimodal distribution. We proposed that the consumption of regulatory protein caused by the addition of inhibitor is not the only reason for pushing cells to the specific state; the change of the intracellular stochasticity is also the main cause for the redistribution. For the level of the inhibitor capable of driving 99% of cells, if there is no consumption of regulatory protein, 88% of cells were guided to the specific state. It implied that cells were pushed, by the inhibitor, to the specific state due to the change of stochasticity.

  13. Tuning structure of oppositely charged nanoparticle and protein complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Sugam, E-mail: sugam@barc.gov.in; Aswal, V. K., E-mail: sugam@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai-400085 (India); Callow, P. [Institut Laue Langevin, DS/LSS, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9 (France)

    2014-04-24

    Small-angle neutron scattering (SANS) has been used to probe the structures of anionic silica nanoparticles (LS30) and cationic lyszyme protein (M.W. 14.7kD, I.P. ∼ 11.4) by tuning their interaction through the pH variation. The protein adsorption on nanoparticles is found to be increasing with pH and determined by the electrostatic attraction between two components as well as repulsion between protein molecules. We show the strong electrostatic attraction between nanoparticles and protein molecules leads to protein-mediated aggregation of nanoparticles which are characterized by fractal structures. At pH 5, the protein adsorption gives rise to nanoparticle aggregation having surface fractal morphology with close packing of nanoparticles. The surface fractals transform to open structures of mass fractal morphology at higher pH (7 and 9) on approaching isoelectric point (I.P.)

  14. Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

    Science.gov (United States)

    Herbschleb-Voogt, E; Ten Kate, J; Meera Khan, P

    1983-01-01

    Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues.

  15. Regulatory CD4 T cells inhibit HIV-1 expression of other CD4 T cell subsets via interactions with cell surface regulatory proteins.

    Science.gov (United States)

    Zhang, Mingce; Robinson, Tanya O; Duverger, Alexandra; Kutsch, Olaf; Heath, Sonya L; Cron, Randy Q

    2018-03-01

    During chronic HIV-1 infection, regulatory CD4 T cells (Tregs) frequently represent the largest subpopulation of CD4 T cell subsets, implying relative resistant to HIV-1. When HIV-1 infection of CD4 T cells was explored in vitro and ex vivo from patient samples, Tregs possessed lower levels of HIV-1 DNA and RNA in comparison with conventional effector and memory CD4 T cells. Moreover, Tregs suppressed HIV-1 expression in other CD4 T cells in an in vitro co-culture system. This suppression was mediated in part via multiple inhibitory surface proteins expressed on Tregs. Antibody blockade of CTLA-4, PD-1, and GARP on Tregs resulted in increased HIV-1 DNA integration and mRNA expression in neighboring CD4 T cells. Moreover, antibody blockade of Tregs inhibitory proteins resulted in increased HIV-1 LTR transcription in co-cultured CD4 T cells. Thus, Tregs inhibit HIV-1 infection of other CD4 T cell subsets via interactions with inhibitory cell surface proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. PipY, a Member of the Conserved COG0325 Family of PLP-Binding Proteins, Expands the Cyanobacterial Nitrogen Regulatory Network

    Directory of Open Access Journals (Sweden)

    José I. Labella

    2017-07-01

    Full Text Available Synechococcus elongatus PCC 7942 is a paradigmatic model organism for nitrogen regulation in cyanobacteria. Expression of genes involved in nitrogen assimilation is positively regulated by the 2-oxoglutarate receptor and global transcriptional regulator NtcA. Maximal activation requires the subsequent binding of the co-activator PipX. PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance, binds to PipX to counteract NtcA activity at low 2-oxoglutarate levels. PII-PipX complexes can also bind to the transcriptional regulator PlmA, whose regulon remains unknown. Here we expand the nitrogen regulatory network to PipY, encoded by the bicistronic operon pipXY in S. elongatus. Work with PipY, the cyanobacterial member of the widespread family of COG0325 proteins, confirms the conserved roles in vitamin B6 and amino/keto acid homeostasis and reveals new PLP-related phenotypes, including sensitivity to antibiotics targeting essential PLP-holoenzymes or synthetic lethality with cysK. In addition, the related phenotypes of pipY and pipX mutants are consistent with genetic interactions in the contexts of survival to PLP-targeting antibiotics and transcriptional regulation. We also showed that PipY overexpression increased the length of S. elongatus cells. Taken together, our results support a universal regulatory role for COG0325 proteins, paving the way to a better understanding of these proteins and of their connections with other biological processes.

  17. Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

    Science.gov (United States)

    Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

    2008-02-22

    One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

  18. Identification and characterization of stable membrane protein complexes

    NARCIS (Netherlands)

    Spelbrink, R.E.J.

    2007-01-01

    Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

  19. Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

    Science.gov (United States)

    Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

    2018-03-01

    The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

  20. Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex.

    Science.gov (United States)

    Sorusch, Nasrin; Bauß, Katharina; Plutniok, Janet; Samanta, Ananya; Knapp, Barbara; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe

    2017-03-15

    The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Topological properties of complex networks in protein structures

    Science.gov (United States)

    Kim, Kyungsik; Jung, Jae-Won; Min, Seungsik

    2014-03-01

    We study topological properties of networks in structural classification of proteins. We model the native-state protein structure as a network made of its constituent amino-acids and their interactions. We treat four structural classes of proteins composed predominantly of α helices and β sheets and consider several proteins from each of these classes whose sizes range from amino acids of the Protein Data Bank. Particularly, we simulate and analyze the network metrics such as the mean degree, the probability distribution of degree, the clustering coefficient, the characteristic path length, the local efficiency, and the cost. This work was supported by the KMAR and DP under Grant WISE project (153-3100-3133-302-350).

  2. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    International Nuclear Information System (INIS)

    Paulin, Sarah; Rosado, Helena; Taylor, Peter W; Jamshad, Mohammed; Dafforn, Timothy R; Garcia-Lara, Jorge; Foster, Simon J; Galley, Nicola F; Roper, David I

    2014-01-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function. (paper)

  3. Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

    Science.gov (United States)

    Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

    2014-07-01

    Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

  4. Predicting protein complexes using a supervised learning method combined with local structural information.

    Science.gov (United States)

    Dong, Yadong; Sun, Yongqi; Qin, Chao

    2018-01-01

    The existing protein complex detection methods can be broadly divided into two categories: unsupervised and supervised learning methods. Most of the unsupervised learning methods assume that protein complexes are in dense regions of protein-protein interaction (PPI) networks even though many true complexes are not dense subgraphs. Supervised learning methods utilize the informative properties of known complexes; they often extract features from existing complexes and then use the features to train a classification model. The trained model is used to guide the search process for new complexes. However, insufficient extracted features, noise in the PPI data and the incompleteness of complex data make the classification model imprecise. Consequently, the classification model is not sufficient for guiding the detection of complexes. Therefore, we propose a new robust score function that combines the classification model with local structural information. Based on the score function, we provide a search method that works both forwards and backwards. The results from experiments on six benchmark PPI datasets and three protein complex datasets show that our approach can achieve better performance compared with the state-of-the-art supervised, semi-supervised and unsupervised methods for protein complex detection, occasionally significantly outperforming such methods.

  5. Revisiting interaction specificity reveals neuronal and adipocyte Munc18 membrane fusion regulatory proteins differ in their binding interactions with partner SNARE Syntaxins.

    Directory of Open Access Journals (Sweden)

    Michelle P Christie

    Full Text Available The efficient delivery of cellular cargo relies on the fusion of cargo-carrying vesicles with the correct membrane at the correct time. These spatiotemporal fusion events occur when SNARE proteins on the vesicle interact with cognate SNARE proteins on the target membrane. Regulatory Munc18 proteins are thought to contribute to SNARE interaction specificity through interaction with the SNARE protein Syntaxin. Neuronal Munc18a interacts with Syntaxin1 but not Syntaxin4, and adipocyte Munc18c interacts with Syntaxin4 but not Syntaxin1. Here we show that this accepted view of specificity needs revision. We find that Munc18c interacts with both Syntaxin4 and Syntaxin1, and appears to bind "non-cognate" Syntaxin1 a little more tightly than Syntaxin4. Munc18a binds Syntaxin1 and Syntaxin4, though it interacts with its cognate Syntaxin1 much more tightly. We also observed that when bound to non-cognate Munc18c, Syntaxin1 captures its neuronal SNARE partners SNAP25 and VAMP2, and Munc18c can bind to pre-formed neuronal SNARE ternary complex. These findings reveal that Munc18a and Munc18c bind Syntaxins differently. Munc18c relies principally on the Syntaxin N-peptide interaction for binding Syntaxin4 or Syntaxin1, whereas Munc18a can bind Syntaxin1 tightly whether or not the Syntaxin1 N-peptide is present. We conclude that Munc18a and Munc18c differ in their binding interactions with Syntaxins: Munc18a has two tight binding modes/sites for Syntaxins as defined previously but Munc18c has just one that requires the N-peptide. These results indicate that the interactions between Munc18 and Syntaxin proteins, and the consequences for in vivo function, are more complex than can be accounted for by binding specificity alone.

  6. Finding low-conductance sets with dense interactions (FLCD) for better protein complex prediction.

    Science.gov (United States)

    Wang, Yijie; Qian, Xiaoning

    2017-03-14

    Intuitively, proteins in the same protein complexes should highly interact with each other but rarely interact with the other proteins in protein-protein interaction (PPI) networks. Surprisingly, many existing computational algorithms do not directly detect protein complexes based on both of these topological properties. Most of them, depending on mathematical definitions of either "modularity" or "conductance", have their own limitations: Modularity has the inherent resolution problem ignoring small protein complexes; and conductance characterizes the separability of complexes but fails to capture the interaction density within complexes. In this paper, we propose a two-step algorithm FLCD (Finding Low-Conductance sets with Dense interactions) to predict overlapping protein complexes with the desired topological structure, which is densely connected inside and well separated from the rest of the networks. First, FLCD detects well-separated subnetworks based on approximating a potential low-conductance set through a personalized PageRank vector from a protein and then solving a mixed integer programming (MIP) problem to find the minimum-conductance set within the identified low-conductance set. At the second step, the densely connected parts in those subnetworks are discovered as the protein complexes by solving another MIP problem that aims to find the dense subnetwork in the minimum-conductance set. Experiments on four large-scale yeast PPI networks from different public databases demonstrate that the complexes predicted by FLCD have better correspondence with the yeast protein complex gold standards than other three state-of-the-art algorithms (ClusterONE, LinkComm, and SR-MCL). Additionally, results of FLCD show higher biological relevance with respect to Gene Ontology (GO) terms by GO enrichment analysis.

  7. Polysaccharide charge density regulating protein adsorption to air/water interfaces by protein/polysaccharide complex formation

    NARCIS (Netherlands)

    Ganzevles, R.A.; Kosters, H.; Vliet, T. van; Stuart, M.A.C.; Jongh, H.H.J. de

    2007-01-01

    Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different

  8. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry

    DEFF Research Database (Denmark)

    Ho, Yuen; Gruhler, Albrecht; Heilbut, Adrian

    2002-01-01

    The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects...... as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were...... identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two...

  9. Conformational Heterogeneity in Antibody-Protein Antigen Recognition IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

    Czech Academy of Sciences Publication Activity Database

    Addis, P. W.; Hall, c. J.; Bruton, S.; Veverka, Václav; Wilkinson, I. C.; Muskett, F. W.; Renshaw, P. S.; Prosser, C. E.; Carrington, B.; Lawson, A. D. G.; Griffin, R.; Taylor, R. J.; Waters, L. C.; Henry, A. J.; Carr, M. D.

    2014-01-01

    Roč. 289, č. 10 (2014), s. 7200-7210 ISSN 0021-9258 Institutional support: RVO:61388963 Keywords : NMR * antibody * protein-protein interaction * protein conformation Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  10. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

    Directory of Open Access Journals (Sweden)

    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  11. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Science.gov (United States)

    Sardiu, Mihaela E; Gilmore, Joshua M; Carrozza, Michael J; Li, Bing; Workman, Jerry L; Florens, Laurence; Washburn, Michael P

    2009-10-06

    Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  12. Determining protein complex connectivity using a probabilistic deletion network derived from quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Mihaela E Sardiu

    2009-10-01

    Full Text Available Protein complexes are key molecular machines executing a variety of essential cellular processes. Despite the availability of genome-wide protein-protein interaction studies, determining the connectivity between proteins within a complex remains a major challenge. Here we demonstrate a method that is able to predict the relationship of proteins within a stable protein complex. We employed a combination of computational approaches and a systematic collection of quantitative proteomics data from wild-type and deletion strain purifications to build a quantitative deletion-interaction network map and subsequently convert the resulting data into an interdependency-interaction model of a complex. We applied this approach to a data set generated from components of the Saccharomyces cerevisiae Rpd3 histone deacetylase complexes, which consists of two distinct small and large complexes that are held together by a module consisting of Rpd3, Sin3 and Ume1. The resulting representation reveals new protein-protein interactions and new submodule relationships, providing novel information for mapping the functional organization of a complex.

  13. Evidence for the robustness of protein complexes to inter-species hybridization.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Leducq

    Full Text Available Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC, which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

  14. Leukemia: Derived heat shock protein gp96-peptide complex ...

    African Journals Online (AJOL)

    Jane

    2011-06-27

    Jun 27, 2011 ... Leukemia is a malignant clonal disease in hematopoietic stem cells that is typically treated with chemotherapy and radiotherapy. However ..... with autologous tumor-derived heatshock protein gp96 after liver resection for ...

