Redox Biology 7 (2016) 68–77 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research paper Deletion of GSTA4-4 results in increased mitochondrial post-translational modification of proteins by reactive aldehydes following chronic ethanol consumption in mice Colin T. Shearn a,n, Kristofer S. Fritz a, Alisabeth H. Shearn b, Laura M. Saba a, Kelly E. Mercer c, Bridgette Engi c, James J. Galligan e, Piotr Zimniak c, David J. Orlicky d, Martin J. Ronis f, Dennis R. Petersen a,nn a Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Anschutz Medical Campus, Aurora, CO, United States b Alpine Achievement Systems Inc., Englewood, CO, United States c Department of Pediatrics, Arkansas Children's Nutrition Center, Little Rock, AR, United States d Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO, United States e Department of Biochemistry, Vanderbilt, Nashville, TN, United States f Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA, United States article info abstract Article history: Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive Received 20 October 2015 electrophilic α/β-unsaturated aldehydes that have the potential to modify proteins. A primary me- Received in revised form chanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic con- 23 November 2015 jugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, Accepted 25 November 2015 À À we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4 / mice is due to Available online 27 November 2015 enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased À À Keywords: hepatic protein carbonylation in GSTA4 / mice as evidenced by increased 4-HNE and MDA im- Ethanol munostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide Lipid peroxidation conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and GSTA4 mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, Protein carbonylation À À 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4 / mice compared to their Liver Oxidative stress respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from fi Mitochondria the mitochondrial fractions revealed an increased propensity for modi cation of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4À/À mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4À/À 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. & 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Abbreviations: ADPH, adipophilin; ALD, alcoholic liver disease; ALT, alanine aminotransferase; CID, collision-induced dissociation; Cyp2E1, Cytochrome P4502E1; ETD, electron transfer dissociation; EtOH, ethanol; GSTA4, glutathione S-transferase isoform A4; 4-HHE, 4-hydroxy-2-hexenal; 4-HNE, 4-hydroxy-2-nonenal; MDA, mal- ondialdehyde; 4-ONE, 4-oxononenal; PF, Pair-fed n Correspondence to: Department of Pharmaceutical Sciences School of Pharmacy University of Colorado Denver Anschutz Medical Campus, 12850 East Montview Blvd Box C238, Building V20 Room 2460B, United States. Fax: þ303 724 7266. nn Correspondence to: Department of Pharmaceutical Sciences. School of Pharmacy. University of Colorado Denver Anschutz Medical Campus, 12850 East Montview Blvd Box C238, Building V20 Room 2131, United States. Fax: þ303 724 7266. E-mail address: [email protected] (C.T. Shearn). http://dx.doi.org/10.1016/j.redox.2015.11.013 2213-2317/& 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). C.T. Shearn et al. / Redox Biology 7 (2016) 68–77 69 1. Introduction concentrations were determined using blood isolated during the dark phase of housing as previously described [21]. Alcoholic liver disease (ALD) is a major contributor of liver failure in the United States today. A common phenotype of ALD is a 2.2. Histological evaluation hepatocellular environment characterized by pronounced lipid accumulation with enhanced oxidative stress [1,2]. In this pro- Formalin fixed slides were immunohistochemically evaluated oxidant environment, increased lipid peroxidation occurs resulting for 4-HNE, adipophilin (ADPH) (Fitzgerald, Ind. Acton, MA), or in the accumulation of reactive aldehydes including 4-hydro- MDA as previously described [6]. xynonenal (4-HNE), 4-oxononenal (4-ONE), acrolein, and mal- ondialdehyde (MDA) [3,4]. Following their production, the afore- 2.3. Biotin hydrazide purification mentioned reactive aldehydes react with DNA as well as Cys, Lys, and His residues within proteins [4]. Total carbonylated proteins were purified from 500 μg cytosolic In chronic ETOH models, we have identified numerous proteins extracts (5PF/5EtOH SV/GSTA4-4À/À), mitochondrial extracts (5PF/ including the lipid phosphatase PTEN, protein kinase AMPKα and 5EtOH SV/GSTA4-4À/À) and microsomal extracts (4PF/4EtOH SV, molecular chaperone Grp78 as targets of electrophilic carbonyla- 3PF/3EtOH GSTA4-4À/À) following biotin hydrazide derivatization tion by reactive aldehydes in vivo [5–7]. Concurrently, using mass (5 mM/2 h/RT/dark) and NaBH4 reduction (10 mM/100 mM NaOH spectrometry and a global proteomic approach, we have char- 1hr/dark). Biotin hydrazide linked carbonylated proteins were in- acterized the “lipid peroxidome” formed during chronic ETOH cubated along with an untreated pooled samples (negative con- administration [8]. From those seminal reports, it was determined trol/background) overnight using Streptavidin HiTrap columns (GE that increased lipid peroxidation due to ethanol consumption re- Biosciences) [8]. Columns were washed 5X in PBS/2 M Urea and sults in increased carbonylation of key proteins involved in cellular eluted with 0.1 M NH4OH. Elutions were dried down using a roto- metabolic pathways involved in fatty acid metabolism, drug me- evaporator, resuspended in 6X SDS PAGE loading buffer and loaded tabolism, oxidative phosphorylation, and the TCA cycle. Interest- on 10% SDS PAGE gels. Gels were run for 20 min at or until the dye ingly, all of these hepatic pathways are reportedly impaired during front migrated approximately 1 cm into the resolving gel. Gels ALD [9–14]. were stained with Coomassie blue overnight and stained 1 cm An important mechanism for removal of toxic lipid aldehydes is bands excised, destained, and digested with trypsin as previously via conjugation with glutathione [15,16] by Glutathione S-trans- described [8]. Peptides were extracted, dried down, and re- ferase A4-4 (GSTA4) [17]. GSTA4 is a phase 2 detoxifying enzyme suspended in 0.1% formic acid in ddH2O before loading on the whose expression is increased in response to oxidative stress. For mass spectrometer. instance, the exposure of GSTA4 transfected HepG2 cells to lipid aldehydes, including 4-HNE results in cellular resistance to toxicity 2.4. LC-MS/MS analysis [18]. In chow-fed SV 129 mice, deletion of GSTA4 results in an age- dependent increased propensity for obesity and increased lipid For LC-MS/MS analysis, 8μl of each peptide mixture in 0.1 M peroxidation, a phenotype not evident in GSTA4À/À C57BL/6J mice formic acid was loaded on a Bruker Amazon Speed LC-MS/MS [17]. Furthermore, consumption of a high fat diet increased adi- (Bruker Daltronics, Billerica, MA). The instrument was operated pocyte oxidative stress and mitochondrial damage. Additionally, using data-dependent alternating collision-induced dissociation deletion of GSTA4-4 significantly enhances protein carbonylation (CID) MS/MS and electron transfer dissociation (ETD) with a and hepatocellular damage following carbon tetrachloride (CCl4) threshold of 10,000 total ion current (TIC). Data analysis was administration, further highlighting the importance of GSTA4 in performed using ProteinScape V3.1.2 (Bruker Daltonics Inc. Bill- removal of lipid aldehydes [19]. erica, MA) and Mascot (v2.1.04, Matrixscience). First a global In this study, we used the GSTA4À/À mouse model treated search for proteins was conducted with a Mascot cutoff score of 15 chronically with ethanol to identify and validate hepatic proteins with the following variable modifications of carbamidomethly that are modified by electrophilic products of lipid peroxidation as (C) and oxidized (M) (Protein identification confidence value of well as evaluate their involvement in the early stages of ALD. The 99%, peptide identification confidence value of 95%,