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.
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 degradative 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.
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