  15. glue protein profiles in the nasuta–albomicans complex

    Indian Academy of Sciences (India)

    . Manasagangotri ... Further, quantitative analysis also shows lack of correlation between the chromosomal ... involving these two races followed by karyotypic screening of hybrid .... The molecular masses of the variable protein fractions were ...

  16. Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

    International Nuclear Information System (INIS)

    Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

    2010-01-01

    Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

  17. Protein complex detection in PPI networks based on data integration and supervised learning method.

    Science.gov (United States)

    Yu, Feng; Yang, Zhi; Hu, Xiao; Sun, Yuan; Lin, Hong; Wang, Jian

    2015-01-01

    Revealing protein complexes are important for understanding principles of cellular organization and function. High-throughput experimental techniques have produced a large amount of protein interactions, which makes it possible to predict protein complexes from protein-protein interaction (PPI) networks. However, the small amount of known physical interactions may limit protein complex detection. The new PPI networks are constructed by integrating PPI datasets with the large and readily available PPI data from biomedical literature, and then the less reliable PPI between two proteins are filtered out based on semantic similarity and topological similarity of the two proteins. Finally, the supervised learning protein complex detection (SLPC), which can make full use of the information of available known complexes, is applied to detect protein complex on the new PPI networks. The experimental results of SLPC on two different categories yeast PPI networks demonstrate effectiveness of the approach: compared with the original PPI networks, the best average improvements of 4.76, 6.81 and 15.75 percentage units in the F-score, accuracy and maximum matching ratio (MMR) are achieved respectively; compared with the denoising PPI networks, the best average improvements of 3.91, 4.61 and 12.10 percentage units in the F-score, accuracy and MMR are achieved respectively; compared with ClusterONE, the start-of the-art complex detection method, on the denoising extended PPI networks, the average improvements of 26.02 and 22.40 percentage units in the F-score and MMR are achieved respectively. The experimental results show that the performances of SLPC have a large improvement through integration of new receivable PPI data from biomedical literature into original PPI networks and denoising PPI networks. In addition, our protein complexes detection method can achieve better performance than ClusterONE.

  18. HTLV-1 Tax protein recruitment into IKKε and TBK1 kinase complexes enhances IFN-I expression.

    Science.gov (United States)

    Diani, Erica; Avesani, Francesca; Bergamo, Elisa; Cremonese, Giorgia; Bertazzoni, Umberto; Romanelli, Maria Grazia

    2015-02-01

    The Tax protein expressed by human T-cell leukemia virus type 1 (HTLV-1) plays a pivotal role in the deregulation of cellular pathways involved in the immune response, inflammation, cell survival, and cancer. Many of these effects derive from Tax multiple interactions with host factors, including the subunits of the IKK-complex that are required for NF-κB activation. IKKɛ and TBK1 are two IKK-related kinases that allow the phosphorylation of interferon regulatory factors that trigger IFN type I gene expression. We observed that IKKɛ and TBK1 recruit Tax into cellular immunocomplexes. We also found that TRAF3, which regulates cell receptor signaling effectors, forms complexes with Tax. Transactivation analyses revealed that expression of Tax, in presence of IKKɛ and TBK1, enhances IFN-β promoter activity, whereas the activation of NF-κB promoter is not modified. We propose that Tax may be recruited into the TBK1/IKKɛ complexes as a scaffolding-adaptor protein that enhances IFN-I gene expression. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Monte Carlo simulations of flexible polyanions complexing with whey proteins at their isoelectric point.

    Science.gov (United States)

    de Vries, R

    2004-02-15

    Electrostatic complexation of flexible polyanions with the whey proteins alpha-lactalbumin and beta-lactoglobulin is studied using Monte Carlo simulations. The proteins are considered at their respective isoelectric points. Discrete charges on the model polyelectrolytes and proteins interact through Debye-Huckel potentials. Protein excluded volume is taken into account through a coarse-grained model of the protein shape. Consistent with experimental results, it is found that alpha-lactalbumin complexes much more strongly than beta-lactoglobulin. For alpha-lactalbumin, strong complexation is due to localized binding to a single large positive "charge patch," whereas for beta-lactoglobulin, weak complexation is due to diffuse binding to multiple smaller charge patches. Copyright 2004 American Institute of Physics

  20. Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

    Science.gov (United States)

    Harner, Max; Neupert, Walter; Deponte, Marcel

    2011-07-15

    The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

  1. Generating functional analysis of complex formation and dissociation in large protein interaction networks

    International Nuclear Information System (INIS)

    Coolen, A C C; Rabello, S

    2009-01-01

    We analyze large systems of interacting proteins, using techniques from the non-equilibrium statistical mechanics of disordered many-particle systems. Apart from protein production and removal, the most relevant microscopic processes in the proteome are complex formation and dissociation, and the microscopic degrees of freedom are the evolving concentrations of unbound proteins (in multiple post-translational states) and of protein complexes. Here we only include dimer-complexes, for mathematical simplicity, and we draw the network that describes which proteins are reaction partners from an ensemble of random graphs with an arbitrary degree distribution. We show how generating functional analysis methods can be used successfully to derive closed equations for dynamical order parameters, representing an exact macroscopic description of the complex formation and dissociation dynamics in the infinite system limit. We end this paper with a discussion of the possible routes towards solving the nontrivial order parameter equations, either exactly (in specific limits) or approximately.

  2. Chaperonin Structure - The Large Multi-Subunit Protein Complex

    Directory of Open Access Journals (Sweden)

    Irena Roterman

    2009-03-01

    Full Text Available The multi sub-unit protein structure representing the chaperonins group is analyzed with respect to its hydrophobicity distribution. The proteins of this group assist protein folding supported by ATP. The specific axial symmetry GroEL structure (two rings of seven units stacked back to back - 524 aa each and the GroES (single ring of seven units - 97 aa each polypeptide chains are analyzed using the hydrophobicity distribution expressed as excess/deficiency all over the molecule to search for structure-to-function relationships. The empirically observed distribution of hydrophobic residues is confronted with the theoretical one representing the idealized hydrophobic core with hydrophilic residues exposure on the surface. The observed discrepancy between these two distributions seems to be aim-oriented, determining the structure-to-function relation. The hydrophobic force field structure generated by the chaperonin capsule is presented. Its possible influence on substrate folding is suggested.

  3. Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye.

    Science.gov (United States)

    Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie J H; Kim, Seonhee; Cho, Seo-Hee

    2015-01-01

    Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.

    Science.gov (United States)

    Tan, Chris Soon Heng; Go, Ka Diam; Bisteau, Xavier; Dai, Lingyun; Yong, Chern Han; Prabhu, Nayana; Ozturk, Mert Burak; Lim, Yan Ting; Sreekumar, Lekshmy; Lengqvist, Johan; Tergaonkar, Vinay; Kaldis, Philipp; Sobota, Radoslaw M; Nordlund, Pär

    2018-03-09

    Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  5. AMMOS2: a web server for protein-ligand-water complexes refinement via molecular mechanics.

    Science.gov (United States)

    Labbé, Céline M; Pencheva, Tania; Jereva, Dessislava; Desvillechabrol, Dimitri; Becot, Jérôme; Villoutreix, Bruno O; Pajeva, Ilza; Miteva, Maria A

    2017-07-03

    AMMOS2 is an interactive web server for efficient computational refinement of protein-small organic molecule complexes. The AMMOS2 protocol employs atomic-level energy minimization of a large number of experimental or modeled protein-ligand complexes. The web server is based on the previously developed standalone software AMMOS (Automatic Molecular Mechanics Optimization for in silico Screening). AMMOS utilizes the physics-based force field AMMP sp4 and performs optimization of protein-ligand interactions at five levels of flexibility of the protein receptor. The new version 2 of AMMOS implemented in the AMMOS2 web server allows the users to include explicit water molecules and individual metal ions in the protein-ligand complexes during minimization. The web server provides comprehensive analysis of computed energies and interactive visualization of refined protein-ligand complexes. The ligands are ranked by the minimized binding energies allowing the users to perform additional analysis for drug discovery or chemical biology projects. The web server has been extensively tested on 21 diverse protein-ligand complexes. AMMOS2 minimization shows consistent improvement over the initial complex structures in terms of minimized protein-ligand binding energies and water positions optimization. The AMMOS2 web server is freely available without any registration requirement at the URL: http://drugmod.rpbs.univ-paris-diderot.fr/ammosHome.php. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Heat shock protein 70 inhibits shrinkage-induced programmed cell death via mechanisms independent of effects on cell volume-regulatory membrane transport proteins

    DEFF Research Database (Denmark)

    Nylandsted, J; Jäättelä, M; Hoffmann, E K

    2004-01-01

    Cell shrinkage is a ubiquitous feature of programmed cell death (PCD), but whether it is an obligatory signalling event in PCD is unclear. Heat shock protein 70 (Hsp70) potently counteracts PCD in many cells, by mechanisms that are incompletely understood. In the present investigation, we found...... that severe hypertonic stress greatly diminished the viability of murine fibrosarcoma cells (WEHI-902) and immortalized murine embryonic fibroblasts (iMEFs). This effect was attenuated markedly by Hsp70 over-expression. To determine whether the protective effect of Hsp70 was mediated via an effect on volume...... regulatory ion transport, we compared regulatory volume decrease (RVD) and increase (RVI) in control WEHI-902 cells and after increasing Hsp70 levels by heat shock or over-expression (WEHI-912). Hsp70 levels affected neither RVD, RVI nor the relative contributions of the Na(+)/H(+)-exchanger (NHE1) and Na...

  7. On the interconnection of stable protein complexes: inter-complex hubs and their conservation in Saccharomyces cerevisiae and Homo sapiens networks.

    Science.gov (United States)

    Guerra, Concettina

    2015-01-01

    Protein complexes are key molecular entities that perform a variety of essential cellular functions. The connectivity of proteins within a complex has been widely investigated with both experimental and computational techniques. We developed a computational approach to identify and characterise proteins that play a role in interconnecting complexes. We computed a measure of inter-complex centrality, the crossroad index, based on disjoint paths connecting proteins in distinct complexes and identified inter-complex hubs as proteins with a high value of the crossroad index. We applied the approach to a set of stable complexes in Saccharomyces cerevisiae and in Homo sapiens. Just as done for hubs, we evaluated the topological and biological properties of inter-complex hubs addressing the following questions. Do inter-complex hubs tend to be evolutionary conserved? What is the relation between crossroad index and essentiality? We found a good correlation between inter-complex hubs and both evolutionary conservation and essentiality.

  8. Comparative evolutionary analysis of protein complexes in E. coli and yeast

    Directory of Open Access Journals (Sweden)

    Ranea Juan AG

    2010-02-01

    Full Text Available Abstract Background Proteins do not act in isolation; they frequently act together in protein complexes to carry out concerted cellular functions. The evolution of complexes is poorly understood, especially in organisms other than yeast, where little experimental data has been available. Results We generated accurate, high coverage datasets of protein complexes for E. coli and yeast in order to study differences in the evolution of complexes between these two species. We show that substantial differences exist in how complexes have evolved between these organisms. A previously proposed model of complex evolution identified complexes with cores of interacting homologues. We support findings of the relative importance of this mode of evolution in yeast, but find that it is much less common in E. coli. Additionally it is shown that those homologues which do cluster in complexes are involved in eukaryote-specific functions. Furthermore we identify correlated pairs of non-homologous domains which occur in multiple protein complexes. These were identified in both yeast and E. coli and we present evidence that these too may represent complex cores in yeast but not those of E. coli. Conclusions Our results suggest that there are differences in the way protein complexes have evolved in E. coli and yeast. Whereas some yeast complexes have evolved by recruiting paralogues, this is not apparent in E. coli. Furthermore, such complexes are involved in eukaryotic-specific functions. This implies that the increase in gene family sizes seen in eukaryotes in part reflects multiple family members being used within complexes. However, in general, in both E. coli and yeast, homologous domains are used in different complexes.

  9. The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism.

    Science.gov (United States)

    McRae, Steven; Iqbal, Jawed; Sarkar-Dutta, Mehuli; Lane, Samantha; Nagaraj, Abhiram; Ali, Naushad; Waris, Gulam

    2016-02-12

    Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with

  10. Formation of protein-birnessite complex: XRD, FTIR, and AFM analysis.

    Science.gov (United States)

    Naidja, A; Liu, C; Huang, P M

    2002-07-01

    Limited information is available on formation chemistry of enzyme-Mn oxide complexes. Adsorption isotherm of protein molecules (tyrosinase) on birnessite (delta-MnO(2)) at pH 6.0 and room temperature (23 degrees C) was of H type, indicating a very high affinity of the enzyme protein molecules to the birnessite mineral surfaces. After thorough washing of the protein-mineral complex with deionized-distilled water, up to 89% of adsorbed protein molecules remained bound to the mineral surfaces. When a high amount of the protein was immobilized, the X-ray diffractogram shows a significant decrease in the intensity of characteristic d-spacings of birnessite. No shift to higher values of the d-spacings of protein-birnessite complex was observed, indicating that the enzyme molecules were not intercalated in the mineral structure but immobilized at the external surfaces and the edges of the mineral oxide. By comparison to the free enzyme, infrared absorption spectra of the protein-birnessite complexes show a shift by up to 11 cm(-1) to lower frequencies in the absorption bands characteristic of amide I and II modes of the polypeptides chains. The mineral surfaces exerted some strain on the protein structure, resulting in an alteration of the protein molecular conformation after binding to the mineral colloid surfaces. In the free state, the globular protein molecules had a spheroid shape with an average cross-sectional diameter of 70+/-6 nm. The unfolding and flattening of the protein molecules after immobilization is clearly shown in atomic force micrographs. Compared to the tyrosinase-birnessite complex, similar FTIR spectra and atomic force micrographs were observed for the pure protein, bovine serum albumin (BSA), after immobilization on birnessite. The information obtained in this study is of fundamental significance for understanding birnessite as an adsorbent of biopolymers and the catalytic role of the enzyme-birnessite complex.

