Inactivation of oxidized andS-nitrosylated mitochondrial... : Hepatology (original) (raw)

Liver Biology and Pathobiology: Pathobiology

Inactivation of oxidized and S -nitrosylated mitochondrial proteins in alcoholic fatty liver of rats

Moon, Kwan-Hoon1; Hood, Brian L.2; Kim, Bong-Jo1; Hardwick, James P.3; Conrads, Thomas P.2; Veenstra, Timothy D.2; Song, Byoung J.1,*

1_Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD_

2_Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., Frederick, MD_

3_Department of Microbiology, Immunology, and Biochemistry, Northeastern Ohio University College of Medicine, Rootstown, OH_

*Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, 9000 Rockville Pike, Bethesda, MD 20892-9410

Email:[email protected]

Received 31 March 2006; Accepted 7 August 2006

Published online in Wiley InterScience (www.interscience.wiley.com).

Grant sponsor: Intramural Research Program of National Institute on Alcohol Abuse and Alcoholism; Grant sponsor: National Cancer Institute, National Institutes of Health; Grant Number: NO1-CO-12400.

Potential conflict of interest: Nothing to report.

Kwan-Hoon Moon and Brian L. Hood equally contributed to this work.

fax: 301-594-3113

Abstract

Increased oxidative/nitrosative stress is a major contributing factor to alcohol-mediated mitochondrial dysfunction. However, which mitochondrial proteins are oxidatively modified under alcohol-induced oxidative/nitrosative stress is poorly understood. The aim of this study was to systematically investigate oxidized and/or S -nitrosylated mitochondrial proteins and to use a biotin-N -maleimide probe to evaluate their inactivation in alcoholic fatty livers of rats. Binge or chronic alcohol exposure significantly elevated nitric oxide, inducible nitric oxide synthase, and ethanol-inducible CYP2E1. The biotin-N -maleimide-labeled oxidized and/or S -nitrosylated mitochondrial proteins from pair-fed controls or alcohol-fed rat livers were subsequently purified with streptavidin-agarose. The overall patterns of oxidized and/or S -nitrosylated proteins resolved by 2-dimensional polyacrylamide gel electrophoresis were very similar in the chronic and binge alcohol treatment groups. Seventy-nine proteins that displayed differential spot intensities from those of control rats were identified by mass spectrometry. These include mitochondrial aldehyde dehydrogenase 2 (ALDH2), ATP synthase, acyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and many proteins involved in chaperone activity, mitochondrial electron transfer, and ion transport. The activity of 3-ketoacyl-CoA thiolase involved in mitochondrial β-oxidation of fatty acids was significantly inhibited in alcohol-exposed rat livers, consistent with hepatic fat accumulation, as determined by biochemical and histological analyses. Measurement of activity and immunoblot results showed that ALDH2 and ATP synthase were also inhibited through oxidative modification of their cysteine or tyrosine residues in alcoholic fatty livers of rats. In conclusion , our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).