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.

Oxidative stress and lipotoxicity

“…In addition, proteomic analysis of lysates from aged murine liver (89), Alzheimer's disease brain (19), and mouse hippocampal neuronal cells (90) suggest targeted carbonylation of critical ER proteins, such as phosphate disulphide isomerase and calreticulin. Proteomic analysis of lysates from a rodent model of ALD confirms the susceptibility of chaperones HSP72, HSP90, and GRP78 to adduction by lipid alde-including in neurodegenerative disease (72,77,104,109), cerebral ischemia-reperfusion injury (77,110), cardiac ischemia-reperfusion injury (111)(112)(113), and in lymphocytes from aged humans (57,106). In-depth study of proteasome oxidation revealed that specific subunits are preferentially modified by reactive lipids and that, depending on which subunit is modified, catalytic activity of some, or all three, of the catalytic peptidase activities of the 20S proteasome can be inhibited (111,114,115).…”

“…As such, identification of specific modified proteins and a better understanding of how the modification affects protein function is a necessary step. To date, several large proteomics studies have aided our understanding of both the specificity and localization of protein carbonyls in tissue, such as adipose, liver, skeletal muscle, plasma, lung, and cardiac tissue (46,48,49,(57)(58)(59)(60)(61). Mass-spectrometry-based approaches have allowed for the identification of hundreds of direct targets of 4-HNE, 4-HHE, and acrolein carbonylation targets with cytosolic, mitochondrial, ER, and nuclear localization.…”

“…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. More recent work has focused on the identification of mitochondrialspecific targets in both mouse and rat models of ALD (57,72). Although the functional consequences of these modifications on mitochondrial dysfunction and the progression of ALD have yet to be determined, it is tempting to speculate that carbonylation of specific mitochondrial proteins plays a central role in regulating hepatocyte metabolic flux in ALD.…”

“…Following this work, Curtis et al (78) showed that silencing of GSTA4 in 3T3-L1 adipocytes led to increased mitochondrial protein carbonylation and impaired mitochondrial function. Recent work by Shearn et al (57) highlights the role of GSTA4 in the liver. In this study, mitochondria were isolated from the livers of GSTA4 / mice treated chronically with ethanol.…”

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

“…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.…”