  11. Protein/polysaccharide complexes at air/water interfaces

    NARCIS (Netherlands)

    Ganzevles, R.A.

    2007-01-01

    KEYWORDS:protein, polysaccharide,In silico analysis, mapping of regulatory elements and corresponding dna-protein interaction in polyphenol oxidase gene promoter from different rice varieties

    International Nuclear Information System (INIS)

    Mahmood, T.; Rehman, M.; Aziz, E.

    2015-01-01

    Polyphenol oxidase (PPO) is an important enzyme that has positive impact regarding plant resistance against different biotic and abiotic stresses. In the present study PPO promoter from six different rice varieties was amplified and then analyzed for cis- and trans-acting elements. The study revealed a total of 79 different cis-acting regulatory elements including 11 elements restricted to only one or other variety. Among six varieties Pakhal-Basmati had highest number (5) of these elements, whereas C-622 and Rachna-Basmati have no such sequences. Rachna-Basmati, IR-36-Basmati and Kashmir- Basmati had 1, 2 and 3 unique elements, respectively. Different elementsrelated to pathogen, salt and water stresses were found, which may be helpful in controlling PPO activity according to changing environment. Moreover, HADDOCK was used to understand molecular mechanism of PPO regulation and it was found that DNA-protein interactions are stabilized by many potential hydrogen bonds. Adenine and arginine were the most reactive residues in DNA and proteins respectively.Structural comparison of different protein-DNA complexes show that even a highly conserved transcriptional factor can adopt different conformations when they contact a different DNA binding sequence, however their stable interactions depend on the number of hydrogen bonds formed and distance. (author)

  12. Carotenoid-protein complexes and their stability towards oxygen and radiation

    International Nuclear Information System (INIS)

    Ramakrishnan, T.V.; Francis, F.J.

    1980-01-01

    Carotenoid-protein complexes isolated from fresh mangoes were found to be more stable to oxygen and radiation when dissolved in water as compared with β-carotene in petroleum ether. Part of the pigment could be released from the complex by gamma irradiation. Observations on the stability of the carotenoid (98% β-carotene) in the complex indicated that the pigment is either associated with the lipid prosthetic group of the protein or loosely attached to the protein by weak hydrophobic bonds. (author)

  13. Rpa4, a homolog of the 34-kilodalton subunit of the replication protein A complex.

    OpenAIRE

    Keshav, K F; Chen, C; Dutta, A

    1995-01-01

    Replication protein A (RPA) is a complex of three polypeptides of 70, 34, and 13 kDa isolated from diverse eukaryotes. The complex is a single-stranded DNA-binding protein essential for simian virus 40-based DNA replication in vitro and for viability in the yeast Saccharomyces cerevisiae. We have identified a new 30-kDa human protein which interacts with the 70- and 13-kDa subunits of RPA, with a yeast two-hybrid/interaction trap method. This protein, Rpa4, has 47% identity with Rpa2, the 34-...

  14. Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells

    DEFF Research Database (Denmark)

    Vandamme, Julien; Völkel, Pamela; Rosnoblet, Claire

    2011-01-01

    Polycomb group (PcG) proteins maintain transcriptional repression of hundreds of genes involved in development, signaling or cancer using chromatin-based epigenetic mechanisms. Biochemical studies in Drosophila have revealed that PcG proteins associate in at least two classes of protein complexes...... known as Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Drosophila core PRC1 is composed of four subunits, Polycomb (Pc), Sex combs extra (Sce), Polyhomeotic (Ph), and Posterior sex combs (Psc). Each of these proteins has multiple orthologs in vertebrates classified respectively as the CBX, RING...... in order to identify interacting partners of CBX family proteins under the same experimental conditions. Our analysis identified with high confidence about 20 proteins co-eluted with CBX2 and CBX7 tagged proteins, about 40 with CBX4, and around 60 with CBX6 and CBX8. We provide evidences that the CBX...

  15. Structural and regulatory diversity shape HLA-C protein expression levels

    DEFF Research Database (Denmark)

    Kaur, Gurman; Gras, Stephanie; Mobbs, Jesse I

    2017-01-01

    expression of HLA-C allomorphs at the cell surface by influencing the structure of the peptide-binding cleft and the diversity of peptides bound by the HLA-C molecules. Together with a phylogenetic analysis, these results highlight the diversity and long-term balancing selection of regulatory factors...

  16. Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

    OpenAIRE

    Maffettone, Carmen; Chen, Guohua; Drozdov, Ignat; Ouzounis, Christos; Pantopoulos, Kostas

    2010-01-01

    Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2(Delta73) (lacking a specific insert of 73 amino acid...

  17. Novel RNA-binding properties of the MTG chromatin regulatory proteins

    NARCIS (Netherlands)

    S. Rossetti (Stefano); L. van Unen (Leontine); N. Sacchi; A.T. Hoogeveen (Andre)

    2008-01-01

    textabstractBackground: The myeloid translocation gene (MTG) proteins are non-DNA-binding transcriptional regulators capable of interacting with chromatin modifying proteins. As a consequence of leukemia-associated chromosomal translocations, two of the MTG proteins, MTG8 and MTG16, are fused to the

  18. Improving prediction of heterodimeric protein complexes using combination with pairwise kernel.

    Science.gov (United States)

    Ruan, Peiying; Hayashida, Morihiro; Akutsu, Tatsuya; Vert, Jean-Philippe

    2018-02-19

    Since many proteins become functional only after they interact with their partner proteins and form protein complexes, it is essential to identify the sets of proteins that form complexes. Therefore, several computational methods have been proposed to predict complexes from the topology and structure of experimental protein-protein interaction (PPI) network. These methods work well to predict complexes involving at least three proteins, but generally fail at identifying complexes involving only two different proteins, called heterodimeric complexes or heterodimers. There is however an urgent need for efficient methods to predict heterodimers, since the majority of known protein complexes are precisely heterodimers. In this paper, we use three promising kernel functions, Min kernel and two pairwise kernels, which are Metric Learning Pairwise Kernel (MLPK) and Tensor Product Pairwise Kernel (TPPK). We also consider the normalization forms of Min kernel. Then, we combine Min kernel or its normalization form and one of the pairwise kernels by plugging. We applied kernels based on PPI, domain, phylogenetic profile, and subcellular localization properties to predicting heterodimers. Then, we evaluate our method by employing C-Support Vector Classification (C-SVC), carrying out 10-fold cross-validation, and calculating the average F-measures. The results suggest that the combination of normalized-Min-kernel and MLPK leads to the best F-measure and improved the performance of our previous work, which had been the best existing method so far. We propose new methods to predict heterodimers, using a machine learning-based approach. We train a support vector machine (SVM) to discriminate interacting vs non-interacting protein pairs, based on informations extracted from PPI, domain, phylogenetic profiles and subcellular localization. We evaluate in detail new kernel functions to encode these data, and report prediction performance that outperforms the state-of-the-art.

  19. Protein complexes and cholesterol in the control of late endosomal dynamicsCholesterol and multi-protein complexes in the control of late endosomal dynamics

    NARCIS (Netherlands)

    Kant, Rik Henricus Nicolaas van der

    2013-01-01

    Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer’s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the

  1. 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...

  2. Implicit solvent simulations of DNA and DNA-protein complexes: Agreement with explicit solvent vs experiment

    Czech Academy of Sciences Publication Activity Database

    Chocholoušová, Jana; Feig, M.

    2006-01-01

    Roč. 110, č. 34 (2006), s. 17240-17251 ISSN 1520-6106 Keywords : implicit solvent * explicit solvent * protein DNA complex Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 4.115, year: 2006

  3. Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

    DEFF Research Database (Denmark)

    Chi, Celestine Ngang; Bach, Anders; Engström, Åke

    2011-01-01

    The E6 protein of human papillomavirus exhibits complex interaction patterns with several host proteins and their roles in HPV mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor......, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues...

  4. Contribution of Human Oral Cells to Astringency by Binding Salivary Protein/Tannin Complexes.

    Science.gov (United States)

    Soares, Susana; Ferrer-Galego, Raúl; Brandão, Elsa; Silva, Mafalda; Mateus, Nuno; Freitas, Victor de

    2016-10-10

    The most widely accepted mechanism to explain astringency is the interaction and precipitation of salivary proteins by food tannins, in particular proline-rich proteins. However, other mechanisms have been arising to explain astringency, such as binding of tannins to oral cells. In this work, an experimental method was adapted to study the possible contribution of both salivary proteins and oral cells to astringency induced by grape seed procyanidin fractions. Overall, in the absence of salivary proteins, the extent of procyanidin complexation with oral cells increased with increasing procyanidin degree of polymerization (mDP). Procyanidin fractions rich in monomers were the ones with the lowest ability to bind to oral cells. In the presence of salivary proteins and for procyanidins with mDP 2 the highest concentrations (1.5 and 2.0 mM) resulted in an increased binding of procyanidins to oral cells. This was even more evident for fractions III and IV at 1.0 mM and upper concentrations. Regarding the salivary proteins affected, it was possible to observe a decrease of P-B peptide and aPRP proteins for fractions II and III. This decrease is greater as the procyanidins' mDP increases. In fact, for fraction IV an almost total depletion of all salivary proteins was observed. This decrease is due to the formation of insoluble salivary protein/procyanidin complexes. Altogether, these data suggest that some procyanidins are able to bind to oral cells and that the salivary proteins interact with procyanidins forming salivary protein/procyanidin complexes that are also able to link to oral cells. The procyanidins that remain unbound to oral cells are able to bind to salivary proteins forming a large network of salivary protein/procyanidin complexes. Overall, the results presented herein provide one more step to understand food oral astringency onset.

  5. The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

    Directory of Open Access Journals (Sweden)

    Kris E Spaeth

    2009-09-01

    Full Text Available In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone. Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion". The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

  6. The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

    Science.gov (United States)

    Miura, Toyokazu; Takeo, Satoru; Ntege, Edward H; Otsuki, Hitoshi; Sawasaki, Tatsuya; Ishino, Tomoko; Takashima, Eizo; Tsuboi, Takafumi

    2018-06-02

    Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. 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.

  8. Research of the complex of functional and technological properties of animal protein

    Directory of Open Access Journals (Sweden)

    Олена Борисівна Дроменко

    2016-12-01

    Full Text Available The analysis of the results of analytical and practical research of the complex of functional and technological properties of animal protein Gelexcel A-95 as the basis for creation of complex functional additives is shown. The regularities of their changes are determined depending on technological factors. Rational parameters of animal protein rehydration, gelation conditions, emulsification for further use in the process of production of meat products are identified

  9. Gel and gel-free approaches for the quantitative characterisation of complex protein mixtures

    CSIR Research Space (South Africa)

    Buthelezi, S

    2012-10-01

    Full Text Available reliable set of methods for profiling proteins in a complex mixture in order to allow for the mining of low abundant species. To achieve this, several fractionation techniques were applied to samples of bovine hepatic tissue. These included two... further separated via low pH reverse phase (RP) chromatography before being introduced for mass spectrometric analysis. MATERIALS AND METHODS Figure 1: Study design to analyse a complex mixture of proteins extracted from hepatic tissue. To determine...

  10. Acute phase proteins in cattle after exposure to complex stress

    DEFF Research Database (Denmark)

    Lomborg, S. R.; Nielsen, L. R.; Heegaard, Peter M. H.

    2008-01-01

    Abstract Stressors such as weaning, mixing and transportation have been shown to lead to increased blood concentrations of acute phase proteins (APP), including serum amyloid A (SAA) and haptoglobin, in calves. This study was therefore undertaken to assess whether SAA and haptoglobin levels...... concentrations of SAA and haptoglobin increased significantly in response to the stressors (P...... in blood mirror stress in adult cattle. Six clinically healthy Holstein cows and two Holstein heifers were transported for four to six hours to a research facility, where each animal was housed in solitary tie stalls. Blood samples for evaluation of leukocyte counts and serum SAA and haptoglobin...

  11. Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS).

    Science.gov (United States)

    Lou, Tzu-Fang; Weidmann, Chase A; Killingsworth, Jordan; Tanaka Hall, Traci M; Goldstrohm, Aaron C; Campbell, Zachary T

    2017-04-15

    RNA-binding proteins (RBPs) collaborate to control virtually every aspect of RNA function. Tremendous progress has been made in the area of global assessment of RBP specificity using next-generation sequencing approaches both in vivo and in vitro. Understanding how protein-protein interactions enable precise combinatorial regulation of RNA remains a significant problem. Addressing this challenge requires tools that can quantitatively determine the specificities of both individual proteins and multimeric complexes in an unbiased and comprehensive way. One approach utilizes in vitro selection, high-throughput sequencing, and sequence-specificity landscapes (SEQRS). We outline a SEQRS experiment focused on obtaining the specificity of a multi-protein complex between Drosophila RBPs Pumilio (Pum) and Nanos (Nos). We discuss the necessary controls in this type of experiment and examine how the resulting data can be complemented with structural and cell-based reporter assays. Additionally, SEQRS data can be integrated with functional genomics data to uncover biological function. Finally, we propose extensions of the technique that will enhance our understanding of multi-protein regulatory complexes assembled onto RNA. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  13. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

    Directory of Open Access Journals (Sweden)

    Jens Milbradt

    2018-01-01

    Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  14. Effects of light and the regulatory B-subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae) infestation.

