Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease.
Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive alpha,beta-unsaturated aldehyde, including 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE). These aldehydes have a documented ability to disrupt protein function following adduct formation with specific residues. Therefore, to identify 4-HNE-modified proteins in a model of ethanol-induced oxidative stress, a proteomic approach was applied to liver fractions prepared from rats fed a combination high-fat/ethanol diet. The results revealed that essential 90-kDa heat shock protein (Hsp90) was consistently modified by 4-HNE in the alcohol-treated animals. In vitro chaperoning experiments using firefly luciferase as a client protein were then performed to assess the functional effect of 4-HNE modification on purified recombinant human Hsp90, modified with concentrations of this aldehyde ranging from 23 to 450 microM. Modification of Hsp90 with 4-ONE also led to significant inhibition of the chaperone. Because 4-HNE and 4-ONE react selectively with Cys, a thiol-specific mechanism of inhibition was suggested by these data. Therefore, thiol sensitivity was confirmed following treatment of Hsp90 with the specific thiol modifier N-ethylmaleimide, which resulted in more than 99% inactivation of the chaperone by concentrations as low as 6 microM (1:1 M ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by liquid chromatography/tandem mass spectrometry peptide analysis identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that 4-HNE consistently modifies Hsp90 in a rat model of alcohol-induced oxidative stress and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.
Carbone
David L
DL
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Doorn
Jonathan A
JA
Kiebler
Zachary
Z
Ickes
Brian R
BR
Petersen
Dennis R
DR
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D004195:Disease Models, Animal; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors*; D015227:Lipid Peroxidation; D008108:Liver Diseases, Alcoholic / Q000378:metabolism*; D008297:Male; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
C403894:4-oxo-2-nonenal; D000447:Aldehydes; D018841:HSP90 Heat-Shock Proteins; C027576:4-hydroxy-2-nonenal