Hsp90 Is Cleaved by Reactive Oxygen Species at a Highly Conserved N-Terminal Amino Acid Motif

Abstract

Hsp90 is an essential chaperone that is necessary for the folding, stability and activity of numerous proteins. In this study, we demonstrate that free radicals formed during oxidative stress conditions can cleave Hsp90. This cleavage occurs through a Fenton reaction which requires the presence of redox-active iron. As a result of the cleavage, we observed a disruption of the chaperoning function of Hsp90 and the degradation of its client proteins, for example, Bcr-Abl, RIP, c-Raf, NEMO and hTert. Formation of Hsp90 protein radicals on exposure to oxidative stress was confirmed by immuno-spin trapping. Using a proteomic analysis, we determined that the cleavage occurs in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90β, and between Ile-131 and Gly-132 in Hsp90α. Given the importance of Hsp90 in diverse biological functions, these findings shed new light on how oxidative stress can affect cellular homeostasis.

-Lapachone Induces NAD(P)H:Quinone Oxidoreductase-1- and Oxidative Stress-Dependent Heat Shock Protein 90 Cleavage and Inhibits Tumor Growth and Angiogenesis

“…Data were from three independent experiments. b-Lapachone Induces Hsp90 Cleavage studies, our results definitely support the statement that Hsp90 represents a new target for oxidant-based anticancer therapies (Clark et al, 2009;Beck et al, 2011aBeck et al, , 2012.…”

“…Given recent findings that highlighted the potential interest of using oxidative stress to target Hsp90 (Beck et al, 2009(Beck et al, , 2011a(Beck et al, ,b, 2012, we asked whether a NQO1-mediated futile redox cycle induced by b-lap could lead to Hsp90 inhibition. In an attempt to address this issue, we examined the effects of b-lap on Hsp90 by Western blotting assays.…”

“…1D), suggesting the minimal role of these two proteases in this process. It was reported that a pan-caspase inhibitor ((benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone, Z-VAD.FMK), a cathepsin inhibitor (ethyl-(1)-(2S,3S)-3-[(S)-methyl-1-(3-methyl-butylcarbamoyl)-butylcarbamoyl]-2-oxiranecarboxylate, E64d), a lysosomal inhibitor (NH 4 Cl), various protease inhibitors (calpeptin, pepstatin, antipain) and proteasome inhibitors were not able to modify both Hsp90 cleavage and the subsequent client protein degradation triggered by oxidation stress (Beck et al, 2009(Beck et al, , 2012. Thus, we assume that Hsp90 cleavage induced by b-lap might occur through a nonenzymatic mechanism; however, the mechanism of Hsp90 cleavage triggered by oxidative stress is still an open question.…”

“…These unique properties of b-lap collectively participated in its exciting antitumor efficacy in our preclinical mouse model in vivo. A recent report documented that oxidative stress generated by ascorbate-driven menadione redox cycling led to Hsp90 cleavage, and the cleavage occurred in a conserved motif of the N-terminal nucleotide binding site, between Ile-126 and Gly-127 in Hsp90b, and between Ile-131 and Gly-132 in Hsp90a, as determined by mass spectrometry analysis (Beck et al, 2012). Because of the similarity of the ROS release pattern between ascorbate/menadione redox cycling and b-lap's futile cycling, we assume that Hsp90 cleavage induced by b-lap may also occur in the N-terminal nucleotide binding site.…”

“…Most of the inhibitors were developed to target the ATP binding site at the N terminus of Hsp90. New approaches have emerged, including inhibition of the C-terminal nucleotide binding pocket of Hsp90, disruption of the interaction between Hsp90 and cochaperones (Li et al, 2009), antagonism of client/Hsp90 associations, interference with post-translational modifications of Hsp90, and Hsp90 deactivation by oxidative stress (Beck et al, 2009(Beck et al, , 2011a(Beck et al, , 2012. In this study, we explored the possible deactivation of Hsp90 by a redox-modulating agent as a likely therapy of human cancers.…”