期刊
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
卷 315, 期 1, 页码 8-15出版社
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS
DOI: 10.1124/jpet.105.088088
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资金
- NIAAA NIH HHS [R01AA09300, F31 AA014308] Funding Source: Medline
- NIEHS NIH HHS [F32 ES11937] Funding Source: Medline
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 mu M. 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 mu M (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.
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