Inactivation of oxidized and S-nitrosylated mitochondrial proteins in alcoholic fatty liver of rats
oxidative stress; nitric-oxide; Gastroenterology & Hepatology; activated receptor-alpha; mouse-liver; Aldehyde dehydrogenase; cytochrome-p450 cyp2e1; dependent hepatotoxicity; ethanol-consumption; hepatic mitochondria; nitrosative stress
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 CYP21. 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) under bar our results help to explain the underlying mechanism for mitochondrial dysfunction and increased susceptibility to alcohol-mediated liver damage.
Moon K H; Hood B L; Kim B J; Hardwick J P; Conrads T P; Veenstra T D; Song B Y J
Hepatology
2006
2006-11
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1002/hep.21372" target="_blank" rel="noreferrer noopener">10.1002/hep.21372</a>
Increased Oxidation And Degradation Of Cytosolic Proteins In Alcohol-exposed Mouse Liver And Hepatoma Cells
alcoholism; apoptosis; Biochemistry & Molecular Biology; CYP2E1; cytochrome p-4502e1; Degradation; disease; ethanol; in-vitro; induced; injury; nitric-oxide; oxidative stress; oxidized proteins; peroxiredoxin; protein; protein oxidation; rat-liver; stress
We recently developed a sensitive method using biotin-N-maleimide (biotin-NM) as a probe to positively identify oxidized mitochondrial proteins. In this study, biotin-NM was used to identify oxidized cytosolic proteins in alcohol-fed mouse livers. Alcohol treatment for 6 wk elevated the levels of CYP2E1 and nitrotyrosine, a marker of oxidative stress. Markedly increased levels of oxidized proteins were detected in alcohol-fed mouse livers compared to pair-fed controls. The biotin-NM-labeled oxidized proteins from alcohol-exposed mouse livers were subsequently purified with streptavidin-agarose and resolved on 2-DE. More than 90 silver-stained protein spots that displayed differential intensities on 2-D gels were identified by MS. Peptide sequence analysis revealed that many enzymes or proteins involved in stress response, chaperone activity, intermediary metabolism, and antioxidant defense systems such as peroxiredoxin were oxidized after alcohol treatment. Smaller fragments of many proteins were repeatedly detected only in alcohol-fed mice, indicating that many oxidized proteins after alcohol exposure were degraded. Immunoblot results showed that the level of oxidized peroxiredoxin (inactivated) was markedly increased in the alcohol-exposed mouse livers and ethanol-sensitive hepatoma cells compared to the corresponding controls. Our results may explain the underlying mechanism for cellular dysfunction and increased susceptibility to other toxic agents following alcohol-mediated oxidative stress.
Kim B J; Hood B L; Aragon R A; Hardwick Jr; Conrads T R; Veenstra T D; Song B J
Proteomics
2006
2006-02
Journal Article or Conference Abstract Publication
<a href="http://doi.org/10.1002/pmic.200500447" target="_blank" rel="noreferrer noopener">10.1002/pmic.200500447</a>