title: Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice.
abstract: Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4(-/-) mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4(-/-) mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4(-)(/-) mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4(-/-) mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4(-/-) PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States. Electronic address: colin.shearn@ucdenver.edu.
authors.forename: Colin T
authors.initials: CT
authors.lastname: Shearn
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
authors.forename: Kristofer S
authors.initials: KS
authors.lastname: Fritz
authors.affiliation: Alpine Achievement Systems Inc., Englewood, CO, United States.
authors.forename: Alisabeth H
authors.initials: AH
authors.lastname: Shearn
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Saba
authors.affiliation: Department of Pediatrics, Arkansas Children's Nutrition Center, Little Rock, AR, United States.
authors.forename: Kelly E
authors.initials: KE
authors.lastname: Mercer
authors.affiliation: Department of Pediatrics, Arkansas Children's Nutrition Center, Little Rock, AR, United States.
authors.forename: Bridgette
authors.initials: B
authors.lastname: Engi
authors.affiliation: Department of Biochemistry, Vanderbilt, Nashville, TN, United States.
authors.forename: James J
authors.initials: JJ
authors.lastname: Galligan
authors.affiliation: Department of Pediatrics, Arkansas Children's Nutrition Center, Little Rock, AR, United States.
authors.forename: Piotr
authors.initials: P
authors.lastname: Zimniak
authors.affiliation: Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Orlicky
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA, United States.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Ronis
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States.
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Petersen
mesh_terms: D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D004195:Disease Models, Animal; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism*; D008108:Liver Diseases, Alcoholic / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism*; D050050:Protein Carbonylation; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational
chemical_list: D000447:Aldehydes; D024101:Mitochondrial Proteins; D020033:Protein Isoforms; C529117:GSTA4-4 protein, mouse; D005982:Glutathione Transferase
keywords: Ethanol; GSTA4; Lipid peroxidation; Liver; Mitochondria; Oxidative stress; Protein carbonylation