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

描述（由申请方提供）：AAA+蛋白酶ClpXP的蛋白质降解在新月柄杆菌中是必需的，并且该蛋白质水解的调节对于该生物体的适当细胞周期进程至关重要。例如，在特定的时间和地点由ClpXP降解主调节因子CtrA对于以及时的方式正确启动DNA复制起着关键作用。虽然底物识别可以在蛋白酶本身的水平上发生，但额外的调节通常以衔接子的形式存在，其增强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，并以受调控的协调方式通过衔接机制特异性降解蛋白质亚群是该项目的中心目标。在目标1，1将使用生物化学测定来鉴定和操纵已知的蛋白水解衔接子的底物谱，该衔接子负责质量控制产品的定向蛋白水解。目的2包括重建的主调节器CtrA的调节降解，并使用生化分离，以分离这一重要的细胞周期调节剂的调制器。最后，目标3侧重于一个通用的方法，其中我将获得通过一个公正的蛋白质组学的方法，利用这些酶的无活性版本，以捕获底物的ClpXP的降解概况。在指导阶段，我将建立在CIpXP基板处理和捕获的初步观察，以开发和验证实现这些目标所需的新技术。对于独立的阶段，我将采用这些技术汇集在一起的分子细节，从体外生化实验与在体内观察这些分子水平的相互作用的细胞后果，以解决如何调节蛋白水解可以影响细胞周期进程等基本过程的具体问题。相关性：该项目有可能通过蛋白质降解酶的作用了解DNA复制和细胞周期正常进展所需的基本调控机制。由于特定的调节蛋白水解对所有生物体中的所有细胞周期过程都至关重要，这项工作将为理解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.