Inactivation of oxidized andS-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 ␤-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).

Oxidative Inactivation of Key Mitochondrial Proteins Leads to Dysfunction and Injury in Hepatic Ischemia Reperfusion

“…4). 23 Consistent with the results shown in Figure 3, only a few biotin-NM labeled oxidized and Snitrosylated proteins were detected in the sham control mouse livers ( Fig. 4A and 4D, respectively).…”

“…22,23,29 Relative purities of the mitochondrial fractions were determined by immunoblot analysis using a specific antibody against mitochondrial α-ATP synthase or cytosolic peroxiredoxin-II. Little contamination with cytosolic proteins in our mitochondrial fractions and vice versa was observed ( Supplementary Fig.…”

“…Other materials and methods not described here were performed as previously described. 23,29,30,33,34 …”

“…To test this hypothesis, we employed a method that uses biotin-N-maleimide (biotin-NM) to detect oxidatively-modified proteins. 23,29,30 In this report, we examined the mechanism of inactivation of oxidatively-modified mitochondrial proteins in a murine model of hepatic I/R injury. Furthermore, by using the superoxide dismutase (SOD) mimetic and peroxynitrite decomposition catalyst (termed "peroxynitrite scavenger") MnTMPyP, 20,31, 32 we demonstrated that elevated oxidative/nitrosative stress plays a critical role in I/Rmediated mitochondrial dysfunction and hepatic damage.…”

Mitochondrial dysfunction and oxidative stress in the pathogenesis of alcohol- and obesity-induced fatty liver diseases

“…These alterations translate into profound modifications to the mitochondrial proteome that encompass not only losses in the 13 mitochondrial encoded polypeptides, but also decreases in numerous nuclear encoded proteins that make up the oxidative phosphorylation complexes [46]. Proteomic analyses have also shown alcohol-dependent changes in mitochondrial matrix enzymes both at the level of abundance and post-translational modifications [46,49,50].…”