Specificity, Regulation and Proteomic Profiling of an Essential AAA+ Protease

AAA+蛋白酶ClpXP的蛋白质降解在新月柄杆菌中是必需的，并且这种降解的调节 蛋白水解是这种生物体的正常细胞周期进程的中心。例如， ClpXP在特定的时间和地点对DNA的正确启动起着关键作用 及时复制。尽管底物识别可以发生在蛋白酶本身的水平上， 另外的调节通常以衔接子的形式存在 并允许电池对底物的选择进行优先化。了解分子机制 ClpXP识别底物（如CtrA），可以以受调节的协调方式特异性降解 通过衔接机制的蛋白质子集是这个项目的中心目标。在目标1中，我建议 解决为什么ClpX在某些生物中是必不可少的，而不是其他生物。目的2包括探索如何调节 ClpX活性调节细胞周期进程中的蛋白水解，并以生物化学方式重建这种调节。 最后，目标3侧重于一种通用方法，其中1将通过以下方法获得ClpXP的降解曲线： 无偏见的蛋白质组学方法利用这些酶的无活性版本来捕获底物。这些 这些实验将使我能够在体内观察到的受调节的蛋白质降解和体外观察到的蛋白质降解之间建立桥梁。 机械生化实验，从而更全面地了解调节作用， 这系统.

Protein degradation by the AAA+ protease CIpXP is essential in Caulobacter crescentus and regulation of this proteolysis is central to the proper cell-cycle progression of this organism. For example, degradation ofthe master regulator CtrA by ClpXP at a specific time and place plays a critical role for proper initiation of DNA replication in a timely fashion. Although substrate recognition can occur at the level ofthe protease itself, additional regulation is often present in the form of adaptors that enhance degradation of particular substrates and allow for prioritization of substrate choice by the cell. Understanding the molecular mechanisms of how ClpXP recognizes substrates (such as CtrA) and can act in a regulated, concerted fashion to specifically degrade subsets of proteins through adaptor mechanisms are the central goals of this project. In Aim 1,1 propose to address why ClpX is essential in some organisms and not others. Aim 2 consists of exploring how modulators of ClpX activity regulate proteolysis during cell-cycle progression and reconstitute this regulation biochemically. Finally, Aim 3 focuses on a general approach in which 1 will obtain degradation profiles of ClpXP through an unbiased proteomic approach utilizing inactive versions of these enzymes to trap substrates. These experiments will allow me to bridge the in vivo observations of regulated protein degradation and the in vitro mechanistic biochemical experiments resulting in a more comprehensive understanding ofthe regulatory role of this system.