Ethanol Metabolism Modifies Hepatic Protein Acylation in Mice

<div><p>Mitochondrial protein acetylation increases in response to chronic ethanol ingestion in mice, and is thought to reduce mitochondrial function and contribute to the pathogenesis of alcoholic liver disease. The mitochondrial deacetylase SIRT3 regulates the acetylation status of several mitochondrial proteins, including those involved in ethanol metabolism. The newly discovered desuccinylase activity of the mitochondrial sirtuin SIRT5 suggests that protein succinylation could be an important post-translational modification regulating mitochondrial metabolism. To assess the possible role of protein succinylation in ethanol metabolism, we surveyed hepatic sub-cellular protein fractions from mice fed a control or ethanol-supplemented diet for succinyl-lysine, as well as acetyl-, propionyl-, and butyryl-lysine post-translational modifications. We found mitochondrial protein propionylation increases, similar to mitochondrial protein acetylation. In contrast, mitochondrial protein succinylation is reduced. These mitochondrial protein modifications appear to be primarily driven by ethanol metabolism, and not by changes in mitochondrial sirtuin levels. Similar trends in acyl modifications were observed in the nucleus. However, comparatively fewer acyl modifications were observed in the cytoplasmic or the microsomal compartments, and were generally unchanged by ethanol metabolism. Using a mass spectrometry proteomics approach, we identified several candidate acetylated, propionylated, and succinylated proteins, which were enriched using antibodies against each modification. Additionally, we identified several acetyl and propionyl lysine residues on the same sites for a number of proteins and supports the idea of the overlapping nature of lysine-specific acylation. Thus, we show that novel post-translational modifications are present in hepatic mitochondrial, nuclear, cytoplasmic, and microsomal compartments and ethanol ingestion, and its associated metabolism, induce specific changes in these acyl modifications. These data suggest that protein acylation, beyond protein acetylation, contributes to the overall metabolic regulatory network and could play an important role in the pathogenesis of alcoholic liver disease.</p> </div

Mitochondria were isolated from livers of wild-type mice fed a standard or ethanol diet for 6-8 weeks and analyzed for mitochondrial protein acetylation (A), propionylation (B), butyrylation (C), and succinylation (D) by western blot analysis with an acyllysine-specific antibody; normalized to total mitochondrial content using anti-electron transfer flavoprotein (ETF); n = 4 mice/condition.

<p>Mitochondria were isolated from livers of wild-type mice fed a standard or ethanol diet for 6-8 weeks and analyzed for mitochondrial protein acetylation (A), propionylation (B), butyrylation (C), and succinylation (D) by western blot analysis with an acyllysine-specific antibody; normalized to total mitochondrial content using anti-electron transfer flavoprotein (ETF); n = 4 mice/condition.</p

Proteomic data summary of all known acylated proteins identified in mitochondrial and non-mitochondrial compartments.

<p>Area represents number of proteins identified in this study (red), compared to previous studies (blue), as acetylated (10:1 scaling), propionylated, and succinylated.</p

Mitochondria were collected from livers of wild-type mice fed a standard or ethanol diet for 1, 3, or 6 weeks and analyzed for total protein acetylation (A), propionylation (B), and succinylation (C) by western blot analysis with acyllysine-specific antibodies; normalized to total mitochondrial content using anti-voltage-dependent anion channel (VDAC); n = 4 mice/condition.

<p>Mitochondria were collected from livers of wild-type mice fed a standard or ethanol diet for 1, 3, or 6 weeks and analyzed for total protein acetylation (A), propionylation (B), and succinylation (C) by western blot analysis with acyllysine-specific antibodies; normalized to total mitochondrial content using anti-voltage-dependent anion channel (VDAC); n = 4 mice/condition.</p

Pathway analyses of acylated proteins.

<p>Unique pathway (in box) and number of acylated proteins identified in that pathway (in parenthesis), divided by major sub-cellular compartments.</p

Mitochondrial extracts were isolated from livers of wild-type or SIRT3KO mice fed a standard or ethanol diet for 6-8 weeks and analyzed for total protein acetylation (A), propionylation (B), butyrylation (C), and succinylation (D) by western blot analysis with an acyllysine-specific antibody; normalized to total mitochondrial content using anti-voltage-dependent anion channel (VDAC); n = 4 mice/condition.

<p>Mitochondrial extracts were isolated from livers of wild-type or SIRT3KO mice fed a standard or ethanol diet for 6-8 weeks and analyzed for total protein acetylation (A), propionylation (B), butyrylation (C), and succinylation (D) by western blot analysis with an acyllysine-specific antibody; normalized to total mitochondrial content using anti-voltage-dependent anion channel (VDAC); n = 4 mice/condition.</p

Mitochondrial Acetylome Analysis in a Mouse Model of Alcohol-Induced Liver Injury Utilizing SIRT3 Knockout Mice

Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD

Heart function as measured by ECHO and invasive left heart catheterization for FXN KO and controls at ages 30, 45 and 65 days.

<p>Heart function as measured by ECHO and invasive left heart catheterization for FXN KO and controls at ages 30, 45 and 65 days.</p

Abnormal cardiac mitochondria ultrastructure is accompanied by respiratory inhibition in FXN KO hearts.

<p>(a) Representative images from electron micrographs of cardiomyocytes viewed at 11,000x. Mitochondrial ultrastructure abnormalities were apparent in FXN KO sections and progressed from days 30 to 65. Findings included matrix density loss, mitochondrial-to-sarcomere disarrangement, and accumulation and clumping of mitochondria. FXN KO day 65 mitochondria demonstrate cristae collapse and dissolution, and hyperdense inclusions. Arrowhead = collapsed cristae; arrow = electron-dense intramitochondrial inclusions. (b) Myofibril:mitochondria area ratios were significantly decreased at day 65 in FXN KO compared to controls (n = 3–8 micrographs per strain at each age). (c) Mitochondrial functional assays demonstrated significantly decreased respiratory control ratios in FXN KO compared to controls. There were 2–4 assay runs on pooled mitochondria from FXN KO (n = 8–12 hearts), or pooled mitochondria from controls (n = 4–6 hearts), with 2–3 pooled hearts per run.</p

FXN KO hearts exhibit features of maladaptive ventricular remodeling.

<p>(a) Cardiac fibrosis is progressive in the FXN KO heart. Ventricular tissue was stained using Masson’s Trichrome to detect blue-staining fibrous tissue. (b) Percent area collagen was significantly increased in FXN KO hearts at postnatal day 65 compared to controls. (c) Evidence of cardiomyocyte degeneration in the FXN KO at day 65 is demonstrated by vacuolation on H&E staining.</p