Increased hepatocellular protein carbonylation in human end-stage alcoholic cirrhosis

Abstract

Objective 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. Methods 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. Results 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.

Differential carbonylation of proteins in end-stage human fatty and nonfatty NASH

“…In addition, immunohistochemical staining for 4-HNE, rabbit polyclonal 16 , 4-oxononenal rabbit polyclonal (4-ONE), acrolein rabbit polyclonal (Cell Sciences, Newburyport, MA), malondialdehyde (MDA) rabbit polyclonal (ABCAM, Billerica, MA), myeloperoxidase (MPO) goat polyclonal (Millipore, Billerica, MA), catalase rabbit polyclonal (SIGMA ALDRICH, St. Louis, MO), GSTπ (rabbit polyclonal MBL Woburn, MA), mitochondrial superoxide dismutase (SOD2) goat polyclonal (ABCAM, Billerica, MA), Thioredoxin (Trx1) rabbit polyclonal (Protein Tech, Rosemont, IL) was completed as previously described 17 . Histologic images were captured on an Olympus BX51 microscope equipped with a four-megapixel Macrofire digital camera (Optronics; Goleta, CA) using the PictureFrame Application 2.3 (Optronics).…”

“…Aldehyde modified proteins from each extract were derivatized using biotin hydrazide (BH; ThermoFisher/Pierce, Waltham, MA; 5mM/2 hrs/RT/dark) followed by NaBH 4 reduction (10mM/100mM NaOH 1hr/dark) followed by Streptavidin purification and trypsin digestion as previously described 16,18 .…”

“…First a global search for proteins was conducted with a Mascot cutoff score of 80 with the following variable modifications of carbamidomethyl (C) and oxidized (M). After the initial search a second search was conducted for post-translational modification by reactive aldehydes using the masses previously described 16 . For this 2 nd iteration, peptide significance required a Mascot score higher than 24 and visual validation of spectra.…”

“…Following their formation, these highly reactive lipid peroxides modify nucleophilic Lys, Cys and His residues on proteins exerting pathophysiological inhibitory effects. Lipid peroxidation/protein aldehyde carbonylation is a validated marker of oxidative stress 7,16,17 . Research in our laboratory has pioneered global proteomic approaches to identify proteins that are post-translationally modified by reactive aldehydes in the liver.…”

“…Research in our laboratory has pioneered global proteomic approaches to identify proteins that are post-translationally modified by reactive aldehydes in the liver. In recent murine and human studies in alcoholic liver disease we have identified 829 and 1244 proteins respectively that are carbonylated during conditions of increased hepatocellular stress 16,18 . In our current study, we utilized LC-MS/MS, immunohistochemistry and Western blotting to determine the impact of human end-stage fatty NASH and nonfatty NASH on protein carbonylation and anti-oxidant defense proteins.…”