Dataset Information

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

ABSTRACT: The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals during cellular stress via several post-translational modifications that change its folding properties, protein-protein interactions and sub-cellular localization. We examined GAPDH properties in acute mouse liver injury due to ethanol and/or acetaminophen (APAP) treatment. Synergistic robust and time-dependent nuclear accumulation and aggregation of GAPDH were observed only in combined, but not individual, ethanol/APAP treatments. The small molecule GAPDH-targeting compound TCH346 partially attenuated liver damage possibly via mitochondrial mechanisms, and independent of nuclear accumulation and aggregation of GAPDH. These findings provide a novel potential mechanism for hepatotoxicity caused by combined alcohol and acetaminophen exposure.

SUBMITTER: Snider NT

PROVIDER: S-EPMC4981434 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

Snider Natasha T NT Portney Daniel A DA Willcockson Helen H HH Maitra Dhiman D Martin Hope C HC Greenson Joel K JK Omary M Bishr MB

PloS one 20160811 8

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) signals during cellular stress via several post-translational modifications that change its folding properties, protein-protein interactions and sub-cellular localization. We examined GAPDH properties in acute mouse liver injury due to ethanol and/or acetaminophen (APAP) treatment. Synergistic robust and time-dependent nuclear accumulation and aggregation of GAPDH were observed only in combined, but not individual, ethanol/AP ...[more]

PMID: 27513663

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Project description:Acetaminophen (APAP) is metabolized in the liver to N-acetyl-p-benzoquinone imine (NAPQI), an electrophilic metabolite known to bind liver proteins resulting in hepatotoxicity. Mammalian thioredoxin reductase (TrxR) is a cellular antioxidant containing selenocysteine (Sec) in its C-terminal redox center, a highly accessible target for electrophilic modification. In the present study, we determined if NAPQI targets TrxR. Hepatotoxicity induced by APAP treatment of mice (300 mg/kg, i.p.) was associated with a marked inhibition of both cytosolic TrxR1 and mitochondrial TrxR2 activity. Maximal inhibition was detected at 1 and 6 h post-APAP for TrxR1 and TrxR2, respectively. In purified rat liver TrxR1, enzyme inactivation was correlated with the metabolic activation of APAP by cytochrome P450, indicating that enzyme inhibition was due to APAP-reactive metabolites. NAPQI was also found to inhibit TrxR1. NADPH-reduced TrxR1 was significantly more sensitive to NAPQI (IC50 = 0.023 μM) than the oxidized enzyme (IC50 = 1.0 μM) or a human TrxR1 Sec498Cys mutant enzyme (IC50 = 17 μM), indicating that cysteine and selenocysteine residues in the redox motifs of TrxR are critical for enzyme inactivation. This is supported by our findings that alkylation of reduced TrxR with biotin-conjugated iodoacetamide, which selectively reacts with selenol or thiol groups on proteins, was inhibited by NAPQI. LC-MS/MS analysis confirmed that NAPQI modified cysteine 59, cysteine 497, and selenocysteine 498 residues in the redox centers of TrxR, resulting in enzyme inhibition. In addition to disulfide reduction, TrxR is also known to mediate chemical redox cycling. We found that menadione redox cycling by TrxR was markedly less sensitive to NAPQI than disulfide reduction, suggesting that TrxR mediates these reactions via distinct mechanisms. These data demonstrate that APAP-reactive metabolites target TrxR, suggesting an additional mechanism by which APAP induces oxidative stress and hepatotoxicity.

Project description:BackgroundAggresomes are collections of intracellular protein aggregates. In liver cells of patients with alcoholic hepatitis, aggresomes appear histologically as cellular inclusions known as Mallory-Denk (M-D) bodies. The proteasome is a multicatalytic intracellular protease that catalyzes the degradation of both normal (native) and abnormal (misfolded and/or damaged) proteins. The enzyme minimizes intracellular protein aggregate formation by rapidly degrading abnormal proteins before they form aggregates. When proteasome activity is blocked, either by specific inhibitors or by intracellular oxidants (e.g., peroxynitrite, acetaldehyde), aggresome formation is enhanced. Here, we sought to verify whether inhibition of proteasome activity by ethanol exposure enhances protein aggregate formation in VL-17A cells, which are recombinant, ethanol-oxidizing HepG2 cells that express both alcohol dehydrogenase (ADH) and cytochrome P450 2E1 (CYP2E1).MethodsWe exposed ethanol-non-oxidizing HepG2 cells (ADH-/CYP2E1-) or ethanol-oxidizing VL-17A (ADH+/CYP2E1+) to varying levels of ethanol for 24 h or 72 h. After these treatments, we stained cells for aggresomes (detected microscopically) and quantified their numbers and sizes. We also conducted flow cytometric analyses to confirm our microscopic findings. Additionally, aggresome content in liver cells of patients with alcohol-induced hepatitis was quantified.ResultsAfter we exposed VL-17A cells to increasing doses of ethanol for 24 h or 72 h, 20S proteasome activity declined in response to rising ethanol concentrations. After 24 h of ethanol exposure, aggresome numbers in VL-17A cells were 1.8-fold higher than their untreated controls at all ethanol concentrations employed. After 72 h of ethanol exposure, mean aggresome numbers were 2.5-fold higher than unexposed control cells. The mean aggregate size in all ethanol-exposed VL-17A cells was significantly higher than in unexposed control cells but was unaffected by the duration of ethanol exposure. Co-exposure of cells to EtOH and rapamycin, the latter an autophagy activator, completely prevented EtOH-induced aggresome formation. In the livers of patients with alcohol-induced hepatitis (AH), the staining intensity of aggresomes was 2.2-fold higher than in the livers of patients without alcohol use disorder (AUD).ConclusionsWe conclude that ethanol-induced proteasome inhibition in ethanol-metabolizing VL-17A hepatoma cells causes accumulation of protein aggregates. Notably, autophagy activation removes such aggregates. The significance of these findings is discussed.

Dataset Information

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

Publications

Ethanol and Acetaminophen Synergistically Induce Hepatic Aggregation and TCH346-Insensitive Nuclear Translocation of GAPDH.

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