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

Crescentus中AAA蛋白酶CIpXP对蛋白质的降解及其调控 蛋白质分解是这种有机体正常的细胞周期进程的中心。例如，降级的 ClpXP在特定时间和地点控制CtrA对DNA的正确启动起着关键作用 及时复制。尽管底物识别可以在蛋白酶本身的水平上发生， 附加调节通常以适配器的形式存在，其增强特定底物的降解 并允许电池对衬底选择进行优先排序。了解分子机制是如何 ClpXP识别底物(如CtrA)，并可以以受监管的、协调一致的方式行动，以专门降解 通过适配机制获得的蛋白质亚集是该项目的中心目标。在目标1中，1提议 阐述为什么ClpX在某些生物体中是必需的，而在其他生物体中不是。目标2包括探索调制器如何 ClpX活性调节细胞周期进程中的蛋白分解，并从生物化学的角度重建这种调节。 最后，目标3侧重于一般方法，在该方法中，1将通过一个 无偏见的蛋白质组学方法，利用这些酶的非活性版本来捕获底物。这些 实验将使我能够在体内观察受调控的蛋白质降解和体外 机械性生化实验使我们更全面地了解了细胞的调节作用 这个系统。

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.