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

蛋白质生物化学部分进行的研究重点是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 依赖性蛋白酶进行的，这些蛋白酶是多组分分子机器。该机器的核心是 ATP 驱动的蛋白质解折叠酶，它结合特定的蛋白质靶标，破坏其结构，并将解折叠的蛋白质转移到紧密相关的自区室化内肽酶的蛋白水解室中。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 蛋白酶进行结构和生化分析，并测定它们在培养细胞中的生物活性。去年，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 的活性位点上，并在 ClpP 成熟过程中自动催化去除。前肽位于 ClpP 室内，如 N 端环位置未改变所示，但在活性位点中未发现，表明底物结合槽在成熟后发生了变化。我们发现，通过用几个不同的残基替换 Asn151 来改变 ClpP 的 P1 结合口袋，对 ClpP 切割的特异性影响很小，表明延伸结合槽中的相互作用在定位肽和确定切割位点中起主导作用。我们通过使用删除了 N 结构域的 ClpX 突变体分离了稳定的 ClpXP 全酶复合物，该突变体还包含催化 Walker B 中的突变。结晶工作在没有 ClpX 的情况下产生了一种新的 ClpP 晶体形式，从而产生了完整的数据我们正在改进，期望它将产生一种新颖的结构。我们还获得了不含 ClpP 的突变 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，并且七聚体保留了肽酶活性，但没有蛋白酶活性。我们生产了允许两个环交联的 ClpP 突变体，并表明活性不需要环的分离，支持通过在 ClpP 室侧面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 末端规则带有不稳定 N 末端残基的蛋白质。结合研究表明，只有一种 ClpS 与 ClpA 六聚体紧密结合并抑制活性，我们正在研究 ClpS 激活 N 端规则蛋白降解的最佳结合。 ClpA 的 N 结构域是可移动的，并且移动性对于活动非常重要。删除 N 结构域和 D1 结构域之间的部分连接子会限制 N 结构域的移动并导致部分活性受损。我们的合作者使用冷冻电子显微镜观察了部分接头缺失的 ClpA N 结构域，并定位在 ClpA D1 结构域的顶端表面上，处于与引入的底物相互作用的位置。连接子区域中的酸性残基突变对活性具有更大的影响，表明连接子与 D1 结构域相互作用并影响底物相互作用和加工。我们生成了在 D1 和 D2 结构域的 Walker B 共有序列中发生改变的 ClpA 突变体，并表明它们结合 ATP 并组装成稳定的复合物。 D2 位点上的核苷酸结合似乎会抑制 D1 位点的活性，这表明 ATP 酶循环期间两个结构域之间的通信可能需要协调底物加工过程中的活性。这些突变体已被纯化，我们的合作者正在筛选结晶条件，这将使我们能够获得有关不同核苷酸状态下 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靶向不完整的蛋白质进行降解。报告这项研究的早期手稿的修订版即将完成，并将在下个月内提交。使用我们实验室制备的特异性抗 SsrA 抗体，我们证实 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 ClpXP。 ATP 依赖性蛋白酶 Lon 和 ClpAP 在降解 SsrA 标记蛋白方面发挥较小作用，并且仅在 ClpX 不存在的情况下才能看到它们的作用。尽管 ClpAP 能够在体外降解这些蛋白质，但 ClpAP 对体内 SsrA 标记蛋白质的降解贡献很小，这表明 ClpAP 参与靶向在体内与 SsrA 标记蛋白质竞争的其他底物。我们开发了在没有 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并分析以确定它们是否具有正常或异常的 N 末端残基。在我们对人 ClpXP 的研究中，我们证实人 ClpP 的过度表达会影响顺铂诱导的细胞凋亡的时间和程度。添加 HCLPP siRNA 16 小时后，HCLPP 蛋白从细胞中丢失，缺乏 hClpP 的细胞在 48 小时后失去线粒体膜完整性并发生细胞凋亡。 HCLPP siRNA 的短期治疗使细胞对顺铂和十字孢菌素（两种诱导细胞凋亡的药物）敏感。 HCLPX siRNA 导致 hClpX 蛋白损失较慢，用 HCLPX siRNA 处理的细胞在 72 小时后开始死亡。 HCLPX siRNA 治疗产生线粒体特异性未折叠蛋白反应，如线粒体 Hsp60 的诱导和 JNK1 和 JNK2 途径的激活所示。报告这些发现的早期手稿的修订版即将完成。我们已经构建了载体，允许突变型 hClpP 的 siRNA 抗性表达，以捕获细胞中的底物，并且正在分离人 ClpXP 的内源底物，并通过串联 HPLC 和质谱法对其进行鉴定。在与确定人类 ClpXP 的靶标和功能相关的工作中，我们与以色列耶路撒冷希伯来大学的 Joseph Orly 博士领导的一个项目进行了合作，研究负责线粒体中类固醇摄取蛋白降解的蛋白酶，线粒体中的类固醇急性调节蛋白或 StAR。 StAR 被 Lon 降解，我们的合作者表明蛋白酶体抑制剂能够通过阻断 Lon 活性来抑制 StAR 的降解。我们提供了纯化的人 ClpXP 来测试其他蛋白酶是否可以靶向 StAR，但结果表明 Lon 是负责的主要酶