All organisms experience environmental stress that can lead to misfolded proteins. The protein quality-control network helps to protect cells from the effects of damaged proteins. This network includes aggregation-prevention proteins, dissaggregases, protein-refolding chaperons, and proteases. Many of these proteins are upregulated when cells are exposed to increased temperatures, and are thus called heat-shock proteins (HSPs). The ubiquitous small HSPs help maintain damaged and/or aggregation-prone proteins in a state that allows them to interact with refolding chaperones and avoid forming large insoluble aggregates. Recent data from the Baker lab has also shown that that E. coli small HSPs (Ibps) are degraded by the proteases Lon and ClpAP. This proposal explores the functions of the E. coli sHSPs and the purpose and mechanism of their newly discovered degradation.
The first aim describes how to elucidate proteins that are robust clients of sHSP in E. coli. I will use a combination of coIPs, westerns, affinity purification, 2D gel analysis and MS/MS to achieve this goal. I will identify proteins that show an increase in association with Ibps in strains lacking one or both of the proteases that degrade these proteins. With a large set of Ibp-clients we maybe able to identify common elements that are used for molecular recognition. This analysis will also increase our understanding of how proteases interact with the other elements of the protein quality-control network. In the second aim Ibp-client complexes will be used to dissect the interplay between sHSPs, damaged proteins, refolding chaperons and proteases. The goal is to determine the fate of Ibp-bound client proteins during Ibp degradation. I will determine if the clients are refolded, or degraded using radioactively labeled clients and Ibps in vitro. Complementary in vivo experiments will be done as well. This work will help define the role of sHSPs and sHSP turnover in the larger protein-quality control network. Finally, how sHSPs are recognized by proteases is unknown. Recent results suggest that Lon recognizes the conserved 1-crystallin domain of sHSPs. Using biophysical techniques and site-directed mutagenesis I will systematically dissect the molecular determinants responsible for recognition of the sHSP by proteases. Understanding if the highly conserved folded 1-crystallin domain is recognized by proteases or if a primary structural motif is recognized will help us understand the specificity of proteases for the sHSP and may highlight an unusual mode of substrate-protease interaction. Understanding the basic principles of Ibp-client interactions as well as Ibp-protease recognition may lead to new insights into the mechanisms that lead to the diseases associated with protein aggregation and ultimately, cellular aging and death.
Formation of aggregates from environmental stresses such as oxidation and high temperature can lead decreased viability of cells and disease in humans. Understanding how biological systems prevent massive protein aggregation and how damaged proteins are recycled may lead to new treatments of disease caused by protein aggregates, or provide new targets for antibiotics, so that bacteria cannot survive mild stresses, such as fever.
|Vieux, Ellen F; Wohlever, Matthew L; Chen, James Z et al. (2013) Distinct quaternary structures of the AAA+ Lon protease control substrate degradation. Proc Natl Acad Sci U S A 110:E2002-8|