    Science.gov (United States)

    Rasool, Brwa; Karpinska, Barbara; Konert, Grzegorz; Durian, Guido; Denessiouk, Konstantin; Kangasjärvi, Saijaliisa; Foyer, Christine H

    2014-01-01

    The interactions between biotic and abiotic stress signaling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP)2A regulatory subunit B'γ (gamma; pp2a-b'γ) or B'ζ (zeta; pp2a-b'ζ1-1 and pp2a-b'ζ 1-2) and in gamma zeta double mutants (pp2a-b'γζ) lacking both subunits. All the mutants except for pp2a-b'ζ 1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b'γ mutant relative to the wild type but not in the pp2a-b'γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b'γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of B-subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonization, depending on the prevailing abiotic stress environment.

  15. Effects of light and the regulatory Beta subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae infestation

    Directory of Open Access Journals (Sweden)

    Brwa eRasool

    2014-08-01

    Full Text Available The interactions between biotic and abiotic stress signalling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP2A regulatory subunit B’γ (gamma; pp2a-b’γ or B’ζ (zeta; pp2a-b’ζ1-1 and pp2a-b’ζ1-2 and in gamma zeta double mutants (pp2a-b’γζ lacking both subunits. All the mutants except for pp2a-b’ζ1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b’γ mutant relative to the wild type but not in the pp2a-b’γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b’γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonisation, depending on the prevailing abiotic stress environment.

  16. Influence of Pea Protein Aggregates on the Structure and Stability of Pea Protein/Soybean Polysaccharide Complex Emulsions

    Directory of Open Access Journals (Sweden)

    Baoru Yin

    2015-03-01

    Full Text Available The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS, and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  17. Influence of pea protein aggregates on the structure and stability of pea protein/soybean polysaccharide complex emulsions.

    Science.gov (United States)

    Yin, Baoru; Zhang, Rujing; Yao, Ping

    2015-03-20

    The applications of plant proteins in the food and beverage industry have been hampered by their precipitation in acidic solution. In this study, pea protein isolate (PPI) with poor dispersibility in acidic solution was used to form complexes with soybean soluble polysaccharide (SSPS), and the effects of PPI aggregates on the structure and stability of PPI/SSPS complex emulsions were investigated. Under acidic conditions, high pressure homogenization disrupts the PPI aggregates and the electrostatic attraction between PPI and SSPS facilitates the formation of dispersible PPI/SSPS complexes. The PPI/SSPS complex emulsions prepared from the PPI containing aggregates prove to possess similar droplet structure and similar stability compared with the PPI/SSPS emulsions produced from the PPI in which the aggregates have been previously removed by centrifugation. The oil droplets are protected by PPI/SSPS complex interfacial films and SSPS surfaces. The emulsions show long-term stability against pH and NaCl concentration changes. This study demonstrates that PPI aggregates can also be used to produce stable complex emulsions, which may promote the applications of plant proteins in the food and beverage industry.

  18. Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

    Science.gov (United States)

    Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

    2010-02-16

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

  19. BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

    Science.gov (United States)

    Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

    2010-01-01

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

  20. A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

    Science.gov (United States)

    Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

    2014-05-01

    The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

  1. 'Fractional recovery' analysis of a presynaptic synaptotagmin 1-anchored endocytic protein complex.

    Directory of Open Access Journals (Sweden)

    Rajesh Khanna

    Full Text Available BACKGROUND: The integral synaptic vesicle protein and putative calcium sensor, synaptotagmin 1 (STG, has also been implicated in synaptic vesicle (SV recovery. However, proteins with which STG interacts during SV endocytosis remain poorly understood. We have isolated an STG-associated endocytic complex (SAE from presynaptic nerve terminals and have used a novel fractional recovery (FR assay based on electrostatic dissociation to identify SAE components and map the complex structure. The location of SAE in the presynaptic terminal was determined by high-resolution quantitative immunocytochemistry at the chick ciliary ganglion giant calyx-type synapse. METHODOLOGY/PRINCIPLE FINDINGS: The first step in FR analysis was to immunoprecipitate (IP the complex with an antibody against one protein component (the IP-protein. The immobilized complex was then exposed to a high salt (1150 mM stress-test that caused shedding of co-immunoprecipitated proteins (co-IP-proteins. A Fractional Recovery ratio (FR: recovery after high salt/recovery with control salt as assayed by Western blot was calculated for each co-IP-protein. These FR values reflect complex structure since an easily dissociated protein, with a low FR value, cannot be intermediary between the IP-protein and a salt-resistant protein. The structure of the complex was mapped and a blueprint generated with a pair of FR analyses generated using two different IP-proteins. The blueprint of SAE contains an AP180/X/STG/stonin 2/intersectin/epsin core (X is unknown and epsin is hypothesized, and an AP2 adaptor, H-/L-clathrin coat and dynamin scission protein perimeter. Quantitative immunocytochemistry (ICA/ICQ method at an isolated calyx-type presynaptic terminal indicates that this complex is associated with STG at the presynaptic transmitter release face but not with STG on intracellular synaptic vesicles. CONCLUSIONS/SIGNIFICANCE: We hypothesize that the SAE serves as a recognition site and also as a

  2. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    Science.gov (United States)

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  3. NMR spectroscopic and analytical ultracentrifuge analysis of membrane protein detergent complexes

    OpenAIRE

    Choe Senyon; Riek Roland; Johnson Casey; Kefala Georgia; Maslennikov Innokentiy; Kwiatkowski Witek

    2007-01-01

    Abstract Background Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization. Results Seeking an effective and standardized means applicable to genomic approaches for the characteriza...

  4. Phosphorylation of protein kinase A (PKA) regulatory subunit RIα by protein kinase G (PKG) primes PKA for catalytic activity in cells.

    Science.gov (United States)

    Haushalter, Kristofer J; Casteel, Darren E; Raffeiner, Andrea; Stefan, Eduard; Patel, Hemal H; Taylor, Susan S

    2018-03-23

    cAMP-dependent protein kinase (PKAc) is a pivotal signaling protein in eukaryotic cells. PKAc has two well-characterized regulatory subunit proteins, RI and RII (each having α and β isoforms), which keep the PKAc catalytic subunit in a catalytically inactive state until activation by cAMP. Previous reports showed that the RIα regulatory subunit is phosphorylated by cGMP-dependent protein kinase (PKG) in vitro , whereupon phosphorylated RIα no longer inhibits PKAc at normal (1:1) stoichiometric ratios. However, the significance of this phosphorylation as a mechanism for activating type I PKA holoenzymes has not been fully explored, especially in cellular systems. In this study, we further examined the potential of RIα phosphorylation to regulate physiologically relevant "desensitization" of PKAc activity. First, the serine 101 site of RIα was validated as a target of PKGIα phosphorylation both in vitro and in cells. Analysis of a phosphomimetic substitution in RIα (S101E) showed that modification of this site increases PKAc activity in vitro and in cells, even without cAMP stimulation. Numerous techniques were used to show that although Ser 101 variants of RIα can bind PKAc, the modified linker region of the S101E mutant has a significantly reduced affinity for the PKAc active site. These findings suggest that RIα phosphorylation may be a novel mechanism to circumvent the requirement of cAMP stimulus to activate type I PKA in cells. We have thus proposed a model to explain how PKG phosphorylation of RIα creates a "sensitized intermediate" state that is in effect primed to trigger PKAc activity.

  5. Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

    Directory of Open Access Journals (Sweden)

    Edyta Marcon

    2014-07-01

    Full Text Available Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

  6. Evidence for the interaction of the regulatory protein Ki-1/57 with p53 and its interacting proteins

    International Nuclear Information System (INIS)

    Nery, Flavia C.; Rui, Edmilson; Kuniyoshi, Tais M.; Kobarg, Joerg

    2006-01-01

    Ki-1/57 is a cytoplasmic and nuclear phospho-protein of 57 kDa and interacts with the adaptor protein RACK1, the transcription factor MEF2C, and the chromatin remodeling factor CHD3, suggesting that it might be involved in the regulation of transcription. Here, we describe yeast two-hybrid studies that identified a total of 11 proteins interacting with Ki-1/57, all of which interact or are functionally associated with p53 or other members of the p53 family of proteins. We further found that Ki-1/57 is able to interact with p53 itself in the yeast two-hybrid system when the interaction was tested directly. This interaction could be confirmed by pull down assays with purified proteins in vitro and by reciprocal co-immunoprecipitation assays from the human Hodgkin analogous lymphoma cell line L540. Furthermore, we found that the phosphorylation of p53 by PKC abolishes its interaction with Ki-1/57 in vitro

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

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    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.

  8. A 3D model of the membrane protein complex formed by the white spot syndrome virus structural proteins.

    Directory of Open Access Journals (Sweden)

    Yun-Shiang Chang

    Full Text Available BACKGROUND: Outbreaks of white spot disease have had a large negative economic impact on cultured shrimp worldwide. However, the pathogenesis of the causative virus, WSSV (whit spot syndrome virus, is not yet well understood. WSSV is a large enveloped virus. The WSSV virion has three structural layers surrounding its core DNA: an outer envelope, a tegument and a nucleocapsid. In this study, we investigated the protein-protein interactions of the major WSSV structural proteins, including several envelope and tegument proteins that are known to be involved in the infection process. PRINCIPAL FINDINGS: In the present report, we used coimmunoprecipitation and yeast two-hybrid assays to elucidate and/or confirm all the interactions that occur among the WSSV structural (envelope and tegument proteins VP51A, VP19, VP24, VP26 and VP28. We found that VP51A interacted directly not only with VP26 but also with VP19 and VP24. VP51A, VP19 and VP24 were also shown to have an affinity for self-interaction. Chemical cross-linking assays showed that these three self-interacting proteins could occur as dimers. CONCLUSIONS: From our present results in conjunction with other previously established interactions we construct a 3D model in which VP24 acts as a core protein that directly associates with VP26, VP28, VP38A, VP51A and WSV010 to form a membrane-associated protein complex. VP19 and VP37 are attached to this complex via association with VP51A and VP28, respectively. Through the VP26-VP51C interaction this envelope complex is anchored to the nucleocapsid, which is made of layers of rings formed by VP664. A 3D model of the nucleocapsid and the surrounding outer membrane is presented.

  9. Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy

    NARCIS (Netherlands)

    de Theije, Chiel C.; Langen, Ramon C. J.; Lamers, Wouter H.; Schols, Annemie M. W. J.; Köhler, S. Eleonore

    2013-01-01

    The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food

  10. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  11. Identification of chromatophore membrane protein complexes formed under different nitrogen availability conditions in Rhodospirillum rubrum

    DEFF Research Database (Denmark)

    Selao, Tiago Toscano; Branca, Rui; Chae, Pil Seok

    2011-01-01

    of two-dimensional Blue Native/SDS-PAGE and NSI-LC-LTQ-Orbitrap mass spectrometry. We have identified several membrane protein complexes, including components of the ATP synthase, reaction center, light harvesting, and NADH dehydrogenase complexes. Additionally, we have identified differentially...

  12. Predicting co-complexed protein pairs using genomic and proteomic data integration

    Directory of Open Access Journals (Sweden)

    King Oliver D

    2004-04-01

    Full Text Available Abstract Background Identifying all protein-protein interactions in an organism is a major objective of proteomics. A related goal is to know which protein pairs are present in the same protein complex. High-throughput methods such as yeast two-hybrid (Y2H and affinity purification coupled with mass spectrometry (APMS have been used to detect interacting proteins on a genomic scale. However, both Y2H and APMS methods have substantial false-positive rates. Aside from high-throughput interaction screens, other gene- or protein-pair characteristics may also be informative of physical interaction. Therefore it is desirable to integrate multiple datasets and utilize their different predictive value for more accurate prediction of co-complexed relationship. Results Using a supervised machine learning approach – probabilistic decision tree, we integrated high-throughput protein interaction datasets and other gene- and protein-pair characteristics to predict co-complexed pairs (CCP of proteins. Our predictions proved more sensitive and specific than predictions based on Y2H or APMS methods alone or in combination. Among the top predictions not annotated as CCPs in our reference set (obtained from the MIPS complex catalogue, a significant fraction was found to physically interact according to a separate database (YPD, Yeast Proteome Database, and the remaining predictions may potentially represent unknown CCPs. Conclusions We demonstrated that the probabilistic decision tree approach can be successfully used to predict co-complexed protein (CCP pairs from other characteristics. Our top-scoring CCP predictions provide testable hypotheses for experimental validation.

  13. Analysis of Native-Like Proteins and Protein Complexes Using Cation to Anion Proton Transfer Reactions (CAPTR)

    Science.gov (United States)

    Laszlo, Kenneth J.; Bush, Matthew F.

    2015-12-01

    Mass spectra of native-like protein complexes often exhibit narrow charge-state distributions, broad peaks, and contributions from multiple, coexisting species. These factors can make it challenging to interpret those spectra, particularly for mixtures with significant heterogeneity. Here we demonstrate the use of ion/ion proton transfer reactions to reduce the charge states of m/ z-selected, native-like ions of proteins and protein complexes, a technique that we refer to as cation to anion proton transfer reactions (CAPTR). We then demonstrate that CAPTR can increase the accuracy of charge state assignments and the resolution of interfering species in native mass spectrometry. The CAPTR product ion spectra for pyruvate kinase exhibit ~30 peaks and enable unambiguous determination of the charge state of each peak, whereas the corresponding precursor spectra exhibit ~6 peaks and the assigned charge states have an uncertainty of ±3%. 15+ bovine serum albumin and 21+ yeast enolase dimer both appear near m/ z 4450 and are completely unresolved in a mixture. After a single CAPTR event, the resulting product ions are baseline resolved. The separation of the product ions increases dramatically after each subsequent CAPTR event; 12 events resulted in a 3000-fold improvement in separation relative to the precursor ions. Finally, we introduce a framework for interpreting and predicting the figures of merit for CAPTR experiments. More generally, these results suggest that CAPTR strongly complements other mass spectrometry tools for analyzing proteins and protein complexes, particularly those in mixtures.

