Probing the mechanism of the Hsp90 chaperone using systematic dominant negative mutational analyses

Univ Of Massachusetts Med Sch Worcester, Worcester MA

Investigators

Linked publications & trials

Abstract

? DESCRIPTION (provided by applicant): Hsp90 is a highly conserved chaperone of great interest because of the unique role it plays in modulating signal transduction. Hsp90 facilitates the maturation of many signaling proteins including a large number of kinases, steroid hormone receptors, and transcription factors to their active states. Importantly, many of the mutated kinases and transcription regulators that are key mediators of cancer depend on Hsp90 for proper folding, resulting in an increased dependence of cancer cells on Hsp90 function. For this reason, Hsp90 is an attractive and promising target for inhibitors of growth of many diverse cancer types. Despite its tremendous clinical importance, the mechanism by which Hsp90 activates its clients remains poorly understood. Investigating the mechanism of Hsp90 is challenging because it is a large, dynamic protein that undergoes dramatic conformational changes mediated by ATP and many transiently interacting co-chaperones. The goal of this research project is to use dominant negative Hsp90 mutants as tools to probe the mechanism used by the chaperone to facilitate client folding. Analysis of dominant negative proteins has historically provided valuable mechanistic information of complex proteins by providing snapshots of dynamic processes. Dominant negative Hsp90 mutants will be systematically identified by measuring the fitness effects of all Hsp90 point mutations in the presence of the wild-type protein in bulk competition experiments. The impact of the dominant negative mutations on Hsp90 structure and conformational dynamics will be assessed using a series of biophysical assays such as fluorescence resonance energy transfer and small-angle X-ray scattering. In addition, the effects of the dominant negative mutations on the ability of Hsp90 to mature model clients will be ascertained. Examination of the dominant negative Hsp90 mutants will help identify intermediates along the client folding pathway, providing novel insight into the mechanism of client activation by Hsp90.