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Peter Chien

Assistant Professor

Office

LSL N325
(413) 545-2310

Focus

Protein degradation and unfolding; protein quality control

Background and Training

PhD: University of California, San Francisco
Postdoctoral training: Massachusetts Institute of Technology

Research Summary

Protein degradation is a powerful mechanism of controlling protein function. 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 elicit can exist only as long as it is necessary. However, as proteolysis is an irreversible event, the cell must also take care to only degrade those factors as needed without disturbing the balance of other proteins. In eukaryotes, exquisite selectivity is generated through cascades of specific molecular events that together yield a polyubiquitination 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 general goals of the Chien lab are to understand how these substrates are recognized in a precise fashion and the impact of substrate degradation on the various regulatory networks of the cell.

The oligomeric AAA+ protease ClpXP (see Figure 1) 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, thus oscillating levels of CtrA regulated by ClpXP activity limit DNA replication to particular phases generating a well-defined cell-cycle. Interestingly, CtrA and ClpXP co-localize to the same region precisely when CtrA is degraded. Proper subcellular localization of ClpXP is dependent on the presence of the response regulator CpdR. Specifically, dephosphorylated CpdR recruits ClpXP to the nascent stalked cell pole and upon CpdR phosphorylation, release of ClpXP from its location results in the rapid accumulation of CtrA. Although CpdR is not essential, deletion of CpdR results in markedly slower degradation of CtrA.
 

 
 

Figure 1. The ClpX hexamer recognizes substrates via exposed degradation tags, processively unfolds them using ATP hydrolysis to power this force, and translocates them to the ClpP peptidase where they are degraded.
 

 

 

 

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 shed light on how cells respond to environmental cues and could potentially lead to development of new antibiotic therapies.

My lab focuses on addressing 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.
 

Figure 2. C. crescentus life cycle. Synchronized populations of C. crescentus progress through a well-defined cell-cycle where morphological transitions from swarmer (SW) to stalked (ST) cells reflect progression from the G1 to S phase. Asymmetric cell division results in a release of a new SW cell and the original mother ST cell. During cell-cycle ClpXP is colocalized with CtrA precisely when CtrA is degraded (shaded regions in cartoon depict CtrA levels as monitored by western blot analysis (bottom)). The recruitment of ClpXP is dependent on the dephosphorylated form of the regulator CpdR which is also localized in a similar fashion as CtrA and ClpXP.

Publications

Rood KL, Clark NE, Stoddard PR, Garman SC, Chien P. Adaptor-Dependent Degradation of a Cell-Cycle Regulator Uses a Unique Substrate Architecture. Structure. 2012 Jun 7. [Epub ahead of print]

Chowdhury, T, Chien, P, Ebrahim, S, Sauer, R.T., Baker, T.  Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species. Protein Sci. Feb;19(2):242-54 (2010). [PubMed]

Kobayashi H, De Nisco NJ, Chien P, Simmons LA, Walker GC. Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection. Mol Microbiol. 73(4):586-600 (2009). [PubMed]

Chien, P., Grant, R.A., Sauer, R.T., Baker, T.A. Structure and Substrate Specificity of an SspB Ortholog: Design Implications for AAA+ Adaptors. Structure. 15(10):1296-305 (2007). [PubMed]

Chien, P., Perchuk, B.S., Laub, M.T., Sauer, R.T., Baker, T.A. Direct and adaptor-mediated substrate recognition by an essential AAA+ protease. PNAS 104(16):6590-5 (2007). [PubMed]