  14. Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

    2015-08-28

    Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.

  15. In situ detection of a heat-shock regulatory element binding protein using a soluble short synthetic enhancer sequence

    Energy Technology Data Exchange (ETDEWEB)

    Harel-Bellan, A; Brini, A T; Farrar, W L [National Cancer Institute, Frederick, MD (USA); Ferris, D K [Program Resources, Inc., Frederick, MD (USA); Robin, P [Institut Gustave Roussy, Villejuif (France)

    1989-06-12

    In various studies, enhancer binding proteins have been successfully absorbed out by competing sequences inserted into plasmids, resulting in the inhibition of the plasmid expression. Theoretically, such a result could be achieved using synthetic enhancer sequences not inserted into plasmids. In this study, a double stranded DNA sequence corresponding to the human heat shock regulatory element was chemically synthesized. By in vitro retardation assays, the synthetic sequence was shown to bind specifically a protein in extracts from the human T cell line Jurkat. When the synthetic enhancer was electroporated into Jurkat cells, not only the enhancer was shown to remain undegraded into the cells for up to 2 days, but also its was shown to bind intracellularly a protein. The binding was specific and was modulated upon heat shock. Furthermore, the binding protein was shown to be of the expected molecular weight by UV crosslinking. However, when the synthetic enhancer element was co-electroporated with an HSP 70-CAT reporter construct, the expression of the reporter plasmid was consistently enhanced in the presence of the exogenous synthetic enhancer.

  16. PRODIGY : a web server for predicting the binding affinity of protein-protein complexes

    NARCIS (Netherlands)

    Xue, Li; Garcia Lopes Maia Rodrigues, João; Kastritis, Panagiotis L; Bonvin, Alexandre Mjj; Vangone, Anna

    2016-01-01

    Gaining insights into the structural determinants of protein-protein interactions holds the key for a deeper understanding of biological functions, diseases and development of therapeutics. An important aspect of this is the ability to accurately predict the binding strength for a given

  17. Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

    Science.gov (United States)

    Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

    2017-07-01

    Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Nanoparticle-Fusion Protein Complexes Protect against Mycobacterium tuberculosis Infection.

    Science.gov (United States)

    Hart, Peter; Copland, Alastair; Diogo, Gil Reynolds; Harris, Shane; Spallek, Ralf; Oehlmann, Wulf; Singh, Mahavir; Basile, Juan; Rottenberg, Martin; Paul, Matthew John; Reljic, Rajko

    2018-03-07

    Tuberculosis (TB) is the leading cause of death from infectious disease, and the current vaccine, Bacillus Calmette-Guerin (BCG), is inadequate. Nanoparticles (NPs) are an emerging vaccine technology, with recent successes in oncology and infectious diseases. NPs have been exploited as antigen delivery systems and also for their adjuvantic properties. However, the mechanisms underlying their immunological activity remain obscure. Here, we developed a novel mucosal TB vaccine (Nano-FP1) based upon yellow carnauba wax NPs (YC-NPs), coated with a fusion protein consisting of three Mycobacterium tuberculosis (Mtb) antigens: Acr, Ag85B, and HBHA. Mucosal immunization of BCG-primed mice with Nano-FP1 significantly enhanced protection in animals challenged with low-dose, aerosolized Mtb. Bacterial control by Nano-FP1 was associated with dramatically enhanced cellular immunity compared to BCG, including superior CD4 + and CD8 + T cell proliferation, tissue-resident memory T cell (Trm) seeding in the lungs, and cytokine polyfunctionality. Alongside these effects, we also observed potent humoral responses, such as the generation of Ag85B-specific serum IgG and respiratory IgA. Finally, we found that YC-NPs were able to activate antigen-presenting cells via an unconventional IRF-3-associated activation signature, without the production of potentially harmful inflammatory mediators, providing a mechanistic framework for vaccine efficacy and future development. Copyright © 2017. Published by Elsevier Inc.

  19. Pancreatic Islet Protein Complexes and Their Dysregulation in Type 2 Diabetes

    DEFF Research Database (Denmark)

    Pedersen, Helle Krogh; Gudmundsdottir, Valborg; Brunak, Søren

    2017-01-01

    Type 2 diabetes (T2D) is a complex disease that involves multiple genes. Numerous risk loci have already been associated with T2D, although many susceptibility genes remain to be identified given heritability estimates. Systems biology approaches hold potential for discovering novel T2D genes...... by considering their biological context, such as tissue-specific protein interaction partners. Pancreatic islets are a key T2D tissue and many of the known genetic risk variants lead to impaired islet function, hence a better understanding of the islet-specific dysregulation in the disease-state is essential...... to unveil the full potential of person-specific profiles. Here we identify 3,692 overlapping pancreatic islet protein complexes (containing 10,805 genes) by integrating islet gene and protein expression data with protein interactions. We found 24 of these complexes to be significantly enriched for genes...

  20. MFIB: a repository of protein complexes with mutual folding induced by binding.

    Science.gov (United States)

    Fichó, Erzsébet; Reményi, István; Simon, István; Mészáros, Bálint

    2017-11-15

    It is commonplace that intrinsically disordered proteins (IDPs) are involved in crucial interactions in the living cell. However, the study of protein complexes formed exclusively by IDPs is hindered by the lack of data and such analyses remain sporadic. Systematic studies benefited other types of protein-protein interactions paving a way from basic science to therapeutics; yet these efforts require reliable datasets that are currently lacking for synergistically folding complexes of IDPs. Here we present the Mutual Folding Induced by Binding (MFIB) database, the first systematic collection of complexes formed exclusively by IDPs. MFIB contains an order of magnitude more data than any dataset used in corresponding studies and offers a wide coverage of known IDP complexes in terms of flexibility, oligomeric composition and protein function from all domains of life. The included complexes are grouped using a hierarchical classification and are complemented with structural and functional annotations. MFIB is backed by a firm development team and infrastructure, and together with possible future community collaboration it will provide the cornerstone for structural and functional studies of IDP complexes. MFIB is freely accessible at http://mfib.enzim.ttk.mta.hu/. The MFIB application is hosted by Apache web server and was implemented in PHP. To enrich querying features and to enhance backend performance a MySQL database was also created. simon.istvan@ttk.mta.hu, meszaros.balint@ttk.mta.hu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

  1. Identification of Protein Complexes from Tandem Affinity Purification/Mass Spectrometry Data via Biased Random Walk.

    Science.gov (United States)

    Cai, Bingjing; Wang, Haiying; Zheng, Huiru; Wang, Hui

    2015-01-01

    Systematic identification of protein complexes from protein-protein interaction networks (PPIs) is an important application of data mining in life science. Over the past decades, various new clustering techniques have been developed based on modelling PPIs as binary relations. Non-binary information of co-complex relations (prey/bait) in PPIs data derived from tandem affinity purification/mass spectrometry (TAP-MS) experiments has been unfairly disregarded. In this paper, we propose a Biased Random Walk based algorithm for detecting protein complexes from TAP-MS data, resulting in the random walk with restarting baits (RWRB). RWRB is developed based on Random walk with restart. The main contribution of RWRB is the incorporation of co-complex relations in TAP-MS PPI networks into the clustering process, by implementing a new restarting strategy during the process of random walk. Through experimentation on un-weighted and weighted TAP-MS data sets, we validated biological significance of our results by mapping them to manually curated complexes. Results showed that, by incorporating non-binary, co-membership information, significant improvement has been achieved in terms of both statistical measurements and biological relevance. Better accuracy demonstrates that the proposed method outperformed several state-of-the-art clustering algorithms for the detection of protein complexes in TAP-MS data.

  2. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    . TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.

  3. Selection on Coding and Regulatory Variation Maintains Individuality in Major Urinary Protein Scent Marks in Wild Mice.

    Directory of Open Access Journals (Sweden)

    Michael J Sheehan

    2016-03-01

    Full Text Available Recognition of individuals by scent is widespread across animal taxa. Though animals can often discriminate chemical blends based on many compounds, recent work shows that specific protein pheromones are necessary and sufficient for individual recognition via scent marks in mice. The genetic nature of individuality in scent marks (e.g. coding versus regulatory variation and the evolutionary processes that maintain diversity are poorly understood. The individual signatures in scent marks of house mice are the protein products of a group of highly similar paralogs in the major urinary protein (Mup gene family. Using the offspring of wild-caught mice, we examine individuality in the major urinary protein (MUP scent marks at the DNA, RNA and protein levels. We show that individuality arises through a combination of variation at amino acid coding sites and differential transcription of central Mup genes across individuals, and we identify eSNPs in promoters. There is no evidence of post-transcriptional processes influencing phenotypic diversity as transcripts accurately predict the relative abundance of proteins in urine samples. The match between transcripts and urine samples taken six months earlier also emphasizes that the proportional relationships across central MUP isoforms in urine is stable. Balancing selection maintains coding variants at moderate frequencies, though pheromone diversity appears limited by interactions with vomeronasal receptors. We find that differential transcription of the central Mup paralogs within and between individuals significantly increases the individuality of pheromone blends. Balancing selection on gene regulation allows for increased individuality via combinatorial diversity in a limited number of pheromones.

  4. Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Arati; Mandon, Elisabet C.; Gilmore, Reid; Rapoport, Tom A. (UMASS, MED); (Harvard-Med)

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71-Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71-Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the endoplasmic reticulum membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome-associated nascent polypeptide chains, also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71-Sec72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  5. Structural analysis of DNA–protein complexes regulating the restriction–modification system Esp1396I

    International Nuclear Information System (INIS)

    Martin, Richard N. A.; McGeehan, John E.; Ball, Neil J.; Streeter, Simon D.; Thresh, Sarah-Jane; Kneale, G. G.

    2013-01-01

    Comparison of bound and unbound DNA in protein–DNA co-crystal complexes reveals insights into controller-protein binding and DNA distortion in transcriptional regulation. The controller protein of the type II restriction–modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein–DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex

  6. Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers

    KAUST Repository

    Jantke, Dominik

    2013-11-01

    Site-directed conjugation of metal centers to proteins is fundamental for biological and bioinorganic applications of transition metals. However, methods for the site-selective introduction of metal centers remain scarce. Herein, we present broadly applicable synthetic strategies for the conjugation of bioactive molecules with a range of organometallic complexes. Following three different synthetic strategies, we were able to synthesize a small library of metal conjugated protein markers featuring different types of protein reactive sites (epoxides, phenylphosphonates, fluorosulfonates and fluorophosphonate groups) as well as different late transition metals (iron, ruthenium, rhodium, palladium and platinum). The products were isolated in moderate to excellent yields and high purity. Furthermore, X-ray diffraction of the metalated protein markers corroborates structural integrity of the metal complex and the protein reactive site. © 2013 Elsevier B.V. All rights reserved.

  7. Synthetic strategies for efficient conjugation of organometallic complexes with pendant protein reactive markers

    KAUST Repository

    Jantke, Dominik; Marziale, Alexander N.; Reiner, Thomas; Kraus, Florian; Herdtweck, Eberhardt; Raba, Andreas; Eppinger, Jö rg

    2013-01-01

    Site-directed conjugation of metal centers to proteins is fundamental for biological and bioinorganic applications of transition metals. However, methods for the site-selective introduction of metal centers remain scarce. Herein, we present broadly applicable synthetic strategies for the conjugation of bioactive molecules with a range of organometallic complexes. Following three different synthetic strategies, we were able to synthesize a small library of metal conjugated protein markers featuring different types of protein reactive sites (epoxides, phenylphosphonates, fluorosulfonates and fluorophosphonate groups) as well as different late transition metals (iron, ruthenium, rhodium, palladium and platinum). The products were isolated in moderate to excellent yields and high purity. Furthermore, X-ray diffraction of the metalated protein markers corroborates structural integrity of the metal complex and the protein reactive site. © 2013 Elsevier B.V. All rights reserved.

  8. Overexpression of KH-type splicing regulatory protein regulates proliferation, migration, and implantation ability of osteosarcoma

    OpenAIRE

    Pruksakorn, Dumnoensun; Teeyakasem, Pimpisa; Klangjorhor, Jeerawan; Chaiyawat, Parunya; Settakorn, Jongkolnee; Diskul-Na-Ayudthaya, Penchatr; Chokchaichamnankit, Daranee; Pothacharoen, Peraphan; Srisomsap, Chantragan

    2016-01-01

    Osteosarcoma is a common malignant bone tumor in children and adolescents. The current 5-year survival rate is ~60% and that seems to be reaching a plateau. In order to improve treatment outcomes of osteosarcoma, a better understanding of tumorigenesis and underlying molecular mechanisms is required for searching out possible new treatment targets. This study aimed to identify the potential proteins involving the pathogenesis of osteosarcoma using a proteomics approach. Proteins extracted fro...

  9. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding.

