Biochemistry of Energy-Dependent (Intracellular) Protein

能量依赖性（细胞内）蛋白质的生物化学

• 批准号: 7337911
• 负责人: MICHAEL MAURIZI
• 金额: --
• 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/7337911
• 关键词: Biochemistry; Energy; Dependent; Intracellular; Protein

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 their homologs in human mitochondria. In the last year, progress has been made in several areas. Based on our crystal structure of ClpP with a covalently bound peptide at the active site, we have generated mutants with altered substrate interactions. Substrates have a mobile binding mode in which polypeptides interact through main chain hydrogen bonding and can slide within an extended active site groove. Only the P1 residue has a defined binding pocket. We have mutated residues within the S1 pocket and found that the activity of ClpP is drastically affected. Studies are underway to determine whether the specificity of cleavage is affected in mutants in which the surface properties of the S1 pocket have been altered. We have initiated rapid kinetic measurements of ClpP and ClpAP peptidase and protease activity in order to determine the initial events in substrate unfolding and translocation and the mechanism by which substrates enter the ClpP chamber. We have obtained biochemical evidence of a dynamic change in ClpP ring contacts that results in dissociation of the tetradecamer into two heptameric rings. This dissociation should reflect a conformational change in ClpP that occurs during the catalytic cycle and could be related to substrate entry into the chamber or release of peptide products from the chamber after degradation. ClpS, an adaptor for ClpA, inhibits ClpA activity in vitro substrates such as GFP-SsrA; however, in vivo, ClpS is required for degradation of a class of substrates called "N-end rule" proteins, which have non-canonical amino terminal residues. Model N-end rule substrates are degraded by ClpA in the presence of ClpS and the N-domain, which is the binding site for ClpS, is required for this degradation. We are in the process of identifying endogenous N-end rule substrates by trapping them in vivo using ClpA and ClpS in combination with inactivated ClpP. In vitro, ClpS inhibits ClpA activity at a stoichiometry of one ClpS per hexamer. We are using a combination of cryo-electron microscopy (in collaboration with A. C. Steven, NIAMS) and biochemical methods to determine the effects of ClpS on the structure of ClpA and the distribution of the N-domains with ClpS bound. A specific anti-SsrA antibody prepared in our laboratory, has been used to measure the half-lives endogenous SsrA-tagged proteins in vivo and to demonstrate that ClpXP plays the major role in degradation of these proteins. Other ATP-dependent proteases (Lon and ClpAP) can degrade SsrA-tagged proteins but their contribution can be seen only in the absence of ClpX. We have found that over expression of many proteins in E. coli results in significant generation of multiple forms of the protein with SsrA-tags. These proteins are mostly degraded by ClpXP but can accumulate to significant levels when ClpXP activity is compromised. In our project aimed at obtaining the crystal structure of ClpA hexamers, we have generated ClpA mutants introducing cysteine residues at the subunit contact points expected in the hexamer.

蛋白质生物化学部分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的细胞调节蛋白的水平中起着关键作用，并且也是蛋白质质量控制系统的重要组成部分。胞质溶胶中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的分子机器。这台机器的核心是ATP驱动的蛋白质解折叠酶，它结合特定的蛋白质靶点，破坏其结构，并将解折叠的蛋白质转位到紧密结合的自区室化内肽酶的蛋白水解室中。我们的研究包括对大肠杆菌的ATP依赖性Clp和Lon蛋白酶的结构和生化分析。coli及其同源物在人线粒体中的表达。去年，在若干领域取得了进展。基于我们的晶体结构的ClpP与共价结合的肽在活性位点，我们已经产生了突变体与改变基板相互作用。底物具有移动的结合模式，其中多肽通过主链氢键相互作用，并且可以在延伸的活性位点凹槽内滑动。只有P1残基具有确定的结合口袋。我们在S1口袋内突变了残基，发现ClpP的活性受到了极大的影响。研究正在进行中，以确定是否切割的特异性在突变体中的S1口袋的表面性质已被改变的影响。我们已经启动了快速动力学测量ClpP和ClpAP肽酶和蛋白酶活性，以确定在底物展开和易位的初始事件和机制，通过该机制，底物进入ClpP室。我们已经获得了ClpP环接触的动态变化的生化证据，其导致十四聚体解离成两个七聚体环。这种解离应反映在催化循环期间发生的ClpP的构象变化，并且可能与底物进入腔室或降解后从腔室释放肽产物有关。ClpS是ClpA的衔接子，在体外底物如GFP-SsrA中抑制ClpA活性;然而，在体内，ClpS是降解一类称为“N-末端规则”蛋白的底物所必需的，所述蛋白具有非规范的氨基末端残基。模型N-末端规则底物在ClpS存在下被ClpA降解，并且N-结构域（其为ClpS的结合位点）是该降解所需的。我们正在通过使用ClpA和ClpS与失活的ClpP组合在体内捕获内源性N-末端规则底物来鉴定内源性N-末端规则底物。在体外，ClpS以每六聚体一个ClpS的化学计量抑制ClpA活性。我们正在使用冷冻电子显微镜（与A。C. Steven，NIAMS）和生物化学方法来确定ClpS对ClpA结构和与ClpS结合的N-结构域分布的影响。在我们的实验室制备的特异性抗SsrA抗体，已被用来测量半衰期内源性SsrA标记的蛋白质在体内，并证明ClpXP在这些蛋白质的降解中起主要作用。其他ATP依赖性蛋白酶（Lon和ClpAP）可以降解SsrA标记的蛋白质，但它们的贡献只能在不存在ClpX的情况下才能看到。我们发现，在E.大肠杆菌中的结果显着产生多种形式的蛋白与SsrA标签。这些蛋白质主要被ClpXP降解，但当ClpXP活性受损时，可以积累到显著水平。在我们旨在获得ClpA六聚体的晶体结构的项目中，我们已经产生了在六聚体中预期的亚基接触点处引入半胱氨酸残基的ClpA突变体。