title: Protein carbonylation in a murine model for early alcoholic liver disease.
abstract: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, University of Colorado-Denver, Aurora, CO 80045, USA.
authors.forename: James J
authors.initials: JJ
authors.lastname: Galligan
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Smathers
authors.forename: Kristofer S
authors.initials: KS
authors.lastname: Fritz
authors.forename: L E
authors.initials: LE
authors.lastname: Epperson
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Hunter
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000171:Acrolein / Q000032:analysis; D000447:Aldehydes / Q000032:analysis; D000818:Animals; D000431:Ethanol / Q000009:adverse effects / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D008099:Liver / Q000378:metabolism* / Q000473:pathology*; D008108:Liver Diseases, Alcoholic / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D008315:Malondialdehyde / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D050050:Protein Carbonylation; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis
chemical_list: D000447:Aldehydes; D005227:Fatty Acids; D011506:Proteins; D017392:Thiobarbituric Acid Reactive Substances; D000431:Ethanol; D008315:Malondialdehyde; D000171:Acrolein; C027576:4-hydroxy-2-nonenal