Chronic Ethanol Metabolism Inhibits Hepatic Mitochondrial Superoxide Dismutase via Lysine Acetylation

Abstract

Background: Chronic ethanol (EtOH) consumption is a major cause of liver disease worldwide. Oxidative stress is a known consequence of EtOH metabolism and is thought to contribute significantly to alcoholic liver disease (ALD). Therefore, elucidating pathways leading to sustained oxidative stress and downstream redox imbalances may reveal how EtOH consumption leads to ALD. Recent studies suggest that EtOH metabolism impacts mitochondrial antioxidant processes through a number of proteomic alterations, including hyperacetylation of key antioxidant proteins.

Methods: To elucidate mechanisms of EtOH-induced hepatic oxidative stress, we investigate a role for protein hyperacetylation in modulating mitochondrial superoxide dismutase (SOD2) structure and function in a 6-week Lieber-DeCarli murine model of EtOH consumption. Our experimental approach includes immunoblotting immunohistochemistry (IHC), activity assays, mass spectrometry, and in silico modeling.

Results: We found that EtOH metabolism significantly increased the acetylation of SOD2 at 2 functionally relevant lysine sites, K68 and K122, resulting in a 40% decrease in enzyme activity while overall SOD2 abundance was unchanged. In vitro studies also reveal which lysine residues are more susceptible to acetylation. IHC analysis demonstrates that SOD2 hyperacetylation occurs near zone 3 within the liver, which is the main EtOH-metabolizing region of the liver.

Conclusions: Overall, the findings presented in this study support a role for EtOH-induced lysine acetylation as an adverse posttranslational modification within the mitochondria that directly impacts SOD2 charge state and activity. Last, the data presented here indicate that protein hyperacetylation may be a major factor contributing to an imbalance in hepatic redox homeostasis due to chronic EtOH metabolism.

Keywords: Alcoholic Liver Disease; Lysine Acetylation; Oxidative Stress; Sirtuin; Superoxide Dismutase.

Figure 1

Chronic ethanol metabolism in a 6-week Lieber-DeCarli model induces steatosis and CYP2E1 expression in liver tissue of alcohol fed mice, as demonstrated by (A) Hematoxylin and eosin staining and (B) CYP2E1 immunohistochemistry. Immunohistochemistry analysis of liver tissue from control- and ethanol-fed mice demonstrates an increase in (C) acetylation of SOD2 K68 and (D) the panlobular distribution of SOD2. CV= central vein, PV=portal vein Scale bar size = 200 micron in (A,B). and 40 micron in (C,D). Quantification of CYP2E1, SOD2-acK68 and SOD2 expression are shown in Supplemental Figure 1.

Figure 2

Chronic ethanol metabolism in a 6-week Lieber-DeCarli model significantly impacts hepatic glutathione homeostasis as determined by (A) a decrease in GSH concentration in ethanol fed group compared to controls (P< 0.05), (B) unchanged GSSG levels between groups, and (C) a significantly decreased redox potential in ethanol-fed animals (P< 0.05). (Mean ± SEM, n=4).

Figure 3

Acetylation of SOD2 is increased in hepatic tissue of ethanol-fed mice in this 6-week chronic ethanol model, as shown via (A) Western blot on liver mitochondrial lysates obtained from 3 controls and 3 ethanol fed mice using specific antibodies against SOD2, SOD2 acetyl-K68, and SOD2 acetyl-K122 and (B) quantification of SOD2 acetyl-K68 (P<0.05) and SOD2 acetyl-K122 (P<0.01). (C) SOD2 activity was significantly decreased in hepatic mitochondrial fractions of ethanol-fed mice when compared to control animals (P<0.05). (Mean ± SEM, n=3).

Figure 4

Two-dimensional SDS-PAGE is utilized to study the impact of ethanol metabolism on the isoelectric point of SOD2. Liver mitochondrial extracts from both control and ethanol-fed mice were separated using 2D electrophoresis and probed with (A) anti-SOD2 and (B) anti-SOD2 ac-K68 antibodies results here reveal that chronic ethanol consumption induces a major shift in the isoelectric point of SOD2 in murine hepatic mitochondria due in large part to protein acetylation and other PTMs.

Figure 5

In vitro acetylation of rSOD2 by acetic anhydride to determine susceptibility of each lysine residue toward acetylation and enzyme inhibition. (A) Western blotting using anti-acetyl-lysine antibody demonstrates an increase in SOD2 acetylation due to non-enzymatic acetylation and Coomassie blue staining shows protein abundance. (B) Acetylated rSOD2 was examined via LC-MS/MS analysis to identify acetylated lysine residues at each concentration of acetic anhydride. (C) SOD2 activity was inhibited as a result of lysine acetylation (P< 0.01). (Mean ± SEM, n=3).

Figure 6

Computational modeling of SOD2 tertiary structure illustrates the experimentally observed sensitivity of lysine residues to in vitro acetylation and supports our finding that one of the regulatory amino acids, K68, was found to have the highest degree of sensitivity to non-enzymatic acetylation.