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

描述(由申请人提供)：由AAA+蛋白水解酶CIpXP降解的蛋白质在新月弯杆菌中是必不可少的，这种蛋白分解的调节是这种生物正确的细胞周期进展的中心。例如，CIpXP在特定时间和地点降解主调控子CtrA，对于及时正确地启动DNA复制起着至关重要的作用。虽然底物识别可以在蛋白酶本身的水平上发生，但额外的调节通常是以适配器的形式存在的，它增强了特定底物的降解，并允许细胞优先选择底物。了解CIpXP如何识别底物(如CtrA)的分子机制，以及如何通过适配器机制以受调控的、协同的方式作用于特定的蛋白质亚集是该项目的中心目标。在目标1中，1将使用生化分析来识别和操纵一种已知的蛋白质分解接头的底物轮廓，该接头负责质量控制产品的直接蛋白质分解。目标2包括重建受调控的主调控子CtrA的降解，并使用生化分级来分离这种重要的细胞周期调节器的调节器。最后，目标3侧重于一种一般的方法，在这种方法中，我将通过一种无偏见的蛋白质组学方法获得CIpXP的降解图谱，该方法利用这些酶的非活性版本来捕获底物。在指导阶段，我将以对CIpXP基板加工和捕获的初步观察为基础，开发和验证实现这些目标所需的新技术。在独立阶段，我将使用这些技术将来自体外生化实验的分子细节与这些分子水平相互作用的细胞后果的体内观察结合在一起，以解决受调控的蛋白质分解如何影响细胞周期进展等基本过程的具体问题。相关性：这个项目有可能通过蛋白质降解酶的作用来理解DNA复制和细胞周期正常进展所需的基本调控机制。由于特定的受调控的蛋白质分解对所有生物体的所有细胞周期过程都是至关重要的，这项工作将为理解当这些过程被破坏时出现的病理后果奠定基础。

DESCRIPTION (provided by applicant): 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 of the master regulator CtrA by CIpXP 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 of the 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 CIpXP 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 will use biochemical assays to identify and manipulate the substrate profile of a known proteolytic adaptor that is responsible for directed proteolysis of quality control products. Aim 2 consists of reconstitution of the regulated degradation of the master regulator CtrA and using biochemical fractionation to isolate modulators of this essential cell-cycle regulator. Finally, Aim 3 focuses on a general approach in which I will obtain degradation profiles of CIpXP through an unbiased proteomic approach utilizing inactive versions of these enzymes to trap substrates. During the mentored phase I will build on preliminary observations of CIpXP substrate processing and trapping to develop and validate the novel technologies needed to accomplish these aims. For the independent phase, I will employ these techniques to bring together the molecular details from in vitro biochemical experiments with in vivo observations of the cellular consequences of these molecular level intereactions to address the specific questions of how regulated proteolysis can impact such fundamental processes as cell-cycle progression. RELEVANCE: This project has the potential of understanding fundamental regulatory mechanism that are needed for DNA replication and proper progression of the cell-cycle through the role of protein degradation enzymes. As specific regulated proteolysis is critical for all cell-cycle processes in all organisms, this work will build foundations for understanding the pathological consequences that emerge when such processes are disrupted.