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).

SummaryA major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.

SummaryBackground & Aims-Ischemia-reperfusion (I/R) is a major mechanism of liver injury following hepatic surgery or transplantation. Despite numerous reports on the role of oxidative/nitrosative stress and mitochondrial dysfunction in hepatic I/R injury, the proteins that are oxidatively-modified during I/R damage are poorly characterized. This study was aimed at investigating the oxidatively-modified proteins underlying the mechanism for mitochondrial dysfunction in hepatic I/R injury. We also studied the effects of a superoxide dismutase mimetic/peroxynitrite scavenger metalloporphyrin MnTMPyP on oxidatively-modified proteins and their functions.

No abstract