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的 ATP 依赖性 Clp 和 Lon 蛋白酶及其在人类线粒体中的同源物进行结构和生化分析。去年，在多个领域取得了进展。基于我们在活性位点具有共价结合肽的 ClpP 晶体结构，我们生成了具有改变的底物相互作用的突变体。底物具有移动结合模式，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 残基具有明确的结合口袋。我们对 S1 口袋内的残基进行了突变，发现 ClpP 的活性受到严重影响。目前正在进行研究以确定 S1 口袋表面特性发生改变的突变体中切割的特异性是否受到影响。我们已经开始对 ClpP 和 ClpAP 肽酶和蛋白酶活性进行快速动力学测量，以确定底物解折叠和易位的初始事件以及底物进入 ClpP 室的机制。我们已经获得了 ClpP 环接触动态变化的生化证据，该变化导致十四聚体解离成两个七聚环。这种解离应反映催化循环期间发生的 ClpP 构象变化，并且可能与底物进入室或降解后肽产物从室中释放有关。 ClpS 是 ClpA 的接头，在体外底物（例如 GFP-SsrA）中抑制 ClpA 活性；然而，在体内，ClpS 是降解一类称为“N 端规则”蛋白质的底物所必需的，该蛋白质具有非规范的氨基末端残基。在存在 ClpS 的情况下，模型 N 端规则底物会被 ClpA 降解，并且 N 结构域（ClpS 的结合位点）是这种降解所必需的。我们正在通过使用 ClpA 和 ClpS 结合灭活的 ClpP 将内源性 N 端规则底物捕获在体内来识别内源性 N 端规则底物。在体外，ClpS 以每六聚体 1 个 ClpS 的化学计量抑制 ClpA 活性。我们结合使用冷冻电子显微镜（与 A. C. Steven, NIAMS 合作）和生化方法来确定 ClpS 对 ClpA 结构和与 ClpS 结合的 N 结构域分布的影响。我们实验室制备的特异性抗 SsrA 抗体已用于测量内源性 SsrA 标签蛋白的体内半衰期，并证明 ClpXP 在这些蛋白的降解中发挥主要作用。其他 ATP 依赖性蛋白酶（Lon 和 ClpAP）可以降解 SsrA 标记的蛋白质，但只有在没有 ClpX 的情况下才能看到它们的贡献。我们发现，许多蛋白质在大肠杆菌中的过度表达会导致带有 SsrA 标签的多种形式的蛋白质的大量产生。这些蛋白质大部分被 ClpXP 降解，但当 ClpXP 活性受到损害时，它们会积累到显着水平。在我们旨在获得 ClpA 六聚体晶体结构的项目中，我们生成了 ClpA 突变体，在六聚体中预期的亚基接触点引入半胱氨酸残基。