The long term goals of the proposed research are to understand sHsp function in vivo and to define further the mechanism of sHsp chaperone activity. The small Hsp (sHsp)/alpha-crystallin family of proteins, which are conserved in both eukaryotes and prokaryotes, are produced at significant levels in cells experiencing heat stress, indicating they have an ancient and conserved role in survival of high temperature. Research indicates that sHsps act as molecular chaperones to prevent irreversible protein aggregation by binding to denaturing proteins and presenting these substrates to other chaperones for ATP-dependent refolding. Members of the sHsp family may also play important roles in the absence of heat stress and in cells stressed by disease. They are found in different cell and tissue types in response to stage of development, differentiation, growth conditions and oncogenic status. Furthermore, sHsps/alpha- crystallins are expressed in cells associated with neurodegenerative diseases, and alpha-crystallin mutations are linked to an inherited desmin myopathy and to a cataract condition, all of which involve protein misfolding. Assays for sHsp chaperone activity have been developed in this laboratory, and detailed structural data on sHsps, using NMR, X-ray and other physical techniques, are being acquired. In addition, a genetic system to analyze sHsp function has been developed; the cyanobacterium Synechocystis, for which the complete genome sequence is known, exhibits a conditional thermotolerance defect in an sHsp deletion mutant. Thus, for the first time in sHsp research, the tools are available to combine genetic, biochemical and structural analysis to study the function of an sHsp in detail. The current research has four specific aims. 1) To define features of the sHsps that are required for activity in vivo by screening for non-functional mutants of Synechocystis Hsp16.6, and to use these mutants to test the validity of the chaperone model for sHsp function. 2) To identify proteins that interact with Synechocystis Hsp16.6 either as substrates or partners in sHsp function. 3) To investigate further the structure of the sHsps as a basis for understanding their molecular mechanism of action. 4) To define substrate binding interactions with the sHsps and to extend the model of sHsp chaperone activity. The proposed studies of these ubiquitous stress proteins will be key to understanding their role in normal and stressed cells.
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