Protein degradation is an essential process for all biological life. Damaged or improperly folded proteins need to be cleared from the cell before they elicit toxic effects. Regulatory proteins need to be degraded so that the response they support exists only as long as it is necessary. However, as proteolysis is an irreversible event, great care must be taken to only degrade those factors as needed without disturbing the balance of other proteins. In eukaryotes, exquisite selectivity is generated through cascading molecular events that together yield a ubiquitination signal which targets a substrate for degradation. As no such system exists in bacteria, the highly specific nature of protein degradation must be accomplished at the level of direct recognition of the substrate or by utilizing auxiliary factors to improve specificity.
The oligomeric AAA+ protease ClpXP is a well- characterized example of an enzyme that exerts post- translational control over a number of pathways. In C. crescentus, the essential response regulator CtrA prevents initiation of DNA replication. Oscillating levels of CtrA are driven in large part through regulated degradation by ClpXP and constrain DNA replication to particular times, thus generating a well-defined cell cycle. Interestingly, CtrA levels are specifically degraded at the swarmer to stalk transition, while ClpX and ClpP levels remain constant, suggesting other factors must serve to regulate degradation during cell-cycle progression. The rate and timing of CtrA degradation is dependent on the response regulator CpdR, which appears to be responsible for localization of ClpXP as well. Specifically, dephosphorylated CpdR recruits ClpXP to the nascent stalked cell pole and upon CpdR phosphorylation, release of ClpXP is coincident with the rapid accumulation of CtrA.
A number of critical unresolved questions emerge from these observations. How does CtrA degradation occur specifically at the G1-S transition? How does dephosphorylated CpdR activate ClpXP and what is the molecular nature of this interaction? Interestingly, although ClpXP is essential, CtrA degradation is not needed for viability. If ClpXP is necessary because of its proteolytic activity, what substrates must be degraded? Because targeted proteolysis is critical for virulence and environmental sensing pathways in many bacteria, a deeper understanding of its regulation will reveal how cells respond to environmental cues and could potentially lead to development of new antibiotic therapies.
We approach these questions using many approaches including biochemistry, structural biology and cell biology. Our ultimate goal is to identify factors needed for the precisely timed degradation of key substrates and to biochemically reconstitute regulated proteolysis using purified components. By understanding how mechanisms specific to our system enforce proper protein lifetimes, we hope to understand how regulated proteolysis is generally controlled. Furthermore, as ClpX is a member of a larger class of other molecular machines whose primary role is to aid in the proper folding of proteins, lessons learned from our studies will also shed light on a broader understanding of energy driven protein folding and unfolding.
Here are some examples of the specific questions that we are trying to address:
What is the essential role of ClpX?
ClpX is essential in C. crescentus, but not in E. coli. However, in vitro experiments suggest both species of ClpX are similarly active for at least some test substrates. What makes ClpX essential in some bacteria and not others? We are exploring this question using genetic complementation studies and more detailed biochemical characterization. More generally this question begins to address how a conserved protein, involved as a hub for many cellular processes, can vary in its necessity for the cell.
How do substrates get degraded at a specific time?
Proteolytic substrates must be recognized and degraded in a timely fashion. In C. crescentus the master regulator CtrA is degraded at a specific time during cell-cycle progression. In vitro experiments suggest that CtrA can be directly recognized by ClpXP with kinetics of degradation rapid enough to satisfy the observed in vivo half-life. Therefore, CtrA degradation is likely limited by the presence of an inhibitor factor as yet to be determined. The goal of this project is to identify this factor through biochemical fractionation and determine its mechanism of action.
How does CpdR regulate ClpX function?
The response regulator CpdR is needed for rapid CtrA degradation in vivo. Both biochemical and in vivo assays suggest CpdR interacts with ClpX, though the mechanism of this interaction is lacking. How does CpdR bind ClpX? How does binding of CpdR affect ClpX activity and function in vitro and in vivo?
What are the cell-cycle regulated ClpXP substrates?
There are only a handful of known ClpXP substrates in Caulobacter. Interestingly, mutant forms of these proteins that are incapable of being degraded do not result in cell death. Therefore, if ClpXP is essential because it must degrade some particular substrate, that substrate has not yet been identified. The aim of this work is to discover novel substrates of ClpXP using an unbiased proteomic survey.