Received for publication April 18, 2005.
Revised June 8, 2005.
Accepted for publication June 8, 2005.

Modification of Hsp90 by 4-Hydroxynonenal in a Rat Model of Chronic Alcoholic Liver Disease

Abstract

Lipid peroxidation during oxidative stress leads to increased concentrations of thiol-reactive , -unsaturated aldehydes 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 the essential heat shock protein 90 (Hsp90) was consistently adducted 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 4ONE 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 (NEM), which resulted in over 99% inactivation of the chaperone by concentrations as low as 6 µM (1:1 molar ratio). Finally, tryptic digest of 4-HNE-modified Hsp90 followed by LC-MS/MS peptide analysis identified Cys 572 as a site for 4-HNE modification. The results presented here thus establish that Hsp90 is consistently modified by 4-HNE in a rat model of alcohol-induced oxidative stress, and that the chaperoning activity of this protein is subject to dysregulation through thiol modification.

Key words: 4-Hydroxynonenal, 4-Oxononenal, Alcoholic Liver Disease, Hsp90, Lipid Peroxidation, Oxidative Stress

This article has been cited by other articles:

				P. A. Grimsrud, H. Xie, T. J. Griffin, and D. A. Bernlohr Oxidative Stress and Covalent Modification of Protein with Bioactive Aldehydes J. Biol. Chem., August 8, 2008; 283(32): 21837 - 21841. [Abstract] [Full Text] [PDF]

				A. Trott, J. D. West, L. Klaic, S. D. Westerheide, R. B. Silverman, R. I. Morimoto, and K. A. Morano Activation of Heat Shock and Antioxidant Responses by the Natural Product Celastrol: Transcriptional Signatures of a Thiol-targeted Molecule Mol. Biol. Cell, March 1, 2008; 19(3): 1104 - 1112. [Abstract] [Full Text] [PDF]

				A. M. Winger, N. L. Taylor, J. L. Heazlewood, D. A. Day, and A. H. Millar The Cytotoxic Lipid Peroxidation Product 4-Hydroxy-2-nonenal Covalently Modifies a Selective Range of Proteins Linked to Respiratory Function in Plant Mitochondria J. Biol. Chem., December 28, 2007; 282(52): 37436 - 37447. [Abstract] [Full Text] [PDF]

				A. T. Jacobs and L. J. Marnett Heat Shock Factor 1 Attenuates 4-Hydroxynonenal-mediated Apoptosis: CRITICAL ROLE FOR HEAT SHOCK PROTEIN 70 INDUCTION AND STABILIZATION OF Bcl-XL J. Biol. Chem., November 16, 2007; 282(46): 33412 - 33420. [Abstract] [Full Text] [PDF]

				J. Whitsett, M. J. Picklo Sr, and J. Vasquez-Vivar 4-Hydroxy-2-Nonenal Increases Superoxide Anion Radical in Endothelial Cells via Stimulated GTP Cyclohydrolase Proteasomal Degradation Arterioscler. Thromb. Vasc. Biol., November 1, 2007; 27(11): 2340 - 2347. [Abstract] [Full Text] [PDF]

				P. A. Grimsrud, M. J. Picklo Sr., T. J. Griffin, and D. A. Bernlohr Carbonylation of Adipose Proteins in Obesity and Insulin Resistance: Identification of Adipocyte Fatty Acid-binding Protein as a Cellular Target of 4-Hydroxynonenal Mol. Cell. Proteomics, April 1, 2007; 6(4): 624 - 637. [Abstract] [Full Text] [PDF]

				B. P. Sampey, D. L. Carbone, J. A. Doorn, D. A. Drechsel, and D. R. Petersen 4-Hydroxy-2-nonenal Adduction of Extracellular Signal-Regulated Kinase (Erk) and the Inhibition of Hepatocyte Erk-Est-Like Protein-1-Activating Protein-1 Signal Transduction Mol. Pharmacol., March 1, 2007; 71(3): 871 - 883. [Abstract] [Full Text] [PDF]

				B. P. Sampey, B. J. Stewart, and D. R. Petersen Ethanol-induced Modulation of Hepatocellular Extracellular Signal-regulated Kinase-1/2 Activity via 4-Hydroxynonenal J. Biol. Chem., January 19, 2007; 282(3): 1925 - 1937. [Abstract] [Full Text] [PDF]

				J. Yan and B. F. Hales Depletion of Glutathione Induces 4-Hydroxynonenal Protein Adducts and Hydroxyurea Teratogenicity in the Organogenesis Stage Mouse Embryo J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 613 - 621. [Abstract] [Full Text] [PDF]