Ethanol, Protein Catabolism and Liver Cell Injury

乙醇、蛋白质分解代谢和肝细胞损伤

• 批准号: 8101956
• 负责人: Terrence M. Donohue
• 金额: $ 26.98万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8101956
• 关键词: ADD-1 protein; Acetaldehyde; Acute; Affect; Alcohol consumption; Alcohol dehydrogenase; Alcoholic Hepatitis; Alcoholic Liver Diseases; Biological; Catabolism; Catalysis; Cell Death; Cell Survival; Cell model; Cells; Chronic; Cirrhosis; Clinical; Complication; Cultured Cells; Cytochrome P-450 CYP2E1; Cytochrome P450; Data; Enzymes; Ethanol; Ethanol Metabolism; Ethanol toxicity; Event; Exhibits; Exposure to; Fatty Liver; Fibrosis; Free Radicals; Generations; Growth; Health; Heavy Drinking; Hepatic; Hepatoblastoma; Hepatocyte; Hepatotoxicity; Human; Inflammatory; Injury; Interferon Type II; Interferons; Investigation; Knowledge; Laboratories; Link; Lipid Peroxides; Liver; Mediating; Mus; Nitric Oxide; Nitrogen; Organ; Oxidative Stress; Oxygen; Peroxonitrite; Phosphorylation; Production; Proteasome Inhibitor; Proteins; Proteolysis; Quality Control; Reaction; Recombinants; Regulation; Research; Rodent; Role; SRE-1 binding protein; STAT1 protein; Signal Transduction; Site; Superoxides; System; Testing; alcohol effect; alcohol exposure; alcohol response; cell injury; chronic alcohol ingestion; cytokine; enzyme activity; genetic regulatory protein; hepatoma cell; hepatotoxin; in vivo; insight; multicatalytic endopeptidase complex; oxidation; prevent; protein aggregate; protein aggregation; protein degradation; protein misfolding; response; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): Ethanol is hepatotoxic because it is metabolically converted to compounds that react with proteins to alter their biological activities. Ethanol oxidation by alcohol dehydrogenase (ADH) and cytochrome P-450 2E1 (CYP2E1) catalyze the conversion of ethanol to acetaldehyde. Catalysis by CYP2E1 also leads to enhanced formation of other reactive species including superoxide and hydroxyethyl radicals, which participate in secondary reactions producing hydroxy radicals, peroxynitrite, and lipid peroxides. These metabolites disrupt the activity of proteins, including the proteasome, a multicatalytic enzyme that is indispensable for degrading damaged and misfolded proteins and for tightly controlling the intracellular content of regulatory proteins. Our research has examined the effects of ethanol on proteasome function and its role in hepatic injury, cell regulation and signal transduction. Theses studies have used recombinant Hep G2 (VL-17A) cells that constitutively express both ADH and CYP2E1 and metabolize ethanol comparably to liver cells. When VL-17A cells are exposed to ethanol for extended periods, proteasome activity decreases and this decrease in enzyme activity is dependent on ethanol metabolism. The importance of this finding is that a decline in proteasome activity can result in a variety of effects, among which are a reduction in cell signaling, stabilization of transcription factors and a significant disruption of protein quality control. For our continued investigations, we propose to examine the possible role of the proteasome in ethanol-elicited steatosis, both in cultured cells and in vivo. Furthermore, we present evidence for the ethanol-induced accumulation of protein aggregates in our cultured hepatoma cell model. We hypothesize that ethanol-induced oxidative stress suppresses proteasome activity in liver cells, thereby affecting the levels of Egr-1 and other regulatory proteins involved in steatosis. Additionally, proteasome suppression disrupts protein quality control, thereby causing intracellular protein aggregation in cells. To test this hypothesis, we propose the following Specific Aims: Aim 1 will characterize the ethanol- elicited induction of the lipogenic transcription factors, Egr-1 and SREBP-1 in ethanol metabolizing cultured cells and determine the essentiality of Egr-1 in ethanol-elicited steatosis. Aim 2 will characterize the ethanol- elicited induction of lipogenic transcription factors in mouse liver. Aim 3 will examine whether protein aggregates that form in response to ethanol exposure are effective substrates or inhibitors of the proteasome and whether such aggregates can be eliminated by proteasome activation. PUBLIC HEALTH RELEVANCE: The insights gained from the proposed investigation will expand our knowledge of the mechanisms of ethanol hepatotoxicity. The proposed experimental approach examines a highly regulated cellular proteolytic system that is a potential therapeutic target to alleviate organ damage caused by heavy alcohol consumption.

