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 report, we describe 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.

Oxidative stress and lipotoxicity

“…In 2012, Curtis et al (52) undertook a proteomics study in which they identified over 200 mitochondrial targets of protein In addition to obesity, elevated protein carbonylation has been observed in human and mouse models of neurodegenerative disease, cardiovascular disease, diabetes, cancer, hepatosteatosis, asthma, and pulmonary disease. including 4HNE, 4ONE, and acrolein modification sites (71). Consistent with the observed mitochondrial dysfunction and lipid accumulation characteristics of this disease model, bioinformatic pathway analysis revealed that fatty acid metabolism, oxidative phosphorylation, glycolysis, and drug metabolism were among the top modified pathways.…”

Identification of 5′ AMP-activated Kinase as a Target of Reactive Aldehydes during Chronic Ingestion of High Concentrations of Ethanol

“…n ϭ 6 mice/group. (MDA), and acrolein are all increased by chronic ETOH consumption after 6 weeks, these effects have not been characterized after 7.5 weeks of ETOH consumption (48). From Fig.…”

“…As a major indicator of oxidative stress and inflammation, 4-HNE is proposed to simultaneously initiate cellular damage as well as promote cellular survival (53). Recent proteomic studies have identified numerous protein targets of 4-HNE in cell culture as well as in a murine model of early ALD (48,54). In cells, concentrations of 4-HNE are highest within membranes with calculated hepatocellular concentrations in CCl 4 -and BrCCl 3 -treated rats of 3.8 and 11.3 mM, respectively (8,55,56).…”

Susceptibility of L-FABP−/− mice to oxidative stress in early-stage alcoholic liver

“…Whereas the animal model used for this current investigation does replicate early ALD, encompassing steatosis and oxidative stress ( 12,13,20 ), it does not encompass progression from the steatosis to steatohepatitis, which would occur using more prolonged ethanol administration paradigms ( 42 ). The present report presents novel data describing the response of L-FABP Ϫ / Ϫ mice to the sustained ingestion of ethanol for six weeks.…”

“…Protein collected from cytosolic fractions was derivatized with biotin hydrazide and stabilized with sodium borohydride as previously described ( 20 ). Samples were denatured, separated via standard SDS-PAGE, and visualized with an anti-biotin antibody (GeneTex, Irvine, CA).…”