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

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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.