Ethanol, Protein Catabolism and Liver Cell Injury

描述(申请人提供)：乙醇具有肝脏毒性，因为它在代谢过程中转化为与蛋白质反应的化合物，从而改变其生物活性。乙醇脱氢酶(ADH)和细胞色素P-450 2E1(细胞色素P-450 2E1)催化乙醇转化为乙醛。CYP2E1的催化作用还会促进其他活性物种的形成，包括超氧化物和羟乙基自由基，它们参与产生羟基自由基、过氧亚硝酸根和过氧化脂质的二次反应。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细胞内调节蛋白质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。这些研究使用了重组的Hep G2(VL-17A)细胞，它既能结构性地表达ADH，又能表达细胞色素P450-2，并能代谢与肝细胞类似的乙醇。当VL-17A细胞长时间接触乙醇时，蛋白酶体活性下降，这种酶活性的下降依赖于乙醇代谢。这一发现的重要性在于，蛋白酶体活性的下降可以导致一系列的影响，其中包括细胞信号的减少，转录因子的稳定和蛋白质质量控制的显著中断。为了继续我们的研究，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，我们提出了乙醇诱导的蛋白质聚集体在我们的培养的肝癌细胞模型中积累的证据。我们假设乙醇诱导的氧化应激抑制肝细胞中蛋白酶体的活性，从而影响与脂肪变性有关的Egr-1和其他调节蛋白的水平。此外，蛋白酶体抑制扰乱了蛋白质质量控制，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标：目的1将表征乙醇诱导的乙醇代谢培养细胞中促脂转录因子Egr-1和SREBP-1的诱导，并确定Egr-1在乙醇诱导的脂肪变性中的重要性。目的2研究乙醇对小鼠肝脏成脂转录因子的诱导作用。目的3将研究酒精暴露后形成的蛋白质聚集体是否是蛋白酶体的有效底物或抑制物，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。拟议的实验方法检查了一个高度调控的细胞蛋白分解系统，这是一个潜在的治疗目标，以减轻大量饮酒造成的器官损伤。

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.