title: Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats.
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 beta-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.
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD, USA.
authors.forename: Kwan-Hoon
authors.initials: KH
authors.lastname: Moon
authors.forename: Brian L
authors.initials: BL
authors.lastname: Hood
authors.forename: Bong-Jo
authors.initials: BJ
authors.lastname: Kim
authors.forename: James P
authors.initials: JP
authors.lastname: Hardwick
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: Byoung J
authors.initials: BJ
authors.lastname: Song
mesh_terms: D000818:Animals; D002492:Central Nervous System Depressants / Q000009:adverse effects*; D003545:Cysteine / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D000431:Ethanol / Q000009:adverse effects*; D005235:Fatty Liver, Alcoholic / Q000209:etiology / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D008301:Maleimides / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000187:drug effects / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D015538:Nitrosation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley; D020539:Sequence Analysis, Protein
chemical_list: D002492:Central Nervous System Depressants; D007527:Isoenzymes; D008301:Maleimides; D024101:Mitochondrial Proteins; D009573:Nitrites; C043592:maleimide; D000431:Ethanol; D019392:Cytochrome P-450 CYP2E1; D019001:Nitric Oxide Synthase; D003545:Cysteine