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: 7677508
• 负责人: Terrence M. Donohue
• 金额: $ 28.35万
• 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 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7677508
• 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; Heavy Drinking; Hepatic; Hepatoblastoma; Hepatocyte; Hepatotoxicity; Human; Inflammatory; Injury; Interferon Type II; Investigation; Knowledge; Laboratories; Link; Lipid Peroxides; Liver; Mediating; Mus; Nitric Oxide; Nitrogen; Organ; Oxidative Stress; Oxygen; Peroxonitrite; Phosphorylation; Primary carcinoma of the liver cells; 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; Toxic effect; 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; public health relevance; 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，因为它会代谢转化为与蛋白质反应以改变其生物活性的化合物。乙醇脱氢酶 (ADH) 和细胞色素 P-450 2E1 (CYP2E1) 催化乙醇氧化为乙醛。 CYP2E1 的催化作用还导致其他活性物质的形成增强，包括超氧化物和羟乙基自由基，它们参与产生羟基自由基、过氧亚硝酸盐和脂质过氧化物的次级反应。这些代谢物会破坏蛋白质的活性，包括蛋白酶体，这是一种多催化酶，对于降解受损和错误折叠的蛋白质以及严格控制调节蛋白的细胞内含量至关重要。我们的研究探讨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。这些研究使用了重组 Hep G2 (VL-17A) 细胞，该细胞组成型表达 ADH 和 CYP2E1，并且代谢乙醇的能力与肝细胞相当。当 VL-17A 细胞长时间暴露于乙醇时，蛋白酶体活性降低，而酶活性的降低取决于乙醇代谢。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、转录因子的稳定以及蛋白质质量控​​制的显着破坏。为了我们的持续研究，我们建议在培养细胞和体内检查蛋白酶体在乙醇引起的脂肪变性中的可能作用。此外，我们提供了在我们的培养肝癌细胞模型中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会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，蛋白酶体抑制会破坏蛋白质质量控​​制，从而导致细胞内蛋白质聚集。为了检验这一假设，我们提出以下具体目标：目标 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 在乙醇中的重要性。引起脂肪变性。目标 2 将描述小鼠肝脏中乙醇诱导的脂肪生成转录因子的特征。目标 3 将检查响应乙醇暴露而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，以及此类聚集体是否可以通过蛋白酶体激活消除。公共健康相关性：从拟议的研究中获得的见解将扩大我们对乙醇肝毒性机制的了解。所提出的实验方法检查了高度调节的细胞蛋白水解系统，该系统是减轻大量饮酒引起的器官损伤的潜在治疗靶点。