Understanding the role of the co-chaperone GCUNC45 in the Hsp90 chaperoning pathway.

The Hsp90 chaperoning pathway plays an important role in pathophysiology of the cardiovascular system. The proper functioning of transcriptional factors such as the steroid receptors is strictly dependent upon their adequate chaperoning through this pathway and molecular chaperones also protect the heart against injury during stress. The progesterone receptor (PR) is thought to initially bind Hsp40 and Hsp70 which subsequently recruits the co-chaperone Hop and Hsp90. Conformational changes in Hsp90, that are dictated by ATP hydrolysis and binding of the co-chaperone p23, convert the PR to a state that is able to bind hormone. Using human cell lines, we have discovered that the novel Hsp90 co-chaperone GCUNC45 is a positive factor in promoting PR function. GCUNC45 interacts directly with Hsp90 and PR and affects PR chaperoning in vitro. In this proposal, we plan to dissect the novel mechanism of action of GCUNC45 and its biological roles in PR chaperoning and trafficking. We will use molecular, biochemical and cell biological methods. Two specific aims are proposed: Aim 1. Explore the biological significance of the selectivity of GCUNC45 for Hsp90 beta and describe key features of this interaction. GCUNC45 is unique in that it is the only known Hsp90 cofactor that distinguishes between the two Hsp90 isoforms alpha and beta. Also, unlike other known TPR co-chaperones, GCUNC45 regulates Hsp90 activities through a unique conformational site that overlaps with the ATP binding site near the N-terminus. This makes this interaction highly sensitive to nucleotides and to Hsp90 inhibitors. We will define the importance of this interaction to Hsp90 functions and dissect the biochemical mechanism and the in vivo relevance of GCUNC45 selectivity for Hsp90 beta. Aim 2. We will characterize and investigate the biological relevance of the novel interaction between GCUNC45 and PR. It appears to involve multiple sites on GCUNC45. We will define more precisely the binding sites, their inter-dependence and the potential role of GCUNC45 in PR transcriptional activity and nuclear localization. These studies will provide valuable information needed to better understand the response of the cardiovascular system to steroids and stress. They will clarify the mechanisms for chaperoning steroid receptors and the functional differences between Hsp90 isoforms, and improve our understanding of the pharmacological actions of Hsp90 inhibitors and ultimately their design. (AHA Program: Scientist Development Grant)