Biochemistry of Energy-Dependent (Intracellular) Protein Degradation

能量依赖性（细胞内）蛋白质降解的生物化学

• 批准号: 7592538
• 负责人: MICHAEL MAURIZI
• 金额: $ 112.49万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592538
• 关键词: 26S proteasome; ATP Hydrolysis; ATP phosphohydrolase; ATP-Dependent Proteases; Active Sites; Acute; Adaptor Signaling Protein; Affect; Antibodies; Antineoplastic Agents; Apical; Apoptosis; Apoptotic; Binding; Binding Sites; Biochemical; Biochemical Genetics; Biochemistry; Biological; Biological Assay; Cell Death; Cells; Cisplatin; Communication; Complex; Condition; Consensus; Coupled; Coupling; Cryoelectron Microscopy; Crystallization; Cultured Cells; Cytosol; Data Set; Diagnostic; E coli Lon protein; Endopeptidases; Enzymes; Escherichia coli; Eukaryota; Eukaryotic Cell; Goals; Growth and Development function; Head; Heart; High Pressure Liquid Chromatography; Holoenzymes; Human; Impairment; In Vitro; Israel; Laboratories; Lead; Learning; Life; Localized; MAPK8 gene; MAPK9 gene; Mammalian Cell; Manuscripts; Mass Spectrum Analysis; Minor; Mitochondria; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Machines; Movement; Mutate; Mutation; N Domain; N-terminal; Nature; New Agents; Nucleotides; Pathway interactions; Peptide Hydrolases; Peptides; Play; Positioning Attribute; Process; Property; Proteasome Inhibitor; Protein Biochemistry; Protein translocation; Proteins; Quality Control; Regulatory Pathway; Reporting; Research; Research Project Grants; Resistance; Resolution; Ribosomes; Role; Screening procedure; Side; Site; Small Interfering RNA; Specificity; Steroids; Structure; Substrate Interaction; Surface; System; Testing; Therapeutic Agents; Time; Translations; Universities; Walkers; Work; antimicrobial; base; crosslink; design; endopeptidase Clp; endopeptidase La; expectation; genetic regulatory protein; in vivo; inhibitor/antagonist; mitochondrial membrane; molecular modeling; mutant; novel; protein degradation; rapid growth; response; unfoldase; uptake; vector