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  10. Co-operative intra-protein structural response due to protein-protein complexation revealed through thermodynamic quantification: study of MDM2-p53 binding

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-10-01

    The p53 protein activation protects the organism from propagation of cells with damaged DNA having oncogenic mutations. In normal cells, activity of p53 is controlled by interaction with MDM2. The well understood p53-MDM2 interaction facilitates design of ligands that could potentially disrupt or prevent the complexation owing to its emergence as an important objective for cancer therapy. However, thermodynamic quantification of the p53-peptide induced structural changes of the MDM2-protein remains an area to be explored. This study attempts to understand the conformational free energy and entropy costs due to this complex formation from the histograms of dihedral angles generated from molecular dynamics simulations. Residue-specific quantification illustrates that, hydrophobic residues of the protein contribute maximum to the conformational thermodynamic changes. Thermodynamic quantification of structural changes of the protein unfold the fact that, p53 binding provides a source of inter-element cooperativity among the protein secondary structural elements, where the highest affected structural elements (α2 and α4) found at the binding site of the protein affects faraway structural elements (β1 and Loop1) of the protein. The communication perhaps involves water mediated hydrogen bonded network formation. Further, we infer that in inhibitory F19A mutation of P53, though Phe19 is important in the recognition process, it has less prominent contribution in the stability of the complex. Collectively, this study provides vivid microscopic understanding of the interaction within the protein complex along with exploring mutation sites, which will contribute further to engineer the protein function and binding affinity.

  11. Regulatory controls on the hydrogeological characterization of a mixed waste disposal site, Radioactive Waste Management Complex, Idaho National Engineering Laboratory

    International Nuclear Information System (INIS)

    Ruebelmann, K.L.

    1990-01-01

    Following the detection of chlorinated volatile organic compounds in the groundwater beneath the SDA in the summer of 1987, hydrogeological characterization of the Radioactive Waste Management Complex (RWMC), Idaho National Engineering Laboratory (INEL) was required by the Resource Conservation and Recovery Act (RCRA). The waste site, the Subsurface Disposal Area (SDA), is the subject of a RCRA Corrective Action Program. Regulatory requirements for the Corrective Action Program dictate a phased approach to evaluation of the SDA. In the first phase of the program, the SDA is the subject of a RCRA Facility Investigation (RIF), which will obtain information to fully characterize the physical properties of the site, determine the nature and extent of contamination, and identify pathways for migration of contaminants. If the need for corrective measures is identified during the RIF, a Corrective Measures Study (CMS) will be performed as second phase. Information generated during the RIF will be used to aid in the selection and implementation of appropriate corrective measures to correct the release. Following the CMS, the final phase is the implementation of the selected corrective measures. 4 refs., 1 fig

  12. Purification and binding analysis of the nitrogen fixation regulatory NifA protein from Azospirillum brasilense

    Directory of Open Access Journals (Sweden)

    L.M.P. Passaglia

    1998-11-01

    Full Text Available NifA protein activates transcription of nitrogen fixation operons by the alternative sigma54 holoenzyme form of RNA polymerase. This protein binds to a well-defined upstream activator sequence (UAS located at the -200/-100 position of nif promoters with the consensus motif TGT-N10-ACA. NifA of Azospirillum brasilense was purified in the form of a glutathione-S-transferase (GST-NifA fusion protein and proteolytic release of GST yielded inactive and partially soluble NifA. However, the purified NifA was able to induce the production of specific anti-A. brasilense NifA-antiserum that recognized NifA from A. brasilense but not from K. pneumoniae. Both GST-NifA and NifA expressed from the E. coli tac promoter are able to activate transcription from the nifHDK promoter but only in an A. brasilense background. In order to investigate the mechanism that regulates NifA binding capacity we have used E. coli total protein extracts expressing A. brasilense nifA in mobility shift assays. DNA fragments carrying the two overlapping, wild-type or mutated UAS motifs present in the nifH promoter region revealed a retarded band of related size. These data show that the binding activity present in the C-terminal domain of A. brasilense NifA protein is still functional even in the presence of oxygen.

  13. MAPA distinguishes genotype-specific variability of highly similar regulatory protein isoforms in potato tuber.

    Science.gov (United States)

    Hoehenwarter, Wolfgang; Larhlimi, Abdelhalim; Hummel, Jan; Egelhofer, Volker; Selbig, Joachim; van Dongen, Joost T; Wienkoop, Stefanie; Weckwerth, Wolfram

    2011-07-01

    Mass Accuracy Precursor Alignment is a fast and flexible method for comparative proteome analysis that allows the comparison of unprecedented numbers of shotgun proteomics analyses on a personal computer in a matter of hours. We compared 183 LC-MS analyses and more than 2 million MS/MS spectra and could define and separate the proteomic phenotypes of field grown tubers of 12 tetraploid cultivars of the crop plant Solanum tuberosum. Protein isoforms of patatin as well as other major gene families such as lipoxygenase and cysteine protease inhibitor that regulate tuber development were found to be the primary source of variability between the cultivars. This suggests that differentially expressed protein isoforms modulate genotype specific tuber development and the plant phenotype. We properly assigned the measured abundance of tryptic peptides to different protein isoforms that share extensive stretches of primary structure and thus inferred their abundance. Peptides unique to different protein isoforms were used to classify the remaining peptides assigned to the entire subset of isoforms based on a common abundance profile using multivariate statistical procedures. We identified nearly 4000 proteins which we used for quantitative functional annotation making this the most extensive study of the tuber proteome to date.

  14. A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?

    Directory of Open Access Journals (Sweden)

    Katrina Brudzynski

    Full Text Available Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002 with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a high molecular weight complexes (230-180 kDa enriched in proteins but possessing a limited reducing activity toward the NBT and (b lower molecular size complexes (110-85 kDa enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the

  15. Adenovirus type 5 DNA-protein complexes from formaldehyde cross-linked cells early after infection

    International Nuclear Information System (INIS)

    Spector, David J.; Johnson, Jeffrey S.; Baird, Nicholas L.; Engel, Daniel A.

    2003-01-01

    We report here the properties of viral DNA-protein complexes that purify with cellular chromatin following formaldehyde cross-linking of intact cells early after infection. The cross-linked viral DNA fractionated into shear-sensitive (S) and shear- resistant (R) components that were separable by sedimentation, which allowed independent characterization. The R component had the density and sedimentation properties expected for DNA-protein complexes and contained intact viral DNA. It accounted for about 50% of the viral DNA recovered at 1.5 h after infection but less than 20% by 4.5 h. The proportion of R component was independent of multiplicity of infection, even at less than one particle per cell. Viral hexon and protein VII, but not protein VI, were detected in the fractions containing the R component. These properties are consistent with those of partially uncoated virions associated with the nuclear envelope. A substantial proportion of the S component viral DNA had the same density as cellular chromatin. Protein VII was the most abundant viral protein present in gradient fractions that contained the S component. Complexes containing USF transcription factor cross-linked to the adenovirus major late promoter were detected by viral chromatin immunoprecipitation of the fractions containing S component. The S component probably contained uncoated nuclear viral DNA that assembles into early viral transcription complexes

  16. Multistructure index in revealing complexity of regulatory mechanisms of human cardiovascular system at rest and orthostatic stress in healthy humans

    Science.gov (United States)

    Makowiec, Danuta; Graff, Beata; Struzik, Zbigniew R.

    2017-02-01

    Biological regulation is sufficiently complex to pose an enduring challenge for characterization of both its equilibrium and transient non-equilibrium dynamics. Two univariate but coupled observables, heart rate and systolic blood pressure, are commonly characterized in the benchmark example of the human cardiovascular regulatory system. Asymmetric distributions of accelerations and decelerations of heart rate, as well as rises and falls in systolic blood pressure, recorded in humans during a head-up tilt test provide insights into the dynamics of cardiovascular response to a rapid, controlled deregulation of the system's homeostasis. The baroreflex feedback loop is assumed to be the fundamental physiological mechanism for ensuring homeostatic blood supply to distant organs at rest and during orthostatic stress, captured in a classical beat-to-beat autoregressive model of baroreflex by de Boer et al. (1987). For model corroboration, a multistructure index statistic is proposed, seamlessly evaluating the size spectrum of magnitudes of neural reflexes such as baroreflex, responsible for maintaining the homeostatic dynamics. The multistructure index exposes a distinctly different dynamics of multiscale asymmetry between results obtained from real-life signals recorded from healthy subjects and those simulated using both the classical and perturbed versions of the model. Nonlinear effects observed suggest the pronounced presence of complex mechanisms resulting from baroreflex regulation when a human is at rest, which is aggravated in the system's response to orthostatic stress. Using our methodology of multistructure index, we therefore show a marked difference between model and real-life scenarios, which we attribute to multiscale asymmetry of non-linear origin in real-life signals, which we are not reproducible by the classical model.

  17. Circulating growth hormone (GH)-binding protein complex: a major constituent of plasma GH in man

    International Nuclear Information System (INIS)

    Baumann, G.; Amburn, K.; Shaw, M.A.

    1988-01-01

    The recent discovery of a specific binding protein for human GH (hGH) in human plasma suggests that hGH circulates in part as a complex in association with the binding protein(s). However, the magnitude of the complexed fraction prevailing under physiological conditions is unknown because of 1) dissociation of the complex during analysis and 2) potential differences in the binding characteristics of radiolabeled and native hGH. We conducted experiments designed to minimize dissociation during analysis (gel filtration in prelabeled columns, frontal analysis, and batch molecular sieving) with both native and radioiodinated hGH. All three methods yielded similar estimates for the complexed fraction. In normal plasma the bound fraction for 22 K hGH averaged 50.1% (range, 39-59%), that for 20 K hGH averaged 28.5% (range, 26-31%). Above a hGH level of about 20 ng/ml the bound fraction declines in concentration-dependent manner due to saturation of the binding protein. We conclude that a substantial part of circulating hGH is complexed with carrier proteins. This concept has important implications for the metabolism, distribution, and biological activity of hGH

  18. Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

    Science.gov (United States)

    Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

    2012-11-30

    Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Structure of the JmjC domain-containing protein NO66 complexed with ribosomal protein Rpl8

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chengliang [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Zhang, Qiongdi [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Hang, Tianrong [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China); Tao, Yue [Shanghai Children’s Medical Center, 1678 Dongfang Road, Pudong, Shanghai 200120, People’s Republic of (China); Ma, Xukai [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Wu, Minhao; Zhang, Xuan, E-mail: xuanzbin@ustc.edu.cn; Zang, Jianye, E-mail: xuanzbin@ustc.edu.cn [University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, People’s Republic of (China); Chinese Academy of Sciences, Hefei, Anhui 230026, People’s Republic of (China)

    2015-08-28

    The structure of the complex of NO66 and Rpl8 was solved in the native state and NO66 recognizes the consensus motif NHXH . Tetramerization is required for efficient substrate binding and catalysis by NO66. The JmjC domain-containing proteins belong to a large family of oxygenases possessing distinct substrate specificities which are involved in the regulation of different biological processes, such as gene transcription, RNA processing and translation. Nucleolar protein 66 (NO66) is a JmjC domain-containing protein which has been reported to be a histone demethylase and a ribosome protein 8 (Rpl8) hydroxylase. The present biochemical study confirmed the hydroxylase activity of NO66 and showed that oligomerization is required for NO66 to efficiently catalyze the hydroxylation of Rpl8. The structures of NO66{sup 176–C} complexed with Rpl8{sup 204–224} in a tetrameric form and of the mutant protein M2 in a dimeric form were solved. Based on the results of structural and biochemical analyses, the consensus sequence motif NHXH recognized by NO66 was confirmed. Several potential substrates of NO66 were found by a BLAST search according to the consensus sequence motif. When binding to substrate, the relative positions of each subunit in the NO66 tetramer shift. Oligomerization may facilitate the motion of each subunit in the NO66 tetramer and affect the catalytic activity.

  20. Integrative analysis of RNA, translation, and protein levels reveals distinct regulatory variation across humans.

    Science.gov (United States)

    Cenik, Can; Cenik, Elif Sarinay; Byeon, Gun W; Grubert, Fabian; Candille, Sophie I; Spacek, Damek; Alsallakh, Bilal; Tilgner, Hagen; Araya, Carlos L; Tang, Hua; Ricci, Emiliano; Snyder, Michael P

    2015-11-01

    Elucidating the consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding, and chromatin have been explored, little is known about global variation in translation and its genetic determinants. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA, translation, and protein levels suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy--many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. © 2015 Cenik et al.; Published by Cold Spring Harbor Laboratory Press.

  1. Compound complex enzymes and proteins of Stipa capillata from Semipalatinsk polygon

    International Nuclear Information System (INIS)

    Sarsenbaev, K.N.; Esnazarov, U.; Sarsenbaeva, M.V.; Seisebaev, A.

    2002-01-01

    The effects of low and high doses of irradiation near Semipalatinsk Atomic lake on the compound complex of different enzymes and proteins of leaves from different population of Stipa capillata are considered. 36 samples of Stipa capillata were analyzed by the iso-electrofocusing methods, native and SDS-electrophoresis. Levels of radioactivity effect on compound complex of peroxidase, esterase, acid phosphates and soluble proteins were found. SDS-PAGE and IEF methods did not show difference in peptides spectra between 36 populations of examined species. It means, that difference between contaminated and non-contaminated populations not so big as was expected. Compound complex soluble protein of Stipa capillata leaves changes under chronic doses of radioactivity. The difference in spectra between control and contaminated leaves make up 3-6 bands. Control leaves have more high molecular weight proteins than contaminated ones. Appearance of new bands is one of ways of plant adaptation. New components of enzymes spectra and soluble proteins were found. It was suggested, that gene mutation or post-translation modification of these proteins are result of chronic irradiation. To prove exactly genetic nature of this alteration aminoacids sequence for these proteins the DNA sequence of different Stipa capillata populations genomes were compared

  2. Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

    Science.gov (United States)

    Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

    2016-11-25

    In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Structure and stability of complexes of agmatine with some functional receptor residues of proteins

    Science.gov (United States)

    Remko, Milan; Broer, Ria; Remková, Anna; Van Duijnen, Piet Th.

