58 research outputs found
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
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
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
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
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 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
Protein Carbonylation in a Murine Model for Early Alcoholic Liver Disease
Hepatic oxidative stress and subsequent lipid peroxidation
are
well-recognized consequences of sustained ethanol consumption. The
covalent adduction of nucleophilic amino acid side-chains by lipid
electrophiles is significantly increased in patients with alcoholic
liver disease (ALD); a global assessment of <i>in vivo</i> protein targets and the consequences of these modifications, however,
has not been conducted. In this article, we describe the identification
of novel protein targets for covalent adduction in a 6-week murine
model for ALD. Ethanol-fed mice displayed a 2-fold increase in hepatic
TBARS, while immunohistochemical analysis for the reactive aldehydes
4-hydroxynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein (ACR), and
malondialdehyde (MDA) revealed a marked increase in the staining of
modified proteins in the ethanol-treated mice. Increased protein carbonyl
content was confirmed utilizing subcellular fractionation of liver
homogenates followed by biotin-tagging through hydrazide chemistry,
where approximately a 2-fold increase in modified proteins was observed
in microsomal and cytosolic fractions. To determine targets of protein
carbonylation, a secondary hydrazide method coupled to a highly sensitive
2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS
or MuDPIT) technique was utilized. Our results have identified 414
protein targets for modification by reactive aldehydes in ALD. The
presence of novel <i>in vivo</i> sites of protein modification
by 4-HNE (2), 4-ONE (4) and ACR (2) was also confirmed in our data
set. While the precise impact of protein carbonylation in ALD remains
unknown, a bioinformatic analysis of the data set has revealed key
pathways associated with disease progression, including fatty acid
metabolism, drug metabolism, oxidative phosphorylation, and the TCA
cycle. These data suggest a major role for aldehyde adduction in the
pathogenesis of ALD
Protein Carbonylation in a Murine Model for Early Alcoholic Liver Disease
Hepatic oxidative stress and subsequent lipid peroxidation
are
well-recognized consequences of sustained ethanol consumption. The
covalent adduction of nucleophilic amino acid side-chains by lipid
electrophiles is significantly increased in patients with alcoholic
liver disease (ALD); a global assessment of <i>in vivo</i> protein targets and the consequences of these modifications, however,
has not been conducted. In this article, we describe the identification
of novel protein targets for covalent adduction in a 6-week murine
model for ALD. Ethanol-fed mice displayed a 2-fold increase in hepatic
TBARS, while immunohistochemical analysis for the reactive aldehydes
4-hydroxynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein (ACR), and
malondialdehyde (MDA) revealed a marked increase in the staining of
modified proteins in the ethanol-treated mice. Increased protein carbonyl
content was confirmed utilizing subcellular fractionation of liver
homogenates followed by biotin-tagging through hydrazide chemistry,
where approximately a 2-fold increase in modified proteins was observed
in microsomal and cytosolic fractions. To determine targets of protein
carbonylation, a secondary hydrazide method coupled to a highly sensitive
2-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS
or MuDPIT) technique was utilized. Our results have identified 414
protein targets for modification by reactive aldehydes in ALD. The
presence of novel <i>in vivo</i> sites of protein modification
by 4-HNE (2), 4-ONE (4) and ACR (2) was also confirmed in our data
set. While the precise impact of protein carbonylation in ALD remains
unknown, a bioinformatic analysis of the data set has revealed key
pathways associated with disease progression, including fatty acid
metabolism, drug metabolism, oxidative phosphorylation, and the TCA
cycle. These data suggest a major role for aldehyde adduction in the
pathogenesis of ALD
Equilibrium binding data utilizing the fluorescent probe ANS.
<p>Values presented are mean ± SEM of two independent experiments consisting of at least 3 replicates. AFU denotes arbitrary fluorescent units.</p
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