描述（由申请人提供）：乙醇具有肝毒性，因为它在代谢过程中转化为与蛋白质反应改变其生物活性的化合物。乙醇通过乙醇脱氢酶（ADH）和细胞色素P-450 2E1 （CYP2E1）氧化催化乙醇转化为乙醛。在CYP2E1的催化作用下，其他活性物质如超氧自由基和羟乙基自由基的生成也会增强，它们参与二次反应，产生羟基自由基、过氧亚硝酸盐和脂质过氧化物。这些代谢物破坏蛋白质的活性，包括蛋白酶体，蛋白酶体是一种多催化酶，对于降解受损和错误折叠的蛋白质以及严格控制细胞内调节蛋白的含量是必不可少的。我们的研究考察了乙醇对蛋白酶体功能的影响及其在肝损伤、细胞调节和信号转导中的作用。这些研究使用重组Hep G2 （VL-17A）细胞，该细胞组成性地表达ADH和CYP2E1，并与肝细胞一样代谢乙醇。当VL-17A细胞长时间暴露于乙醇中时，蛋白酶体活性降低，这种酶活性的降低依赖于乙醇代谢。这一发现的重要性在于，蛋白酶体活性的下降会导致多种影响，其中包括细胞信号传导的减少、转录因子的稳定和蛋白质质量控制的显著破坏。对于我们继续的研究，我们建议在培养细胞和体内研究蛋白酶体在乙醇诱导的脂肪变性中的可能作用。此外，我们在培养的肝癌细胞模型中提供了乙醇诱导的蛋白质聚集体积累的证据。我们假设乙醇诱导的氧化应激抑制肝细胞中的蛋白酶体活性，从而影响Egr-1和其他参与脂肪变性的调节蛋白的水平。此外，蛋白酶体抑制破坏了蛋白质质量控制，从而导致细胞内蛋白质聚集。为了验证这一假设，我们提出了以下具体目的：目的1将表征乙醇诱导的脂肪生成转录因子Egr-1和SREBP-1在乙醇代谢培养细胞中的诱导作用，并确定Egr-1在乙醇诱导的脂肪变性中的重要性。目的2将描述乙醇诱导的小鼠肝脏脂肪生成转录因子。目的3将研究在乙醇暴露下形成的蛋白质聚集体是否是蛋白酶体的有效底物或抑制剂，以及这种聚集体是否可以通过蛋白酶体激活来消除。公共卫生相关性：从拟议的调查中获得的见解将扩大我们对乙醇肝毒性机制的了解。提出的实验方法研究了一个高度调节的细胞蛋白水解系统，这是一个潜在的治疗靶点，可以减轻大量饮酒引起的器官损伤。

项目成果

Cellular steatosis in ethanol oxidizing-HepG2 cells is partially controlled by the transcription factor, early growth response-1.

• DOI: 10.1016/j.biocel.2012.10.002
• 发表时间: 2013-02
• 期刊: INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
• 影响因子: 4
• 作者: Thomes, Paul G.;Osna, Natalia A.;Davis, John S.;Donohue, Terrence M., Jr.
• 通讯作者: Donohue, Terrence M., Jr.

Ethanol Exposure to Ethanol-Oxidizing HEPG2 Cells Induces Intracellular Protein Aggregation.

• DOI: 10.3390/cells12071013
• 发表时间: 2023-03-26
• 期刊: Cells
• 影响因子: 6

Early growth response-1 contributes to steatosis development after acute ethanol administration.

• DOI: 10.1111/j.1530-0277.2011.01681.x
• 发表时间: 2012-05
• 期刊: Alcoholism, clinical and experimental research
• 作者: Donohue TM Jr;Osna NA;Trambly CS;Whitaker NP;Thomes PG;Todero SL;Davis JS
• 通讯作者: Davis JS

Ethanol-induced oxidant stress modulates hepatic autophagy and proteasome activity.

• DOI: 10.1016/j.redox.2014.10.006
• 发表时间: 2014
• 期刊: REDOX BIOLOGY
• 影响因子: 11.4
• 作者: Donohue, Terrence M., Jr.;Thomes, Paul G.
• 通讯作者: Thomes, Paul G.