Seventy kD heat shock protein (hsp7O) family members are essential genes in all species. They exert their catalytic function by coupling the binding and release of substrate proteins to the binding and hydrolysis of ATP. They play a vital role in the folding of new proteins into their biologically active forms and in transporting them into organelles within the cell. The cellular response to hyperthermia and oxidative stress is mediated by the hsp7O family, which recognize unfolded polypeptide chains and participate in their refolding. We are investigating the structural and functional aspects of the interaction between hsp7O and its substrate proteins. (i) The relationship between molecular structure and function will be investigated by constructing deletion mutants of a murine hsp7O.1 gene and analyzing their biochemical function using cell lysates and reconstituted in vitro systems. Hsp7O is a multi-domain protein and our objective is to map the locations and interactions between the domains responsible for ATP binding, peptide binding, calmodulin binding, nuclear localization and other functions. (ii) Kinetic studies of 2 members of the hsp7O family - the heat induced hsp7O.1 and the constitutive hsc7O - will be carried out. Purified recombinant hsp7O proteins will be used to measure the rates of ATP hydrolysis, ATP/ADP exchange, the binding constants for ATP and ADP and how peptide binding, calmodulin and other important domains alter these parameters. (iii) Association of substrate proteins with hsp7O may require recognition motifs in the sequence of substrate proteins that become accessible only during cell metabolism and stress. We will study the sequence and secondary structure dependence of hsp7O recognition with a series of synthetic peptides of defined sequence and with proteins that associate with hsp7O, including the p53 tumor antigen. (iv) To distinguish the relative contributions of different hsp7O family members during, for instance, thermotolerance, expression of specific hsp7O species will be antagonized with antisense RNA. Understanding of the molecular mechanisms underlying hsp7O function has clinical implications in understanding the nature of treatments such as hyperthermia and responses to it such as thermotolerance, and important ramifications for tracing essential functions in cells, such as the intracellular pathways of protein folding.
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