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 in vivo 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 in vivo 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.