Isolation of an inhibitor of hsp70 activity

Hsp70 and a related protein called HscTO help the cell survive stresses by bind ing to partially denatured proteins and assisting to refold these proteins into more stable native structures. The objective of these studies was to characterize a novel cardiac Hsp70 interacting protein cDNA (HspBPl) recently isolated using the yeast two-hybrid system. The derived amino sequence is unique and therfore represents a potentially new regulator of Hsp70. Northern blots of RNA from human tissues indicated that HspBPl mRNA has a size of approximately 1.7 kb and is present in all tissues analyzed but was most abundant in heart and skeletal muscle. This cDNA was expressed in bacteria as a fusion protein containing a 6X His tag and yielded a protein of approximately 42 kDa in size. The ATPase domain of Hsp70 (amino acids 1-351) demonstrated binding to His-tagged HspBPl using a Ni²⁺ affinity resin. Further experiments demonstrated binding to Hsp70 and HscTO in a total heart extract. The binding of HspBPl to the ATPase domain of Hsp70 prompted us to examine the affect on Hsp70 ATPase activity. HspBPl (8μM) inhibited approximately 90% of the Hsp40 activated Hsp70 ATPase. The Hsp40 activated ATPase activity is essential for the renaturation activity of Hsp70, therefore, the effect of HspBPl on renaturation of lucifrrase in reticulocyte lysate was examined. HspBPl in hibited renaturation with half-maximal inhibition at 2μM. These preliminary data indicate that we have identified a novel Hsp70 interacting protein that in hibits Hsp70 chaperone activity. Supported by the American Heart Association, Arizona Affiliate.