    2017-04-01

    The paper reports the results of a theoretical study of the conformational behavior and basicity of biogenic amine agmatine. The complexes modelling of agmatine - protein interaction are also under scrutiny of our investigation using the Becke3LYP and B97D levels of the density functional theory. The relative stabilities (Gibbs energies) of individual complexes are by both DFT methods described equally. Hydration has a dramatic effect on the hydrogen bonded complexes studied. The pairing acidic carboxylate group with different agmatine species resulted in charged hydrogen bond complexes containing negatively charged acetate species acting as proton acceptors.

  4. The KYxxL motif in Rad17 protein is essential for the interaction with the 9–1–1 complex

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan); Ikeuchi, Masayoshi; Nakayama, Yuji [Department of Biochemistry & Molecular Biology, Kyoto Pharmaceutical University, Kyoto 607-8414 (Japan); Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp [Laboratory of Molecular Cell Biology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675 (Japan)

    2016-09-02

    ATR-dependent DNA damage checkpoint is the major DNA damage checkpoint against UV irradiation and DNA replication stress. The Rad17–RFC and Rad9–Rad1–Hus1 (9–1–1) complexes interact with each other to contribute to ATR signaling, however, the precise regulatory mechanism of the interaction has not been established. Here, we identified a conserved sequence motif, KYxxL, in the AAA+ domain of Rad17 protein, and demonstrated that this motif is essential for the interaction with the 9–1–1 complex. We also show that UV-induced Rad17 phosphorylation is increased in the Rad17 KYxxL mutants. These data indicate that the interaction with the 9–1–1 complex is not required for Rad17 protein to be an efficient substrate for the UV-induced phosphorylation. Our data also raise the possibility that the 9–1–1 complex plays a negative regulatory role in the Rad17 phosphorylation. We also show that the nucleotide-binding activity of Rad17 is required for its nuclear localization. - Highlights: • We have identified a conserved KYxxL motif in Rad17 protein. • The KYxxL motif is crucial for the interaction with the 9–1–1 complex. • The KYxxL motif is dispensable or inhibitory for UV-induced Rad17 phosphorylation. • Nucleotide binding of Rad17 is required for its nuclear localization.

  5. Identification of Novel Targets of the Human Cell Cycle Regulatory Protein Cdc34

    Science.gov (United States)

    1999-07-01

    centrifugal elutriation, with a purity of -80% as shown by micro - ethyl acetate and separated on thin-layer chromatography plates (Whatman, scopic...Spain. (2) Servicio Bioquimica, Hosp. La Paz. Madrid. ICER protein is elevated in mHR6b-/- (murine Rad6B) fibroblasts Spain. and in human cells

  6. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    Energy Technology Data Exchange (ETDEWEB)

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  7. Determinants of RNA binding and translational repression by the Bicaudal-C regulatory protein.

    Science.gov (United States)

    Zhang, Yan; Park, Sookhee; Blaser, Susanne; Sheets, Michael D

    2014-03-14

    Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.

  8. Expression of cytoskeleton regulatory protein Mena in human hepatocellular carcinoma and its prognostic significance.

    Science.gov (United States)

    Hu, Kunpeng; Wang, Jiani; Yao, Zhicheng; Liu, Bo; Lin, Yuan; Liu, Lei; Xu, Lihua

    2014-05-01

    The molecular mechanisms of the development and progression of hepatocellular carcinoma (HCC) are poorly understood. The main objective of this study was to analyze the expression of Enabled [mammalian Ena (Mena)] protein and its clinical significance in human HCC. The Mena expression was examined at mRNA and protein levels by real-time quantitative polymerase chain reaction and Western blotting analysis in ten paired HCC tissues and the adjacent normal tissues. The expression of Mena protein in 81 specimens of HCC tissues was determined by immunohistochemistry. Associations of Mena expression with the clinicopathological features were analyzed, and prognosis of HCC patients was evaluated. The result shows the expression of Mena mRNA and protein was higher in HCC than in the adjacent normal tissues in ten paired samples. Mena was mainly accumulated in the cytoplasm of tumor cells and over-expressed in 40.74% (33/81) patients by immunohistochemical staining. Over-expression of Mena was significantly associated with poor cellular differentiation (P = 0.025), advanced tumor stage (P = 0.003) and worse disease-free survival (DFS, P Mena is an independent prognostic factor for DFS in multivariate analysis (HR 2.309, 95% CI 1.104-4.828; P = 0.026). Mena is up-regulated in HCC and associated with tumor differentiation and clinical stage. Mena may be an independent prognostic marker for DFS of HCC patients.

  9. Biochemical characterization of the prolyl 3-hydroxylase 1.cartilage-associated protein.cyclophilin B complex.

    Science.gov (United States)

    Ishikawa, Yoshihiro; Wirz, Jackie; Vranka, Janice A; Nagata, Kazuhiro; Bächinger, Hans Peter

    2009-06-26

    The rough endoplasmic reticulum-resident protein complex consisting of prolyl 3-hydroxylase 1 (P3H1), cartilage-associated protein (CRTAP), and cyclophilin B (CypB) can be isolated from chick embryos on a gelatin-Sepharose column, indicating some involvement in the biosynthesis of procollagens. Prolyl 3-hydroxylase 1 modifies a single proline residue in the alpha chains of type I, II, and III collagens to (3S)-hydroxyproline. The peptidyl-prolyl cis-trans isomerase activity of cyclophilin B was shown previously to catalyze the rate of triple helix formation. Here we show that cyclophilin B in the complex shows peptidyl-prolyl cis-trans isomerase activity and that the P3H1.CRTAP.CypB complex has another important function: it acts as a chaperone molecule when tested with two classical chaperone assays. The P3H1.CRTAP.CypB complex inhibited the thermal aggregation of citrate synthase and was active in the denatured rhodanese refolding and aggregation assay. The chaperone activity of the complex was higher than that of protein-disulfide isomerase, a well characterized chaperone. The P3H1.CRTAP.CypB complex also delayed the in vitro fibril formation of type I collagen, indicating that this complex is also able to interact with triple helical collagen and acts as a collagen chaperone.

  10. Imaging and structural studies of DNA–protein complexes and membrane ion channels

    KAUST Repository

    Marini, Monica; Limongi, Tania; Falqui, Andrea; Genovese, Alessandro; Allione, Marco; Moretti, Manola; Lopatin, Sergei; Tirinato, Luca; Das, Gobind; Torre, Bruno; Giugni, Andrea; Cesca, F.; Benfenati, F.; Di Fabrizio, Enzo M.

    2017-01-01

    In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABA(A) channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 angstrom, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

  11. Imaging and structural studies of DNA–protein complexes and membrane ion channels

    KAUST Repository

    Marini, Monica

    2017-01-17

    In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABA(A) channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 angstrom, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.

  12. GBA manager: an online tool for querying low-complexity regions in proteins.

    Science.gov (United States)

    Bandyopadhyay, Nirmalya; Kahveci, Tamer

    2010-01-01

    Abstract We developed GBA Manager, an online software that facilitates the Graph-Based Algorithm (GBA) we proposed in our earlier work. GBA identifies the low-complexity regions (LCR) of protein sequences. GBA exploits a similarity matrix, such as BLOSUM62, to compute the complexity of the subsequences of the input protein sequence. It uses a graph-based algorithm to accurately compute the regions that have low complexities. GBA Manager is a user friendly web-service that enables online querying of protein sequences using GBA. In addition to querying capabilities of the existing GBA algorithm, GBA Manager computes the p-values of the LCR identified. The p-value gives an estimate of the possibility that the region appears by chance. GBA Manager presents the output in three different understandable formats. GBA Manager is freely accessible at http://bioinformatics.cise.ufl.edu/GBA/GBA.htm .

  13. Lysine acetylation targets protein complexes and co-regulates major cellular functions

    DEFF Research Database (Denmark)

    Choudhary, Chuna Ram; Kumar, Chanchal; Gnad, Florian

    2009-01-01

    Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600......, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other...

  14. Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry.

    Science.gov (United States)

    Dubois, Marie-Line; Bastin, Charlotte; Lévesque, Dominique; Boisvert, François-Michel

    2016-09-02

    The extensive identification of protein-protein interactions under different conditions is an important challenge to understand the cellular functions of proteins. Here we use and compare different approaches including affinity purification and purification by proximity coupled to mass spectrometry to identify protein complexes. We explore the complete interactome of the minichromosome maintenance (MCM) complex by using both approaches for all of the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair, and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

  15. Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11.

    Science.gov (United States)

    Herrera, María Georgina; Pignataro, María Florencia; Noguera, Martín Ezequiel; Cruz, Karen Magalí; Santos, Javier

    2018-05-16

    Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.

  16. Transcriptional analysis of the jamaicamide gene cluster from the marine cyanobacterium Lyngbya majuscula and identification of possible regulatory proteins

    Directory of Open Access Journals (Sweden)

    Dorrestein Pieter C

    2009-12-01

    Full Text Available Abstract Background The marine cyanobacterium Lyngbya majuscula is a prolific producer of bioactive secondary metabolites. Although biosynthetic gene clusters encoding several of these compounds have been identified, little is known about how these clusters of genes are transcribed or regulated, and techniques targeting genetic manipulation in Lyngbya strains have not yet been developed. We conducted transcriptional analyses of the jamaicamide gene cluster from a Jamaican strain of Lyngbya majuscula, and isolated proteins that could be involved in jamaicamide regulation. Results An unusually long untranslated leader region of approximately 840 bp is located between the jamaicamide transcription start site (TSS and gene cluster start codon. All of the intergenic regions between the pathway ORFs were transcribed into RNA in RT-PCR experiments; however, a promoter prediction program indicated the possible presence of promoters in multiple intergenic regions. Because the functionality of these promoters could not be verified in vivo, we used a reporter gene assay in E. coli to show that several of these intergenic regions, as well as the primary promoter preceding the TSS, are capable of driving β-galactosidase production. A protein pulldown assay was also used to isolate proteins that may regulate the jamaicamide pathway. Pulldown experiments using the intergenic region upstream of jamA as a DNA probe isolated two proteins that were identified by LC-MS/MS. By BLAST analysis, one of these had close sequence identity to a regulatory protein in another cyanobacterial species. Protein comparisons suggest a possible correlation between secondary metabolism regulation and light dependent complementary chromatic adaptation. Electromobility shift assays were used to evaluate binding of the recombinant proteins to the jamaicamide promoter region. Conclusion Insights into natural product regulation in cyanobacteria are of significant value to drug discovery

  17. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

    KAUST Repository

    Lengsfeld, Bettina M.; Berry, Kayla N.; Ghosh, Sharmistha; Takahashi, Masateru; Francis, Nicole J.

    2012-01-01

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  18. Large, dynamic, multi-protein complexes: a challenge for structural biology

    Czech Academy of Sciences Publication Activity Database

    Rozycki, B.; Bouřa, Evžen

    2014-01-01

    Roč. 26, č. 46 (2014), 463103/1-463103/11 ISSN 0953-8984 R&D Projects: GA MŠk LO1302 EU Projects: European Commission(XE) 333916 - STARPI4K Institutional support: RVO:61388963 Keywords : protein structure * multi-protein complexes * hybrid methods of structural biology Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.346, year: 2014

  19. Fanconi Anemia Proteins FANCA, FANCC, and FANCG/XRCC9 Interact in a Functional Nuclear Complex

    OpenAIRE

    Garcia-Higuera, Irene; Kuang, Yanan; Näf, Dieter; Wasik, Jennifer; D’Andrea, Alan D.

    1999-01-01

    Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A to H). Three FA genes, corresponding to complementation groups A, C, and G, have been cloned, but their cellular function remains unknown. We have previously demonstrated that the FANCA and FANCC proteins interact and form a nuclear complex in normal cells, suggesting that the proteins cooperate in a nuclear function. In this report, we demonstrate that the recently clone...

  20. A Polycomb complex remains bound through DNA replication in the absence of other eukaryotic proteins

    KAUST Repository

    Lengsfeld, Bettina M.

    2012-09-17

    Propagation of chromatin states through DNA replication is central to epigenetic regulation and can involve recruitment of chromatin proteins to replicating chromatin through interactions with replication fork components. Here we show using a fully reconstituted T7 bacteriophage system that eukaryotic proteins are not required to tether the Polycomb complex PRC1 to templates during DNA replication. Instead, DNA binding by PRC1 can withstand passage of a simple replication fork.

  1. A conserved endoplasmic reticulum membrane protein complex (EMC facilitates phospholipid transfer from the ER to mitochondria.

    Directory of Open Access Journals (Sweden)

    Sujoy Lahiri

    2014-10-01

    Full Text Available Mitochondrial membrane biogenesis and lipid metabolism require phospholipid transfer from the endoplasmic reticulum (ER to mitochondria. Transfer is thought to occur at regions of close contact of these organelles and to be nonvesicular, but the mechanism is not known. Here we used a novel genetic screen in S. cerevisiae to identify mutants with defects in lipid exchange between the ER and mitochondria. We show that a strain missing multiple components of the conserved ER membrane protein complex (EMC has decreased phosphatidylserine (PS transfer from the ER to mitochondria. Mitochondria from this strain have significantly reduced levels of PS and its derivative phosphatidylethanolamine (PE. Cells lacking EMC proteins and the ER-mitochondria tethering complex called ERMES (the ER-mitochondria encounter structure are inviable, suggesting that the EMC also functions as a tether. These defects are corrected by expression of an engineered ER-mitochondrial tethering protein that artificially tethers the ER to mitochondria. EMC mutants have a significant reduction in the amount of ER tethered to mitochondria even though ERMES remained intact in these mutants, suggesting that the EMC performs an additional tethering function to ERMES. We find that all Emc proteins interact with the mitochondrial translocase of the outer membrane (TOM complex protein Tom5 and this interaction is important for PS transfer and cell growth, suggesting that the EMC forms a tether by associating with the TOM complex. Together, our findings support that the EMC tethers ER to mitochondria, which is required for phospholipid synthesis and cell growth.

