Mitochondrial Acetylation and Acetylome Dynamics in Alcoholic Liver Disease assessed with Heavy Water

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Summary Alcoholic liver disease (ALD) is a major cause of liver-related death. Alcohol intake is associated with hyperacetylation of hepatic mitochondrial proteins and impaired mitochondrial function. However, the significance of mitochondrial acetylation has been debated because of the low stoichiometry of acetylation. We propose that changes in acetylome dynamics, rather than levels, are the determinant of mitochondrial function. The rationale is that hepatic mitochondria may respond to alcohol-induced stress via acetyl-transfer dependent enzymatic inhibition (short-term regulation) and/or the acetyl-transfer independent regulation of protein stability (long-term regulation). To assess the impact of these acetylation-mediated changes, we will quantify mitochondrial acetylome dynamics in ALD mice liver in vivo using a stable isotope-resolved high- resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method relies on our technical advancement that dissects the isotope-labeling of acetyl moiety and peptide backbone. Acetylation turnover is determined based on labeling of acetyl moiety. The effect of acetylation on protein stability is assessed by comparisons of the half-lives of the intact native peptide and acetylated peptide fragments without acetyl moiety. We will use this method and genetic and pharmacological tools to study the role of altered acetylome dynamics in ALD mice livers. Experiments will be performed in collaboration with The Northern Ohio Alcohol Center (see LOS of Dr. Nagy) using an established mouse model of ALD induced with a Lieber-DeCarli diet that results in hepatic hyperacetylation and mitochondrial dysfunction. Aim 1 will determine if alcohol-induced acetylation alters the stability of hepatic mitochondrial proteins and mitochondrial respiration. Aim 2 will assess the impact of alcohol consumption on the acetylation turnover of mitochondrial proteins and the consequence of altered acetyl transfer on regulations of metabolic and antioxidant enzymes. Our novel tools and collaborative expertise will enable us to investigate the mechanisms of alcohol-induced mitochondrial dysfunction. Completion of this project will uncover a novel role of altered acetylation dynamics in the alcoholic liver and help to identify acetylation as a therapeutic target for the treatment of ALD.