Modification of the Mitochondrial Proteome in Response to the Stress of Ethanol-dependent Hepatotoxicity

Abstract

Mitochondria are particularly susceptible to increased formation of reactive oxygen and nitrogen species in the cell that can occur in response to pathological and xenobiotic stimuli. Proteomics can give insights into both mechanism of pathology and adaptation to stress. Herein we report the use of proteomics to evaluate alterations in the levels of mitochondrial proteins following chronic ethanol exposure in an animal model. Forty-three proteins showed differential expression, 13 increased and 30 decreased, as a consequence of chronic ethanol. Of these proteins, 25 were not previously known to be affected by chronic ethanol emphasizing the power of proteomic approaches in revealing global responses to stress. Both nuclear and mitochondrially encoded gene products of the oxidative phosphorylation complexes in mitochondria from ethanol-fed rats were decreased suggesting an assembly defect in this integrated metabolic pathway. Moreover mtDNA damage was increased by ethanol demonstrating that the effects of ethanol consumption extend beyond the proteome to encompass mtDNA. Taken together, we have demonstrated that chronic ethanol consumption extends to a modification of the mitochondrial proteome far broader than realized previously. These data also suggest that the response of mitochondria to stress may not involve non-discriminate changes in the proteome but is restricted to those metabolic pathways that have a direct role in a specific pathology.Chronic ethanol consumption causes liver damage by a complex process thought to involve oxidative and nitrosative stress, hypoxia, up-regulation of proinflammatory cytokines, and defects in energy metabolism (1-3). As both a source for the formation and target of modifications mediated by reactive oxygen and nitrogen species, the mitochondrion is recognized as a site critical in the cellular stress response induced by chronic ethanol exposure. Increased mitochondrial production of reactive oxygen species (4 -6), oxidation of mitochondrial proteins (7-10), depressed oxidative phosphorylation activity (11-13), and disrupted fatty acid metabolism (14 -16) occur following consumption of alcohol, indicating that ethanol induces significant changes in mitochondria physiology. These responses are also accompanied by a profound increase in the sensitivity of the respiratory chain to inhibition by NO, which we propose plays a key role in contributing to the hypoxia associated with ethanol-dependent hepatotoxicity (17). While mechanisms responsible for ethanol-induced mitochondrial dysfunction have been investigated, the impact of chronic ethanol consumption on the overall content of mitochondrial proteins, the "mitochondrial proteome," has not been studied. Earlier studies by Cunningham and colleagues (18,19) have demonstrated that ethanol consumption decreases the synthesis of the 13 mitochondrially encoded proteins that comprise respiratory complexes I, III, and IV and the ATP synthase. It is proposed that the inhibition of mitochondrial protein synthesis following chronic etha...

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

“…19 Furthermore, increased degradation of oxidatively modified proteins may contribute to the reduced levels of several hepatic mitochondrial proteins such as ATP synthase and 3-ketoacyl-CoA thiolase in alcohol-exposed rats, as previously reported. 27,28 Our 2-D PAGE and MS analyses also revealed that many protein spots, though identified as being the same protein, exhibited different pI values that possessed the same or similar apparent masses. For example, several proteins with apparently different pI values were each identified as ALDH2 (spots 3,6-8,11,13,53,54), ATP synthase (spots 6-13), 3-ketoacyl-CoA thiolase (spots 15-24), electron-transfer flavoprotein ␤ subunit (spots 26-30 and 32-34), and MDH (spots 55-67).…”

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

“…Moreover, this decrease in ADP-stimulated respiration is linked to decreased electron transport in all segments of the respiratory chain as chronic alcohol consumption decreases the activities of all the respiratory complexes, except complex II [41,42]. Several laboratories have presented strong evidence that inhibition of mitochondrial protein synthesis [43] linked to mtDNA damage [44][45][46] and ribosomal defects [47,48] contribute, in part, to decreased functioning of the oxidative phosphorylation system following chronic alcohol consumption. 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].…”

“…Several laboratories have presented strong evidence that inhibition of mitochondrial protein synthesis [43] linked to mtDNA damage [44][45][46] and ribosomal defects [47,48] contribute, in part, to decreased functioning of the oxidative phosphorylation system following chronic alcohol consumption. 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].…”

“…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].…”

“…Studies have suggested that Complex II-linked respiration may lead to increased production of O 2 • − within Complex I as a consequence of reverse electron transfer [65,66]. It is proposed that reverse electron transfer may contribute to increased mitochondrial production of O 2 • − due to chronic alcohol-mediated defects within Complex I [43,46]. This would have the effect to compromise the functioning of the oxidative phosphorylation system as a result of the loss of mitochondrial gene products that make up segments of the respiratory chain; subsequently leading to more ROS production.…”