Research conducted in the Biochemistry of Proteins Section is focused on the function and control of protein degradation in bacterial and human cells. Intracellular protein degradation plays a critical part in controlling the levels of important cellular regulatory proteins and is an essential component of the protein quality control system as well. Most protein degradation within the cytosol is carried out by ATP-dependent proteases, which are multi-component molecular machines. The heart of the machine is an ATP-driven protein unfoldase that binds a specific protein target, disrupts its structure, and translocates the unfolded protein into the proteolytic chamber of a tightly associated self-compartmentalized endopeptidase. Our studies encompass structural and biochemical analysis of the ATP-dependent Clp and Lon proteases from E. coli and from human mitochondria and assay of their biological activities in cultured cells. In the last year, we have continued analysis of the crystal structure of ClpP and have determined the structure of a mutant of ClpP that retains the pro-peptide. The pro-peptide binds in the active site of ClpP and is removed autocatalytically during maturation of ClpP. The pro-peptide was inside the ClpP chamber, as indicated by the unchanged position of the N-terminal loop but was not found in the active site, indicating that the substrate binding groove is changed after maturation. We found that changing the P1 binding pocket of ClpP by replacing Asn151 with several different residues has very little influence on the specificity of cleavage by ClpP, indicating that interactions in the extended binding groove play the dominant role in positioning peptides and determining the site of cleavage. We have isolated stable holoenzyme complexes of ClpXP by using a mutant of ClpX deleted for the N-domain also containing a mutation in the catalytic Walker B. Crystallization efforts yielded a novel crystal form of ClpP in the absence of ClpX, which yielded a complete data set that we are refining in the expectation that it will lead to a novel structure. We also obtained crystals of the mutant ClpX but without ClpP, and are optimizing conditions to obtain crystals with good diffraction properties. In other studies, we found that the two rings of ClpP separate at low concentrations and that peptidase activity is retained by the heptamer although not protease activity. We produced mutants of ClpP that allow the two rings to be cross-linked and have shown that separation of the rings is not required for activity, supporting the model that release of products occurs by opening of smaller exit channels in the sides of the ClpP chamber. In studies of ClpA, we found that the N-domains are needed for activity against specific substrates, in particular proteins bearing destabilizing N-terminal residues according to the N-end rule. Binding studies indicate that only one ClpS binds tightly to the ClpA hexamer and inhibits activity and we are in the process of studying the optimum binding of ClpS for activation of N-end rule protein degradation. The N-domains of ClpA are mobile and that mobility is important for activity. Deleting part of the linker between the N- and D1 domains restricts movement of the N-domains and causes a partial impairment of activity. The N-domains of ClpA with the partial linker deletion have been visualized by our collaborators using cryo electron microscopy and localize on the apical surface of the ClpA D1 domain in a position to interact with incoming substrates. Mutating the acidic residues in the linker region has an even greater effect on activity, suggesting that the linker interacts with the D1 domain and affects substrate interaction and processing. We generated ClpA mutants altered in the Walker B consensus of the D1 and D2 domains and showed that they bind ATP and assemble into stable complexes. Binding of nucleotide at the D2 site appears to inhibit activity at the D1 site, suggesting that communication between the two domains during the ATPase cycle may be needed to coordinate the activity during substrate processing. These mutants have been purified and our collaborators are screening for crystallization conditions that will allow us to obtained detailed structural information about the conformation of ClpA in different nucleotide states. We completed our studies of accumulation and degradation of endogenous SsrA-tagged proteins, which are produced to relieve ribosome stalling during translation and to target the incomplete proteins for degradation. A revised version of an earlier manuscript reporting this study is nearing completion and will be submitted within the next month. Using the specific anti-SsrA antibody prepared in our laboratory, we confirmed that ClpXP plays the major role in degradation of SsrA-tagged proteins and that the adaptor protein, SspB, helps target those proteins to ClpXP. The ATP-dependent proteases Lon and ClpAP have a minor role in degrading SsrA-tagged proteins and their contributions are seen only in the absence of ClpX. The small contribution of ClpAP to degradation of SsrA-tagged proteins in vivo despite being able to degrade these proteins in vitro indicates that ClpAP is engaged in targeting other substrates that out compete SsrA-tagged proteins in vivo. We developed vectors to express ClpA and ClpP in the absence of ClpX and will isolate and identify substrates for ClpAP from E. coli cells. These proteins will be identified and also analyzed to determine if they have normal or abnormal N-terminal residues. In our studies of human ClpXP, we have confirmed that over expression of human ClpP affects the timing and extent of cisplatin-induced apoptosis. HCLPP protein is lost from cells after 16 h following the addition of HCLPP siRNA, and the cells lacking hClpP lose mitochondrial membrane integrity and undergo apoptotic cell death after 48 h. Short term treatment with HCLPP siRNA sensitizes the cells to both cisplatin and to staurosporin, two agents that induce apoptosis. HCLPX siRNA leads to a slower loss of hClpX protein, and cells treated with HCLPX siRNA begin to die after 72 h. Treatment HCLPX siRNA produces a mitochondria-specific unfolded protein response, as shown by induction of mitochondrial Hsp60 and activation of the JNK1 and JNK2 pathways. A revised version of an earlier manuscript reporting these findings is nearing completion. We have constructed vectors that allow siRNA-resistant expression of mutant hClpP for trapping substrates in cells and are in the process of isolating endogenous substrates of human ClpXP and identifying them by tandem HPLC and mass spectrometry. In work related to identifying the targets and function of human ClpXP, we collaborated on a project headed by Dr. Joseph Orly, at the Hebrew University of Jerusalem, Israel, in studying the proteases responsible for degradation of the steroid uptake protein in mitochondria, the steroid acute regulatory protein or StAR, in mitochondria. StAR is degraded by Lon and our collaborators showed that proteasome inhibitors are capable of inhibiting degradation of StAR by blocking Lon activity. We provided purified human ClpXP to test whether additional proteases can target StAR but the results showed that Lon is the major enzyme responsible