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: 9404927
• 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/9404927
• 关键词: Adrenodoxin; Affect; Alcohol consumption; Alcoholic Liver Diseases; Alcoholic liver damage; Alcohols; Animal Experiments; Antioxidants; Applications Grants; Biological Markers; 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; 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; Oxidases; Oxidative Stress; Oxidative Stress Induction; PKA inhibitor; Pancreatitis; Pathology; Patients; Peptide Signal Sequences; Pharmaceutical Preparations; Phosphorylation Site; Play; Post-Translational Protein Processing; Production; Productivity; Protein Analysis; 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; clinical development; clinically significant; coronary fibrosis; cost; cytochrome c oxidase; drug metabolism; endopeptidase La; enzyme activity; global health; health management; 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; prevent; problem drinker; promoter; protein degradation; public health relevance; 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(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，如果不治疗，可能会导致肝癌。CYP2E1是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质的氧化损伤是酒精性肝病(ALD)的致病因素。本研究的主要目的是探讨酒精所致线粒体功能障碍的机制，并开发抗氧化剂和酶抑制剂以减少酒精所致的肝损伤。最近的研究表明，线粒体靶向的CYP2E1(mt-CYP2E1)比内质网靶向的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含量比率的扭曲。结果强烈表明，携带更强大的mt-靶向CYP2E1突变的携带者更容易发生酒精中毒。我们的合作研究还表明，细胞色素C氧化酶(CcO)、线粒体ALDH2(乙醛脱氢酶)和mtTFA(线粒体转录因子A)的稳定水平是mt-CYP2E1增强酒精损伤的关键靶点。目前的双重研究建议是为了验证一个主要假设，即人mt-CYP2E1在增强ALD中起关键作用，CcO、ALDH2和mtTFA是一些直接靶点，如以下目标所概述：1)我们建议使用IPSC(诱导多能干细胞)技术生成的肝细胞来确定mtCYP2E1的代谢活性在酒精毒性和诱导氧化应激中的作用。我们将结合使用转基因肝细胞、最先进的ROS检测系统和mt靶向抗氧化剂(Mito-CP和Mito-B)和CYP2E1抑制剂(Mito-二烯丙基硫化物，Mito-D)来记录mt-CYP2E1在毒性中的作用。2)Aim1的体外数据将得到体内研究的补充，该研究使用最近开发的表达人类变异形式的CYP2E1(L32N和W23/30R)的人源化小鼠模型，目的是建立人类对酒精肝毒性的遗传易感性之间的联系。这个 将研究Mito-CP、Mito-B和Mito-D减轻或逆转酒精毒性的能力。来自酒精中毒患者的肝脏将被分析，以将mtCYP2E1含量与疾病的程度和进展速度联系起来。3)利用IPSC肝细胞和人源化小鼠模型，研究酒精介导的线粒体PKA活化机制，以及LON蛋白酶介导的CcO、ALDH2和mtTFA蛋白降解机制。这些结果应该证明线粒体在各种疾病条件下产生ROS的新机制，以及预防或缓解应激条件的有效手段。