Abstract
Chronic ethanol consumption is a significant source of morbidity and mortality worldwide and contributes to approximately 6% of global deaths. Understanding mechanisms of hepatic mitochondrial dysfunction resulting from ethanol metabolism remains a key area of interest. Our findings demonstrate that liver physiology is altered by ethanol-induced mitochondrial protein hyperacetylation. Since oxidative stress is a critical marker of early-stage ethanol toxicity in the liver, scrutiny is focused on the impact of ethanol toxicity on the acetylation status of mitochondrial antioxidant proteins and overall antioxidant status. We used a mouse model that employs a Lieber-DeCarli liquid diet to examine the impact of chronic ethanol consumption in a high-fat Western diet. Towards the goal of elucidating mechanisms of alcohol toxicity, we integrated a multi-omics approach utilizing techniques such as 1D- and 2D-SDS-PAGE followed by Western blot, immunohistochemistry (IHC), enzyme activity assays, quantitative acetylomics via HPLC-MS/MS, and computational modeling. These proteomic techniques were employed to assess changes in the acetylation status of individual lysine residues on mitochondrial antioxidant proteins from control and ethanol-fed mice. The results of our studies indicate that, as a result of chronic ethanol consumption, the global acetylation of SOD2 lysine residues was increased in liver by 270% and 400%, as quantified by Western blot and LC-MS/MS, respectively. Western blot analysis also showed an approximate 400% increase in SOD2 K68 acetylation, which is known to inhibit enzyme activity. IHC analysis revealed that ethanol-induced acetylation of SOD2-K68 is significantly increased in the zone 3 in liver, which is generally known to be hypoxic. Our findings also correlate with a decreased ratio of GSH/GSSG as a consequence of ethanol toxicity. These acetyl-related changes were then correlated with alterations in SOD2 enzymatic structure and activity. Interestingly, chronic ethanol consumption shifted the isoelectric point of SOD2 toward a more negative state as demonstrated by 2D- electrophoresis. Ethanol-induced acetylation in vivo also decreased SOD2 activity by 40%. Through the integration of enzyme assays, mass spectrometry, and computational biology, our goal is to further elucidate mechanisms of nutrient disruption and oxidative stress due to alcohol toxicity.