CYP2E1 Mediated Mitochondrial Injury and Cell Damage in Alcohol Liver Disease

CYP2E1 介导的酒精性肝病中的线粒体损伤和细胞损伤

• 批准号: 9003017
• 负责人: NARAYAN G AVADHANI
• 金额: $ 52.08万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-05 至 2019-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003017
• 关键词: Adrenodoxin; Affect; Alcohol consumption; Alcoholic Liver Diseases; Alcoholic liver damage; Alcohols; Animal Experiments; Antioxidants; Applications Grants; Bortezomib; CYP2E1 gene; Cells; Characteristics; Collaborations; Complement; Complex; Coumarins; Cyclic AMP-Dependent Protein Kinases; Cytochromes; Data; Defect; Detection; Development; Disease; Electron Transport; Endocrine system; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Ethanol Metabolism; Ethanol toxicity; Exhibits; Family; Fibroblasts; Functional disorder; Future; Gene Expression; Genetic Predisposition to Disease; Health; Health Care Costs; Heavy Drinking; Hepatocyte; Hepatotoxicity; High Pressure Liquid Chromatography; Human; Human Genetics; Hypoxia; Immune System Diseases; In Vitro; Infarction; Injury; Intervention; Laboratories; Lead; Left; Link; Liver; Liver diseases; Malignant neoplasm of liver; Measures; Mediating; Membrane; Membrane Proteins; Messenger RNA; Metabolic; Mitochondria; Mitochondrial DNA; Mitochondrial Proteins; Molecular; Molecular Target; Mus; Mutation; NADPH-Ferrihemoprotein Reductase; Nuclear; Oxidative Stress; Oxidative Stress Induction; PKA inhibitor; Pancreatitis; Pathology; Patients; Peptide Hydrolases; Peptide Signal Sequences; Pharmaceutical Preparations; Phosphorylation Site; Play; Post-Translational Protein Processing; Production; Productivity; Proteins; Proteolysis; Reproductive system; Research Personnel; Respiration; Respiratory Chain; Role; Sampling; Small Interfering RNA; Stress; System; Systems Analysis; Testing; Tissues; Toxic effect; Transgenic Mice; Variant; alcohol intervention; alcohol response; aldehyde dehydrogenases; allyl sulfide; antioxidant enzyme; cell injury; clinically significant; coronary fibrosis; cost; cytochrome c oxidase; drug metabolism; enzyme activity; global health; human disease; humanized mouse; hydroethidine; in vivo; induced pluripotent stem cell; inhibitor/antagonist; innovation; insight; knock-down; liver injury; mitochondrial dysfunction; mouse model; mtTF1 transcription factor; mutant; novel; overexpression; oxidative damage; potential biomarker; prevent; problem drinker; promoter; protein degradation; screening; stem cell technology; subcellular targeting; transcription factor

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.

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一个主要健康问题，如果不及时治疗，可能导致肝癌。CYP 2 E1是参与酒精代谢的两种主要酶之一，已知其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质的氧化损伤是酒精性肝病（ALD）的促成因素。本文的主要目的是研究酒精诱导的线粒体功能障碍的机制，并开发抗氧化剂和酶抑制剂以最大限度地减少酒精诱导的肝损伤。 最近的研究表明，线粒体靶向的CYP 2 E1（mt-CYP 2 E1）比ER靶向的CYP 2 E1（mc-CYP 2 E1）调节更严重的酒精毒性。对人肝脏样本的分析还发现了独特的CYP 2 E1人变体形式，其亚细胞靶向改变导致mt-CYP 2 E1：mc-CYP 2 E1含量比发生偏移。结果强烈表明，更强大的mt靶向CYP 2 E1突变的携带者更容易发生酒精诱导的毒性。我们的合作研究还表明，稳态水平的细胞色素c氧化酶（CcO），线粒体ALDH 2（醛脱氢酶）和mtTFA（线粒体转录因子A）的mt-CYP 2 E1增强酒精损伤的关键目标。本双重研究者提案旨在检验以下主要假设：人mt-CYP 2 E1在增强ALD中起关键作用，CcO、ALDH 2和mtTFA是以下目的中概述的一些直接靶点：1）我们建议使用由iPSC产生的肝细胞（诱导多能干细胞）技术，用于确定mtCYP 2 E1代谢活性在酒精毒性和氧化应激诱导中的作用。我们将使用基因修饰肝细胞、最先进的ROS检测系统和mt靶向抗氧化剂（Mito-CP和Mito-B）和CYP 2 E1抑制剂（Mito-diallylsulfide，Mito-D）的组合来记录mt-CYP 2 E1在毒性中的作用。2)Aim 1的体外数据将通过使用最近开发的表达CYP 2 E1人类变体形式（L32 N和W23/30 R）的人源化小鼠模型的体内研究进行补充，目的是建立人类遗传易感性与酒精肝毒性的联系。的 将研究Mito-CP、Mito-B和Mito-D减轻或逆转酒精毒性的能力。将分析酒精中毒患者的肝脏，以将mtCYP 2 E1含量与疾病程度和进展速度相关联。3)使用iPSC肝细胞和人源化小鼠模型，我们将确定酒精介导的线粒体PKA活化和LON蛋白酶介导的CcO、ALDH 2和mtTFA蛋白降解的机制。这些结果应该记录在各种疾病条件下线粒体ROS产生的新机制以及预防或减轻应激条件的有效手段。