Structure and function of ClpXP

项目摘要 AAA+蛋白水解酶去除有毒蛋白并调节许多其他重要的细胞过程 促进健康和预防疾病。与此同时，蛋白质的降解必须是 受到严格的监管。AAA+蛋白水解酶通过其内部的 蛋白水解室，通过狭窄的通道进入，不包括天然折叠的蛋白质。 这种机制保护大多数蛋白质免受意外降解，并需要特定的 底物被识别、展开，然后转移到降解室中。在……里面 例如，AAA+ClpXP蛋白酶，ClpX的环六聚体使用ATP的能量 将特定的目标蛋白解开，并将它们转移到ClpP中进行降解。 ClpXP是最具特性的AAA+蛋白水解酶之一，也是其他ATP- 依赖的蛋白水解酶和AAA+重塑机制。这些以三磷酸腺苷为燃料的酶 细胞中各种各样的机械重塑、运输和调节任务。在哺乳动物身上， 线粒体ClpP的缺失会导致不孕、听力损失和生长缺陷，而 线粒体ClpX在血红素生物合成中起着重要作用。细菌ClpXP可以促进 并且是结核分枝杆菌有效的抗生素靶点。取得了实质性进展 了解大肠杆菌Clpxp的一般生化和结构特征 和其他AAA+酶，但关于分子的重要和基本问题 这些机器的机械装置仍然存在。例如，尚不清楚ClpX如何识别许多 N端和C端退化的类别，ClpX是否相对于ClpP旋转 在正常运行期间，校对是否有助于确保降级特异性，如何 多个底物链可以同时通过ClpX通道转运，什么 在机械展开过程中，细节与多肽底物相互作用，是否 ATP水解可以在螺旋ClpX环中的多个位置发生，也可以只在一个或几个位置发生 特殊职位，以及详细的ATPase循环如何与机械工作相结合。这个 本提案中描述的实验将解决这些问题，并提供概念性的 适用于研究整个AAA+超家族机器的框架。

Project Summary AAA+ proteases remove toxic proteins and regulate many other important cellular processes that promote health and prevent disease. At the same time, protein degradation must be carefully regulated. AAA+ proteases assemble into multi-subunit structures with an internal proteolytic chamber, accessible through narrow channels that exclude natively folded proteins. This mechanism protects most proteins from unintended degradation and requires specific substrates to be recognized, unfolded, and then translocated into the degradation chamber. In the AAA+ ClpXP protease, for example, a ring hexamer of ClpX uses the energy of ATP hydrolysis to unfold specific target proteins and translocate them into ClpP for degradation. ClpXP is one of the best-characterized AAA+ proteases and is a paradigm for other ATP- dependent proteases and AAA+ remodeling machines. These ATP-fueled enzymes perform a wide variety of mechanical remodeling, transport, and regulatory tasks in the cell. In mammals, loss of mitochondrial ClpP results in infertility, hearing loss, and growth defects, whereas mitochondrial ClpX plays an important role in heme biosynthesis. Bacterial ClpXP can promote pathogenesis and is a validated antibiotic target in M. tuberculosis. Substantial progress has been made in understanding the general biochemical and structural features of E. coli ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these machines remain. For example, it is not known how ClpX identifies many classes of N-terminal and C-terminal degrons, whether ClpX rotates with respect to ClpP during normal function, whether proofreading helps ensure degradation specificity, how multiple substrate chains can be simultaneously translocated through the ClpX channel, what the detailed and interacts with polypeptide substrates during mechanical unfolding, whether ATP hydrolysis can occur at multiple positions in the spiral ClpX ring or only at one or a few special positions, and how the detailed ATPase cycle is coupled to mechanical work. The experiments described in this proposal will address these questions and provide a conceptual framework applicable to studies of the entire superfamily of AAA+ machines.