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Borner GH Antrobus R Hirst J Bhumbra GS Kozik P Jackson LP Sahlender DA Robinson MS 《The Journal of cell biology》2012,197(1):141-160
Despite recent advances in mass spectrometry, proteomic characterization of transport vesicles remains challenging. Here, we describe a multivariate proteomics approach to analyzing clathrin-coated vesicles (CCVs) from HeLa cells. siRNA knockdown of coat components and different fractionation protocols were used to obtain modified coated vesicle-enriched fractions, which were compared by stable isotope labeling of amino acids in cell culture (SILAC)-based quantitative mass spectrometry. 10 datasets were combined through principal component analysis into a "profiling" cluster analysis. Overall, 136 CCV-associated proteins were predicted, including 36 new proteins. The method identified >93% of established CCV coat proteins and assigned >91% correctly to intracellular or endocytic CCVs. Furthermore, the profiling analysis extends to less well characterized types of coated vesicles, and we identify and characterize the first AP-4 accessory protein, which we have named tepsin. Finally, our data explain how sequestration of TACC3 in cytosolic clathrin cages causes the severe mitotic defects observed in auxilin-depleted cells. The profiling approach can be adapted to address related cell and systems biological questions. 相似文献
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Structure of foot-and-mouth disease virus RNA-dependent RNA polymerase and its complex with a template-primer RNA 总被引:15,自引:0,他引:15
Ferrer-Orta C Arias A Perez-Luque R Escarmís C Domingo E Verdaguer N 《The Journal of biological chemistry》2004,279(45):47212-47221
Genome replication in picornaviruses is catalyzed by a virally encoded RNA-dependent RNA polymerase, termed 3D. The enzyme performs this operation, together with other viral and probably host proteins, in the cytoplasm of their host cells. The crystal structure of the 3D polymerase of foot-and-mouth disease virus, one of the most important animal pathogens, has been determined unliganded and bound to a template-primer RNA decanucleotide. The enzyme folds in the characteristic fingers, palm and thumb subdomains, with the presence of an NH2-terminal segment that encircles the active site. In the complex, several conserved amino acid side chains bind to the template-primer, likely mediating the initiation of RNA synthesis. The structure provides essential information for studies on RNA replication and the design of antiviral compounds. 相似文献
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Structural analysis of the hepatitis C virus RNA polymerase in complex with ribonucleotides 总被引:12,自引:0,他引:12
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We report here the results of a systematic high-resolution X-ray crystallographic analysis of complexes of the hepatitis C virus (HCV) RNA polymerase with ribonucleoside triphosphates (rNTPs) and divalent metal ions. An unexpected observation revealed by this study is the existence of a specific rGTP binding site in a shallow pocket at the molecular surface of the enzyme, 30 A away from the catalytic site. This previously unidentified rGTP pocket, which lies at the interface between fingers and thumb, may be an allosteric regulatory site and could play a role in allowing alternative interactions between the two domains during a possible conformational change of the enzyme required for efficient initiation. The electron density map at 1.7-A resolution clearly shows the mode of binding of the guanosine moiety to the enzyme. In the catalytic site, density corresponding to the triphosphates of nucleotides bound to the catalytic metals was apparent in each complex with nucleotides. Moreover, a network of triphosphate densities was detected; these densities superpose to the corresponding moieties of the nucleotides observed in the initiation complex reported for the polymerase of bacteriophage phi6, strengthening the proposal that the two enzymes initiate replication de novo by similar mechanisms. No equivalent of the protein stacking platform observed for the priming nucleotide in the phi6 enzyme is present in HCV polymerase, however, again suggesting that a change in conformation of the thumb domain takes place upon template binding to allow for efficient de novo initiation of RNA synthesis. 相似文献
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Wei C Yang J Zhu J Zhang X Leng W Wang J Xue Y Sun L Li W Wang J Jin Q 《Journal of proteome research》2006,5(8):1860-1865
Shigella flexneri is the causative agent of most shigellosis cases in developing countries. We used different proteolytic enzymes to selectively shave the protruding proteins on the surface of purified bacterial membrane sheets or vesicles, and recovered peptides were subsequently identified using 2-D LC-MS/MS. As a result, a total of 666 proteins were unambiguously assigned, including 159 integral membrane proteins, 35 outer membrane proteins and 114 proteins previously annotated as hypothetical. The former had an average grand average hydrophobicity score of 0.362 and were predicted to separate within a pH range of 4.1-10.6 with molecular mass 8-148 kDa, which represents the largest validated set of integral membrane proteins in this organism to date. A functional classification revealed that a large proportion of the identified proteins were involved in cell envelope biogenesis and energy production and conversion. For the first time, this work provides a global view of the S. flexneri 2a membrane subproteome. 相似文献
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Mänz B Götz V Wunderlich K Eisel J Kirchmair J Stech J Stech O Chase G Frank R Schwemmle M 《The Journal of biological chemistry》2011,286(10):8414-8424
To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFNα/β receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses. 相似文献