  2. The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

    Science.gov (United States)

    de Winter, J P; van der Weel, L; de Groot, J; Stone, S; Waisfisz, Q; Arwert, F; Scheper, R J; Kruyt, F A; Hoatlin, M E; Joenje, H

    2000-11-01

    Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.

  3. Polymorphisms of twenty regulatory proteins between Mycobacterium tuberculosis and Mycobacterium bovis

    Science.gov (United States)

    Mycobacterium tuberculosis and Mycobacterium bovis are responsible for tuberculosis in humans or animals, respectively. Both species are closely related and belong to the Mycobacterium tuberculosis complex (MTC). M. tuberculosis is the most ancient species from which M. bovis and the other members o...

  4. The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF₁.

    Science.gov (United States)

    Robinson, Graham C; Bason, John V; Montgomery, Martin G; Fearnley, Ian M; Mueller, David M; Leslie, Andrew G W; Walker, John E

    2013-02-13

    The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α(DP)- and β(DP)-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β(E)-subunits. The nucleotide binding site in β(E)-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.

  5. The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

    Science.gov (United States)

    Robinson, Graham C.; Bason, John V.; Montgomery, Martin G.; Fearnley, Ian M.; Mueller, David M.; Leslie, Andrew G. W.; Walker, John E.

    2013-01-01

    The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF1 has been determined at 2.5 Å resolution. The inhibitory region of IF1 from residues 1 to 36 is entrapped between the C-terminal domains of the αDP- and βDP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F1-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic βE-subunits. The nucleotide binding site in βE-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule. PMID:23407639

  6. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

    Science.gov (United States)

    Rudolph, Michael C; Monks, Jenifer; Burns, Valerie; Phistry, Meridee; Marians, Russell; Foote, Monica R; Bauman, Dale E; Anderson, Steven M; Neville, Margaret C

    2010-12-01

    The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase (Fasn), insulin-induced gene 1 (Insig1), mitochondrial citrate transporter (Slc25a1), and stearoyl-CoA desaturase 2 (Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α (Acaca) and ATP citrate lyase (Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

  7. 3D pressure field in lipid membranes and membrane-protein complexes

    DEFF Research Database (Denmark)

    Ollila, O H Samuli; Risselada, H Jelger; Louhivuori, Martti

    2009-01-01

    We calculate full 3D pressure fields for inhomogeneous nanoscale systems using molecular dynamics simulation data. The fields represent systems with increasing level of complexity, ranging from semivesicles and vesicles to membranes characterized by coexistence of two phases, including also...... a protein-membrane complex. We show that the 3D pressure field is distinctly different for curved and planar bilayers, the pressure field depends strongly on the phase of the membrane, and that an integral protein modulates the tension and elastic properties of the membrane....

  8. Brownian dynamics of a protein-polymer chain complex in a solid-state nanopore

    Science.gov (United States)

    Wells, Craig C.; Melnikov, Dmitriy V.; Gracheva, Maria E.

    2017-08-01

    We study the movement of a polymer attached to a large protein inside a nanopore in a thin silicon dioxide membrane submerged in an electrolyte solution. We use Brownian dynamics to describe the motion of a negatively charged polymer chain of varying lengths attached to a neutral protein modeled as a spherical bead with a radius larger than that of the nanopore, allowing the chain to thread the nanopore but preventing it from translocating. The motion of the protein-polymer complex within the pore is also compared to that of a freely translocating polymer. Our results show that the free polymer's standard deviations in the direction normal to the pore axis is greater than that of the protein-polymer complex. We find that restrictions imposed by the protein, bias, and neighboring chain segments aid in controlling the position of the chain in the pore. Understanding the behavior of the protein-polymer chain complex may lead to methods that improve molecule identification by increasing the resolution of ionic current measurements.

  9. Computer simulation of protein—carbohydrate complexes: application to arabinose-binding protein and pea lectin

    Science.gov (United States)

    Rao, V. S. R.; Biswas, Margaret; Mukhopadhyay, Chaitali; Balaji, P. V.

    1989-03-01

    The CCEM method (Contact Criteria and Energy Minimisation) has been developed and applied to study protein-carbohydrate interactions. The method uses available X-ray data even on the native protein at low resolution (above 2.4 Å) to generate realistic models of a variety of proteins with various ligands. The two examples discussed in this paper are arabinose-binding protein (ABP) and pea lectin. The X-ray crystal structure data reported on ABP-β- L-arabinose complex at 2.8, 2.4 and 1.7 Å resolution differ drastically in predicting the nature of the interactions between the protein and ligand. It is shown that, using the data at 2.4 Å resolution, the CCEM method generates complexes which are as good as the higher (1.7 Å) resolution data. The CCEM method predicts some of the important hydrogen bonds between the ligand and the protein which are missing in the interpretation of the X-ray data at 2.4 Å resolution. The theoretically predicted hydrogen bonds are in good agreement with those reported at 1.7 Å resolution. Pea lectin has been solved only in the native form at 3 Å resolution. Application of the CCEM method also enables us to generate complexes of pea lectin with methyl-α- D-glucopyranoside and methyl-2,3-dimethyl-α- D-glucopyranoside which explain well the available experimental data in solution.

  10. Cooperation of TOM and TIM23 complexes during translocation of proteins into mitochondria.

    Science.gov (United States)

    Waegemann, Karin; Popov-Čeleketić, Dušan; Neupert, Walter; Azem, Abdussalam; Mokranjac, Dejana

    2015-03-13

    Translocation of the majority of mitochondrial proteins from the cytosol into mitochondria requires the cooperation of TOM and TIM23 complexes in the outer and inner mitochondrial membranes. The molecular mechanisms underlying this cooperation remain largely unknown. Here, we present biochemical and genetic evidence that at least two contacts from the side of the TIM23 complex play an important role in TOM-TIM23 cooperation in vivo. Tim50, likely through its very C-terminal segment, interacts with Tom22. This interaction is stimulated by translocating proteins and is independent of any other TOM-TIM23 contact known so far. Furthermore, the exposure of Tim23 on the mitochondrial surface depends not only on its interaction with Tim50 but also on the dynamics of the TOM complex. Destabilization of the individual contacts reduces the efficiency of import of proteins into mitochondria and destabilization of both contacts simultaneously is not tolerated by yeast cells. We conclude that an intricate and coordinated network of protein-protein interactions involving primarily Tim50 and also Tim23 is required for efficient translocation of proteins across both mitochondrial membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

    Science.gov (United States)

    Gao, Yang; Westfield, Gerwin; Erickson, Jon W; Cerione, Richard A; Skiniotis, Georgios; Ramachandran, Sekar

    2017-08-25

    The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G T ). This results in the dissociation of G T into its component α T -GTP and β 1 γ 1 subunit complex. Structural information for the Rho*-G T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G T *) comprising a Gα T /Gα i1 chimera (α T *) and β 1 γ 1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα T * is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β 2 -adrenergic receptor-G S complex, including a flexible α T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

    Science.gov (United States)

    Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

    2016-08-01

    Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

  13. A Novel Interaction of Ecdysoneless (ECD) Protein with R2TP Complex Component RUVBL1 Is Required for the Functional Role of ECD in Cell Cycle Progression.

    Science.gov (United States)

    Mir, Riyaz A; Bele, Aditya; Mirza, Sameer; Srivastava, Shashank; Olou, Appolinaire A; Ammons, Shalis A; Kim, Jun Hyun; Gurumurthy, Channabasavaiah B; Qiu, Fang; Band, Hamid; Band, Vimla

    2015-12-28

    Ecdysoneless (ECD) is an evolutionarily conserved protein whose germ line deletion is embryonic lethal. Deletion of Ecd in cells causes cell cycle arrest, which is rescued by exogenous ECD, demonstrating a requirement of ECD for normal mammalian cell cycle progression. However, the exact mechanism by which ECD regulates cell cycle is unknown. Here, we demonstrate that ECD protein levels and subcellular localization are invariant during cell cycle progression, suggesting a potential role of posttranslational modifications or protein-protein interactions. Since phosphorylated ECD was recently shown to interact with the PIH1D1 adaptor component of the R2TP cochaperone complex, we examined the requirement of ECD phosphorylation in cell cycle progression. Notably, phosphorylation-deficient ECD mutants that failed to bind to PIH1D1 in vitro fully retained the ability to interact with the R2TP complex and yet exhibited a reduced ability to rescue Ecd-deficient cells from cell cycle arrest. Biochemical analyses demonstrated an additional phosphorylation-independent interaction of ECD with the RUVBL1 component of the R2TP complex, and this interaction is essential for ECD's cell cycle progression function. These studies demonstrate that interaction of ECD with RUVBL1, and its CK2-mediated phosphorylation, independent of its interaction with PIH1D1, are important for its cell cycle regulatory function. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  14. A simple and reliable approach to docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints

    International Nuclear Information System (INIS)

    Tang, Chun; Clore, G. Marius

    2006-01-01

    A simple and reliable approach for docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints (as few as three from a single point) in combination with a novel representation for an attractive potential between mapped interaction surfaces is described. Unambiguous assignments of very sparse intermolecular NOEs are obtained using a reverse labeling strategy in which one the components is fully deuterated with the exception of selective protonation of the δ-methyl groups of isoleucine, while the other component is uniformly 13 C-labeled. This labeling strategy can be readily extended to selective protonation of Ala, Leu, Val or Met. The attractive potential is described by a 'reduced' radius of gyration potential applied specifically to a subset of interfacial residues (those with an accessible surface area ≥ 50% in the free proteins) that have been delineated by chemical shift perturbation. Docking is achieved by rigid body minimization on the basis of a target function comprising the sparse NOE distance restraints, a van der Waals repulsion potential and the 'reduced' radius of gyration potential. The method is demonstrated for two protein-protein complexes (EIN-HPr and IIA Glc -HPr) from the bacterial phosphotransferase system. In both cases, starting from 100 different random orientations of the X-ray structures of the free proteins, 100% convergence is achieved to a single cluster (with near identical atomic positions) with an overall backbone accuracy of ∼2 A. The approach described is not limited to NMR, since interfaces can also be mapped by alanine scanning mutagenesis, and sparse intermolecular distance restraints can be derived from double cycle mutagenesis, cross-linking combined with mass spectrometry, or fluorescence energy transfer

  15. A simple and reliable approach to docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Chun; Clore, G. Marius [National Institutes of Health, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases (United States)], E-mail: mariusc@intra.niddk.nih.gov

    2006-09-15

    A simple and reliable approach for docking protein-protein complexes from very sparse NOE-derived intermolecular distance restraints (as few as three from a single point) in combination with a novel representation for an attractive potential between mapped interaction surfaces is described. Unambiguous assignments of very sparse intermolecular NOEs are obtained using a reverse labeling strategy in which one the components is fully deuterated with the exception of selective protonation of the {delta}-methyl groups of isoleucine, while the other component is uniformly {sup 13}C-labeled. This labeling strategy can be readily extended to selective protonation of Ala, Leu, Val or Met. The attractive potential is described by a 'reduced' radius of gyration potential applied specifically to a subset of interfacial residues (those with an accessible surface area {>=} 50% in the free proteins) that have been delineated by chemical shift perturbation. Docking is achieved by rigid body minimization on the basis of a target function comprising the sparse NOE distance restraints, a van der Waals repulsion potential and the 'reduced' radius of gyration potential. The method is demonstrated for two protein-protein complexes (EIN-HPr and IIA{sup Glc}-HPr) from the bacterial phosphotransferase system. In both cases, starting from 100 different random orientations of the X-ray structures of the free proteins, 100% convergence is achieved to a single cluster (with near identical atomic positions) with an overall backbone accuracy of {approx}2 A. The approach described is not limited to NMR, since interfaces can also be mapped by alanine scanning mutagenesis, and sparse intermolecular distance restraints can be derived from double cycle mutagenesis, cross-linking combined with mass spectrometry, or fluorescence energy transfer.

  16. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    Science.gov (United States)

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  17. Radioiodination of the protein complex of the VA-MENGOC-BC vaccine

    International Nuclear Information System (INIS)

    Caso, R.; Lastre, M.; Alvarez, L.

    1996-01-01

    In this work was made the labelling of the protein complex of the vaccine VA-MEMGOC-BC with I-125 in order to study its immunological responses. These proteins were in both forms: dissolved and conjugated with polisacarids of the C-group. There were used three methods of iodination: chloramine-T iodogen and lactoperoxidase. Was found out that dissolved proteins can be iodinated using these methods with 0,1 mCi of I-125, and the obtained specific activities were similar

  18. The Drosophila IKK-related kinase (Ik2 and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

    Directory of Open Access Journals (Sweden)

    Shaanan Boaz

    2008-09-01

    Full Text Available Abstract Background IkappaB kinases (IKKs regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The Drosophila genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that ik2 regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since spn-F mutants display oocyte defects similar to those of ik2 mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2. Results We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization. Conclusion We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during Drosophila oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.

  19. Electrospray droplet exposure to organic vapors: metal ion removal from proteins and protein complexes.