Mechanistic Studies of the Hsp70 Chaperone System

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DESCRIPTION (provided by applicant): The Hsp70 protein family is a diverse and ubiquitous group of molecular chaperones. There are multiple representatives of the Hsp70 family in all living organisms. Their cellular roles include assistance in protein folding, protein targeting and translocation, the assembly and disassembly of macromolecular complexes, protein transport to appropriate cellular regions, and the delivery of proteins to a proteolytic system for their disposal. The roles of Hsp70s in the prevention of protein aggregation, the refolding of denatured proteins, and the disposal of irreversibly damaged proteins are cytoprotective. Within the nervous system, Hsp70s assist in the prevention of neurodegeneration which results from amyloid aggregates, as well as play a fundamental role in synaptic vesicle recycling. Overexpression of Hsp70s protects cells against the damaging effects of a variety of stressors (i.e. inflammation, ischemia, and free radical oxygen species). Consequently, there is considerable interest in elucidating their mechanism. The basic features of the Hsp70 cycle has been known for some time. ATP binding to the ATPase domain of the Hsp70 induces an allosteric change in the protein substrate binding domain, resulting in the release of substrate, thus setting the stage for the binding of a new substrate molecule. ATP hydrolysis, and the subsequent release of Pi, results in the formation of a stable ADP*Hsp70*substrate complex. The energy released from ATP hydrolysis can be harnessed to drive protein translocation and macromolecular complex remodeling reactions. The multiple steps involved in the Hsp70 cycle are regulated by cochaperones that increase the rate of ATP hydrolysis and deliver various substrates to the Hsp70, or increase the rate ofADP/ATP exchange. However, the precise mechanisms of these processes, chiefly the allosteric mechanism, remain elusive. To alleviate these deficiencies in our understanding, I will characterize the structures of an Hsp70 in distinct, allosteric states. Both mutational and biochemical experiments will be used to investigate the observed intermolecular interactions and mechanistic hypotheses inferred from inspection of these structures.