CYP2E1 Mediated Mitochondrial Injury and Cell Damage in Alcohol Liver Disease

描述（由申请人提供）：过量饮酒导致酒精性肝病是世界范围内的主要健康问题，如果不及时治疗，可能导致肝癌。CYP2E1是参与酒精代谢的两种主要酶之一，已知可能通过诱导氧化组织损伤而导致酒精性肝损伤。最近的研究表明，线粒体DNA/膜损伤和线粒体蛋白的氧化损伤是酒精性肝病（ALD）的促进因素。本研究的主要目的是研究酒精诱导线粒体功能障碍的机制，并开发抗氧化剂和酶抑制剂，以尽量减少酒精引起的肝损伤。最近的研究表明，线粒体靶向CYP2E1 （mt-CYP2E1）比内质网靶向CYP2E1 （mc-CYP2E1）调节更严重的酒精毒性。对人类肝脏样本的分析还发现了独特的人类CYP2E1变异形式，其亚细胞靶向改变，导致mt-CYP2E1:mc-CYP2E1含量比例的倾斜。结果强烈表明，携带更强的靶向mt的CYP2E1突变的人更容易发生酒精诱导的毒性。我们的合作研究还表明，细胞色素c氧化酶（CcO）、线粒体醛脱氢酶（ALDH2）和线粒体转录因子A （mtTFA）的稳态水平是mt- cyp2e1增强的酒精损伤的关键靶点。目前，两位研究者的建议是验证人类mt-CYP2E1在增强ALD中起关键作用的主要假设，以及CcO， ALDH2和mtTFA是一些直接目标，如下所述目的：1)我们建议使用iPSC（诱导多能干细胞）技术产生的肝细胞来确定mtCYP2E1代谢活性在酒精毒性和诱导氧化应激中的作用。我们将使用转基因肝细胞、最先进的ROS检测系统、mt靶向抗氧化剂（Mito-CP和Mito-B）和CYP2E1抑制剂（mito -双烯丙基硫醚，Mito-D）的组合来记录mt-CYP2E1在毒性中的作用。2) Aim1的体外数据将通过最近开发的表达人类CYP2E1变体（L32N和W23/30R）的人源化小鼠模型进行体内研究来补充，目的是建立人类对酒精肝毒性遗传易感性的联系。这个

DESCRIPTION (provided by applicant): Excessive alcohol consumption leading to alcohol liver disease is a major health issue world-wide, and if left untreated, can lead to liver cancer. CYP2E1, one of the two major enzymes involved in alcohol metabolism is known to contribute to alcohol liver injury possibly by inducing oxidative tissue damage. More recent studies suggest that mitochondrial DNA/membrane damage and oxidative damage to mitochondrial proteins are contributing factors in alcohol liver disease (ALD). A major objective here is to investigate the mechanisms of alcohol induced mitochondrial dysfunction and develop antioxidants and enzyme inhibitors to minimize alcohol induced liver damage. Recent studies show that mitochondria targeted CYP2E1 (mt-CYP2E1) modulates more severe alcohol toxicity than the ER targeted CYP2E1 (mc-CYP2E1). Analysis of human liver samples also identified unique human variant forms of CYP2E1 with altered subcellular targeting that result in a skewing of the mt-CYP2E1:mc-CYP2E1 content ratio. Results strongly suggest that carriers of more robust mt-targeting CYP2E1 mutations are more prone to alcohol induced toxicity. Our collaborative studies also suggest that steady state levels of cytochrome c oxidase (CcO), mitochondrial ALDH2 (aldehyde dehydrogenase) and mtTFA (mitochondrial transcription factor A) are critical targets of mt-CYP2E1-potentiated alcohol damage. The present dual investigator proposal is to test the major hypothesis that human mt-CYP2E1 plays a critical role in potentiating ALD and that CcO, ALDH2 and mtTFA are some of the immediate targets as outlined in the following Aims: 1) We propose to use hepatocytes generated by iPSC (induced pluripotent stem cell) technology for defining the role of metabolic activity of mtCYP2E1 in alcohol toxicity and induction of oxidative stress. We will use a combination of genetically modified hepatocytes, state of the art ROS detection systems and mt-targeted antioxidants (Mito-CP and Mito-B) and CYP2E1 inhibitor (Mito-diallylsulfide, Mito-D) for documenting the role of mt-CYP2E1 in toxicity. 2) The in vitro data in Aim1 will be complemented by in vivo studies using recently developed humanized mouse models expressing human variant forms of CYP2E1 (L32N and W23/30R) with the aim of establishing a link for human genetic susceptibility to alcohol liver toxicity. The ability of Mito-CP, Mito-B and Mito-D in alleviating or reversing alcohol toxicity will be investigated. Livers from alcoholic patients will be analyzed to correlate mtCYP2E1 contents with the extent of disease and rate of progression. 3) Using iPSC hepatocytes and humanized mouse models, we will define the mechanism of alcohol mediated mitochondrial PKA activation, and LON protease mediated degradation of CcO, ALDH2 and mtTFA proteins. These results should document novel mechanisms of mitochondrial ROS production under various disease conditions and effective means of preventing or alleviating the stress conditions.