Abstract
Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive α,β-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 μM. 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 μM (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.
Footnotes
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This work was supported by National Institutes of Health Grants NIAAA R01AA09300, NIEHS F32 ES11937 (J.A.D.), and NIAAA F31 AA014308 (D.L.C.). This work was presented in part at the 44th annual meeting of the Society of Toxicology, March 6–10, New Orleans, LA.
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doi:10.1124/jpet.105.088088.
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ABBREVIATIONS: Hsp, heat shock protein; ALD, alcoholic liver disease; 4-HNE, 4-hydroxy-2-nonenal; 4-ONE, 4-oxo-2-nonenal; RRL, rabbit reticulocyte lysate; ALT, alanine aminotransferase; NEM, N-ethylmaleimide; LC-MS/MS, liquid chromatography/tandem mass spectrometry; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
- Received April 15, 2005.
- Accepted June 8, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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