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

在蛋白质生物化学部门进行的研究主要集中在细菌和人类细胞中蛋白质降解的功能和控制。细胞内蛋白质降解在控制重要细胞调节蛋白的水平方面起着关键作用，也是蛋白质质量控制系统的重要组成部分。细胞质内的大多数蛋白质降解是由atp依赖的蛋白酶进行的，这是一种多组分的分子机器。机器的核心是atp驱动的蛋白质展开酶，它结合特定的蛋白质靶标，破坏其结构，并将未折叠的蛋白质易位到紧密相关的自区隔内肽酶的蛋白质水解室中。我们的研究包括对来自大肠杆菌和人类线粒体的atp依赖性Clp和Lon蛋白酶的结构和生化分析，以及它们在培养细胞中的生物活性测定。在过去的一年里，我们继续分析了ClpP的晶体结构，并确定了保留前肽的ClpP突变体的结构。前肽结合在ClpP的活性位点，并在ClpP成熟过程中自动催化去除。前肽位于ClpP腔室内，n端环位置不变，但未在活性位点发现，表明成熟后底物结合槽发生了变化。我们发现，用几种不同的残基替换Asn151改变ClpP的P1结合袋对ClpP切割的特异性影响很小，这表明扩展结合槽中的相互作用在肽定位和确定切割位点中起主导作用。我们通过使用n结构域缺失的ClpX突变体分离出了稳定的ClpXP全酶复合物，该突变体也包含催化Walker b中的突变。在缺乏ClpX的情况下，结晶工作产生了一种新的ClpP晶体形式，这产生了一个完整的数据集，我们期望它将导致一种新的结构。我们还获得了不含ClpP的突变体ClpX晶体，并正在优化条件以获得具有良好衍射性能的晶体。在其他研究中，我们发现ClpP的两个环在低浓度下分离，肽酶活性被七聚体保留，而不是蛋白酶活性。我们生产了允许两个环交联的ClpP突变体，并表明活性不需要分离环，这支持了通过在ClpP腔室两侧打开较小的出口通道来释放产物的模型。在ClpA的研究中，我们发现n结构域是针对特定底物的活性所必需的，特别是根据n端规则携带不稳定n端残基的蛋白质。结合研究表明，只有一个ClpS与ClpA六聚体紧密结合并抑制活性，我们正在研究激活n端规则蛋白降解的ClpS的最佳结合方式。ClpA的n结构域是可移动的，这种可移动性对活性很重要。删除N-和D1结构域之间的部分连接物限制了N-结构域的运动，并导致部分活性受损。我们的合作者使用低温电子显微镜观察了ClpA部分连接体缺失的n结构域，并将其定位于ClpA D1结构域的顶端表面，以便与传入的底物相互作用。突变连接体区域的酸性残基对活性的影响更大，这表明连接体与D1结构域相互作用并影响底物的相互作用和加工。我们产生了改变了D1和D2结构域Walker B共识的ClpA突变体，并表明它们结合ATP并组装成稳定的复合物。核苷酸在D2位点的结合似乎抑制了D1位点的活性，这表明在atp酶循环过程中，两个结构域之间的通信可能需要在底物加工过程中协调活性。这些突变体已经被纯化，我们的合作者正在筛选结晶条件，这将使我们能够获得关于不同核苷酸状态下ClpA构象的详细结构信息。我们完成了内源性ssra标记蛋白的积累和降解研究，这些蛋白的产生是为了缓解翻译过程中核糖体的停滞，并针对不完全蛋白进行降解。报告这项研究的早期手稿的修订版即将完成，将于下个月提交。使用我们实验室制备的特异性抗ssra抗体，我们证实了ClpXP在ssra标记蛋白的降解中起主要作用，并且接头蛋白SspB有助于将这些蛋白靶向到ClpXP。atp依赖性蛋白酶Lon和ClpAP在降解ssra标记的蛋白中起次要作用，并且仅在缺乏ClpX的情况下才能看到它们的作用。尽管在体外能够降解这些蛋白，但ClpAP对体内ssra标记蛋白降解的贡献很小，这表明ClpAP在体内参与靶向与ssra标记蛋白竞争的其他底物。我们开发了在缺乏ClpX的情况下表达ClpA和ClpP的载体，并将从大肠杆菌细胞中分离和鉴定ClpAP的底物。这些蛋白质将被鉴定和分析，以确定它们是否有正常或异常的n端残基。在我们对人ClpXP的研究中，我们已经证实，人ClpP的过表达会影响顺铂诱导的细胞凋亡的时间和程度。在加入HCLPP siRNA后16小时，细胞中失去HCLPP蛋白，缺乏HCLPP的细胞在48小时后失去线粒体膜的完整性并发生凋亡细胞死亡。短期使用HCLPP siRNA治疗可使细胞对顺铂和司他罗菌素这两种诱导细胞凋亡的药物敏感。HCLPX siRNA导致HCLPX蛋白较慢的丢失，用HCLPX siRNA处理的细胞在72小时后开始死亡。HCLPX siRNA处理产生线粒体特异性的未折叠蛋白反应，如诱导线粒体Hsp60和激活JNK1和JNK2途径所示。报告这些发现的早期手稿的修订版本即将完成。我们已经构建了允许突变hClpP抗sirna表达的载体，用于捕获细胞中的底物，并正在分离人ClpXP的内源性底物，并通过串联HPLC和质谱法对其进行鉴定。在确定人类ClpXP的靶点和功能的相关工作中，我们与以色列耶路撒冷希伯来大学的Joseph Orly博士合作，研究了线粒体中负责降解类固醇摄取蛋白的蛋白酶，即线粒体中的类固醇急性调节蛋白（StAR）。StAR被Lon降解，我们的合作者发现蛋白酶体抑制剂能够通过阻断Lon活性来抑制StAR的降解。我们提供纯化的人ClpXP来测试是否有其他蛋白酶可以靶向StAR，但结果表明Lon是主要的酶