Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis

• Published in: Free radical biology & medicine Vol. 89; pp. 1144 - 1153
• Main Authors: Shearn, C.T., Orlicky, D.J., Saba, L.M., Shearn, A.H., Petersen, Dennis R.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.12.2015
• Subjects: 4-HNE; Adult; Alcoholic liver disease; Biomarkers - chemistry; Biomarkers - metabolism; Blotting, Western; Case-Control Studies; Chromatography, Liquid - methods; Female; Humans; Liver; Liver Cirrhosis, Alcoholic - diagnosis; Liver Cirrhosis, Alcoholic - metabolism; Male; Middle Aged; Oxidative stress; Protein Carbonylation; Protein Processing, Post-Translational; Reactive aldehydes; Tandem Mass Spectrometry - methods; Oxidative stress; Reactive aldehydes; HHE; Liver; ALT; EtOH; MDA; Alcoholic liver disease; ETD; 4-HNE; Cyp2E1; ALD; CID; 4-ONE
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2015.10.420

Oxidative stress is a significant contributing factor in the pathogenesis of alcoholic liver disease (ALD). In the murine models of chronic alcohol consumption, induction of oxidative stress results in increased peroxidation of polyunsaturated fatty acids to form highly reactive electrophilic α/β unsaturated aldehydes that post-translationally modify proteins altering activity. Data are presented here suggesting that oxidative stress and the resulting carbonylation of hepatic proteins is an ongoing process involved in alcohol-induced cirrhosis. Using age-matched pooled hepatic tissue obtained from healthy humans and patients with end stage cirrhotic ALD, overall carbonylation was assessed by immunohistochemistry and LC-MS/MS of streptavidin purified hepatic whole cell extracts treated with biotin hydrazide. Identified carbonylated proteins were further evaluated using bioinformatics analyses. Using immunohistochemistry and Western blotting, protein carbonylation was increased in end stage ALD occurring primarily in hepatocytes. Mass spectrometric analysis revealed a total of 1224 carbonylated proteins in normal hepatic and end-stage alcoholic cirrhosis tissue. Of these, 411 were unique to cirrhotic ALD, 261 unique to normal hepatic tissue and 552 common to both groups. Bioinformatic pathway analysis of hepatic carbonylated proteins revealed a propensity of long term EtOH consumption to increase post-translational carbonylation of proteins involved in glutathione homeostatic, glycolytic and cytoskeletal pathways. Western analysis revealed increased expression of GSTA4 and GSTπ in human ALD. Using LC-MS/MS analysis, a nonenaldehyde post-translational modification was identified on Lysine 235 of the cytoskeletal protein vimentin in whole cell extracts prepared from human end stage ALD hepatic tissue. Conclusions: These studies are the first to use LC-MS/MS analysis of carbonylated proteins in human ALD and begin exploring possible mechanistic links with end-stage alcoholic cirrhosis and oxidative stress. [Display omitted] •We demonstrate increased production of reactive aldehydes in human ALD.•Using LC-MS/MS we identify 411 carbonylated proteins unique to human ALD.•Pathway analysis revealed a propensity for adduction of GSH homeostatic proteins.•Using MS/MS, a nonenaldehyde adduct was identified on vimentin residue Lys235.•Reactive aldehyde modification of proteins contributes to pathogenesis of human ALD.