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Radiation-induced genomic instability is a well-studied phenomenon that is measured as mitotically heritable genetic alterations observed in the progeny of an irradiated cell. The mechanisms that perpetuate this instability are unclear; however, a role for chronic oxidative stress has consistently been demonstrated. In the chromosomally unstable LS12 cell line, oxidative stress and genomic instability were correlated with mitochondrial dysfunction. To clarify this mitochondrial dysfunction and gain insight into the mechanisms underlying radiation-induced genomic instability we have evaluated the mitochondrial subproteome and performed quantitative mass spectrometry analysis of LS12 cells. Of 98 quantified mitochondrial proteins, 17 met criteria for fold changes and reproducibility; and 11 were statistically significant in comparison with the stable parental GM10115 cell line. Previous observations implicated defects in the electron transport chain (ETC) in the LS12 cell mitochondrial dysfunction. Proteomic analysis supports these observations, demonstrating significantly reduced levels of mitochondrial cytochrome c, the intermediary between complexes III and IV of the ETC. Results also suggest that LS12 cells compensate for ETC dysfunction and oxidative stress through increased levels of tricarboxylic acid cycle enzymes and upregulation of proteins that protect against oxidative stress and apoptosis. More than one cellular defect is likely to contribute to the genomic instability phenotype, and evaluation of gene and microRNA expression suggests that epigenetics play a role in the phenotype. These data suggest that LS12 cells have adapted mechanisms that allow survival under suboptimal conditions of oxidative stress and compromised mitochondrial function to perpetuate genomic instability. 相似文献
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RNA polymerase of influenza virus. III. Isolation of RNA polymerase-RNA complexes from influenza virus PR8 总被引:3,自引:0,他引:3
Ribonucleoprotein (RNP) cores with RNA-synthesizing activity were prepared in two fractions, M protein-free and M protein-associated, from detergent-treated influenza virus PR8 by centrifugation through a discontinuous triple gradient of cesium sulfate, glycerol, and NP-40. The M-free RNP was fractionated by phosphocellulose column chromatography into two major RNP forms, A and B, which differed in the content of P proteins, while the M-associated RNP gave only the low P-content Form-B RNP. Starting from the high P-content Form-A RNP, an RNA-P proteins complex virtually free from NP protein was isolated by cesium sulfate equilibrium centrifugation. The complex, containing only three P proteins (P1, P2, and P3), was still active in catalyzing RNA synthesis in vitro without addition of exogenous template, indicating that NP protein is not required for the catalysis of RNA synthesis. RNA synthesis by the isolated RNA-P proteins complex was dependent on either ApG or capped RNA primers, and required four ribonucleoside triphosphates as substrates. The RNA product in this reaction was hybridizable to viral RNA. A complex of one each of the three P proteins was separated from RNA by glycerol gradient centrifugation after ribonuclease treatment or cesium chloride equilibrium centrifugation. 相似文献
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Anti-hepatitis C virus activity of tamoxifen reveals the functional association of estrogen receptor with viral RNA polymerase NS5B 总被引:1,自引:0,他引:1
Watashi K Inoue D Hijikata M Goto K Aly HH Shimotohno K 《The Journal of biological chemistry》2007,282(45):32765-32772
Hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma. HCV genome replication occurs in the replication complex (RC) around the endoplasmic reticulum membrane. However, the mechanisms regulating the HCV RC remain widely unknown. Here, we used a chemical biology approach to show that estrogen receptor (ESR) is functionally associated with HCV replication. We found that tamoxifen suppressed HCV genome replication. Part of ESRalpha resided on the endoplasmic reticulum membranes and interacted with HCV RNA polymerase NS5B. RNA interference-mediated knockdown of endogenous ESRalpha reduced HCV replication. Mechanistic analysis suggested that ESRalpha promoted NS5B association with the RC and that tamoxifen abrogated NS5B-RC association. Thus, ESRalpha regulated the presence of NS5B in the RC and stimulated HCV replication. Moreover, the ability of ESRalpha to regulate NS5B was suggested to serve as a potential novel target for anti-HCV therapeutics. 相似文献
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Mosley RT Edwards TE Murakami E Lam AM Grice RL Du J Sofia MJ Furman PA Otto MJ 《Journal of virology》2012,86(12):6503-6511
The replication of the hepatitis C viral (HCV) genome is accomplished by the NS5B RNA-dependent RNA polymerase (RdRp), for which mechanistic understanding and structure-guided drug design efforts have been hampered by its propensity to crystallize in a closed, polymerization-incompetent state. The removal of an autoinhibitory β-hairpin loop from genotype 2a HCV NS5B increases de novo RNA synthesis by >100-fold, promotes RNA binding, and facilitated the determination of the first crystallographic structures of HCV polymerase in complex with RNA primer-template pairs. These crystal structures demonstrate the structural realignment required for primer-template recognition and elongation, provide new insights into HCV RNA synthesis at the molecular level, and may prove useful in the structure-based design of novel antiviral compounds. Additionally, our approach for obtaining the RNA primer-template-bound structure of HCV polymerase may be generally applicable to solving RNA-bound complexes for other viral RdRps that contain similar regulatory β-hairpin loops, including bovine viral diarrhea virus, dengue virus, and West Nile virus. 相似文献