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

基本信息

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
项目类别：
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

项目摘要

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）：导致酒精肝病的过度饮酒是全球一个主要的健康问题，如果不治疗，可能会导致肝癌。 CYP2E1是涉及酒精代谢的两种主要酶之一，可能通过诱导氧化组织损伤而导致酒精肝损伤。最近的研究表明，线粒体DNA/膜损伤和对线粒体蛋白的氧化损伤是酒精肝病（ALD）的促成因素。这里的一个主要目的是研究酒精诱导的线粒体功能障碍的机制，并产生抗氧化剂和酶抑制剂，以最大程度地减少酒精诱导的肝损伤。最近的研究表明，线粒体靶向CYP2E1（MT-CYP2E1）比靶向的CYP2E1（MC-CYP2E1）调节更严重的酒精毒性。人肝样品的分析还鉴定了CYP2E1的独特人类变体形式，其亚细胞靶向改变，从而导致MT-CYP2E1：MC-CYP2E1含量率偏斜。结果强烈表明，更健壮的MT靶向CYP2E1突变更容易引起酒精诱导的毒性。我们的协作研究还表明，细胞色素C氧化酶（CCO），线粒体ALDH2（醛脱氢酶）和MTTFA（线粒体转录因子A）是MT-CYP2E1-抑制酒精损害的关键靶标。目前的双重研究者的建议是测试主要的假设，即人MT-CYP2E1在增强ALD中起着至关重要的作用，并且CCO，CCO，ALDH2和MTTFA是以下目的概述的一些直接靶标：1）我们建议使用IPSC生成的hepatocyte（启用plulipot plulipot plulipot plulipot and pluripot plulipot cy）（定义plulipot plulipot cy）（ MTCYP2E1在酒精毒性和诱导氧化应激的诱导中。我们将结合遗传修饰的肝细胞，最先进的ROS检测系统以及MT靶向的抗氧化剂（Mito-CP和Mito-B）和CYP2E1抑制剂（Mito-Dallylsulfide，Mito-D），以记录MT-CYP2E1在毒性中的作用。 2）AIM1中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人性化小鼠模型表达了人类变异形式的CYP2E1（L32N和W23/30R），目的是建立人类遗传易感性对酒精肝毒性的联系。这将研究MITO-CP，MITO-B和MITO-D减轻或逆转酒精毒性的能力。将分析来自酒精患者的肝脏，以将MTCYP2E1含量与疾病和进展率相关。 3）使用IPSC肝细胞和人源化小鼠模型，我们将定义酒精介导的线粒体PKA激活的机制，以及LON蛋白酶介导的CCO，ALDH2和MTTFA蛋白的降解。这些结果应记录在各种疾病条件下线粒体ROS产生的新机制，以及预防或减轻压力条件的有效手段。