Exploring Hsp90 as a possible pseudo-substrate receptor for E3 ligases.

Abstract

Hsp90 is a chaperone protein responsible for aiding in the proper folding of its client proteins, particularly in response to cellular stresses such as heat shock. It has been demonstrated in human cell lines that inhibition of Hsp90's ATPase activity, which is required for its chaperone activity, leads to proteolytic degradation of c-Raf, which is an oncogenic kinase in the MAPK/ERK pathway and a known client protein of Hsp90. In eukaryotes, the primary mechanism of protein degradation is through the ubiquitin-proteasome system, in which E3 ligases post-translationally modify substrate proteins with ubiquitin, which marks the protein for degradation by the proteasome. It has been shown that siRNA silencing of the E3 ligases HECTD3 and Cullin5 in Hsp90-inhibited cells leads to recovery of c-Raf, suggesting that HECTD3 and Cullin5 are responsible for initiating the degradation of c-Raf. Furthermore, it has been shown that increasing concentrations of the Hsp90 inhibitor AUY922 causes HECTD3 to pull down both c-Raf and Hsp90, implying that HECTD3, and perhaps also Cullin5, directly interact with Hsp90 in order to immediately ubiquitinate misfolded Hsp90 clients and mark them for proteasomal degradation. In order to investigate the validity of this model, we are expressing these proteins in vitro and will report which proteins directly interact with which, with the goal of reconstituting the full E3 ligase complex responsible for c-Raf ubiquitination.