Role of Mitochondria Targeted CYP2E1 and HO-1 in Alcohol Mediated Tissue Injury

线粒体靶向 CYP2E1 和 HO-1 在酒精介导的组织损伤中的作用

• 批准号: 8119383
• 负责人: NARAYAN G AVADHANI
• 金额: $ 33.72万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8119383
• 关键词: Affect; Alcohols; Animal Model; Antioxidants; Attenuated; Biogenesis; COS Cells; Cell model; Cells; Complex; Cytochromes; Data; Destinations; Ectopic Expression; Electron Transport; Electrons; Enzymes; Ethanol; Family; Fluorescent Probes; Functional disorder; Genetic; Heart; Heart Injuries; Hemeproteins; Hepatic; Human; Human Genetics; Hypoxia; In Vitro; Individual; Injury; Intervention; Laboratories; Lesion; Lipid Peroxides; Literature; Liver; Mammalian Cell; Measurement; Mediating; Membrane; Messenger RNA; Microsomes; Mitochondria; Mitochondrial DNA; Molecular; Molecular Target; Mutation; Myocardial; Myocardial Ischemia; Oxidases; Oxidative Stress; Pathology; Patients; Peptide Signal Sequences; Peptides; Perfusion; Pharmacogenetics; Phenotype; Play; Post-Translational Protein Processing; Production; Proteins; Rattus; Reperfusion Injury; Reperfusion Therapy; Research Project Grants; Role; Sampling; Signal Transduction; Signaling Molecule; Structure; System; Testing; Therapeutic Intervention; Tissue Banking; Tissue Banks; Tissues; Toxic effect; Variant; Work; Yeasts; alcohol abuse therapy; alcohol measurement; alcohol risk; base; cytochrome P-450 CYP3A1 (rat); cytochrome c oxidase; design; genetic variant; heme a; heme oxygenase-1; human tissue; in vivo; inhibitor/antagonist; insight; mitochondrial dysfunction; non-alcoholic; oxidative damage; prevent; problem drinker; prognostic; protein complex; respiratory; rho; stable cell line; therapeutic target; tool

DESCRIPTION (provided by applicant): Cytochrome P4502E1 (CYP2E1) and inducible heme oxygenase 1 (HO-1) and a number of other proteins are bimodally targeted to mitochondria in addition to their well-established ER or cytoplasmic destination. Work in the PI's laboratory has led to the discovery of a new family of "chimeric" signals, which direct the bimodal targeting of CYPs, GSTs, and a number of other signaling molecules to more than one subcellular compartment. Mitochondria-targeted CYP2E1 and HO-1 induced oxidative stress, reduced activity of cytochrome c oxidase and other complexes and induced rho zero phenotypes in yeast. A pharmacogenetic screen of human liver bank showed inter-individual variations in mt-CYP2E1 levels which is likely to be associated with mutations that affect targeting efficiency of the protein. The objective of this application is to initiate new studies on the genetic basis for increased mitochondrial import of CYP2E1 and HO-1 and the role of these proteins in alcohol-mediated liver and heart injury using a multi-prong approach: 1) Investigate the mechanistic roles of mitochondrial CYP2E1 and HO-1 in ROS production, modulating the structure/function of cytochrome c oxidase and also other electron transfer complexes. Ethanol mediated ROS production and mt-dysfunction will be studied in stable cell lines expressing predominantly mt- or predominantly mc-targeted enzymes, and the effects of specific inhibitors of CYP2E1 and HO-1 in attenuating the damage will be tested. We will use a newly developed mitochondria-specific mito-DEPMPO EPR probe and also fluorescent probes for ROS measurement. Mitochondrial oxidative stress, mtDNA damage and functional parameters in cells treated with alcohol will be studied. In parallel yeast expression system will be used to assess cell toxicity. 2) Investigate the levels of mt-CYP2E1, and mt-HO-1 in the livers and hearts of human tissue bank from alcoholic and non-alcoholic patients. We will investigate the structure/function of CcO, mtDNA integrity and mt function in these patient samples. We will also analyze the 5' or 3' ends of mRNAs encoding the signal regions of the two proteins to identify human variants with altered mt-targeting efficiency to understand the genetic basis of inter-individual variations in mt contents of these proteins in the liver and heart tissues and to assess any correlation between mt-protein levels and alcohol mediated tissue injury. 3) Evaluate the ability of mitochondria-targeted antioxidant, Mito-Q, and inhibitors of CYP2E1, and HO-1 in attenuating alcohol-mediated lesions in two metabolically distinct tissues, the liver and heart. We will study hepatic lesions, mt-dysfunction, myocardial ischemia/reperfusion injury in alcohol-treated or alcohol + LPS treated rats. Another objective is to develop molecular interventions in the form of mt-targeted peptides that are designed to interfere with the catalytic activities of mt-CYP2E1, HO-1 with a view to reduce or prevent alcoholic tissue damage. Myocardial ischemia/reperfusion will be carried out using Langendorff perfusion system. These results should provide valuable new insights on genetic basis of alcohol induced mitochondrial toxicity and tissue damage, in addition to help identifying important molecular targets for developing therapeutic interventions. The objective of this application is to initiate studies on the role of bimodally targeted CYP2E1 and HO-1 in alcohol induced toxicity to the liver and heart using cell and animal models. Human genetic variant forms of CYP2E1 and HO-1 with vastly increased mitochondrial targeting will be investigated to understand the genetic basis for inter-individual variations in alcohol induced toxicity. The proposed research project is based on the hypothesis that mitochondrially targeted CYP2E1 and HO-1 contribute to ROS production, oxidative stress and mitochondrial dysfunction as important factors leading to tissue injury.

描述（由申请人提供）：细胞色素 P4502E1 (CYP2E1) 和诱导型血红素加氧酶 1 (HO-1) 以及许多其他蛋白质除了其公认的 ER 或细胞质目的地外，还双峰靶向线粒体。 PI 实验室的工作发现了一种新的“嵌合”信号家族，该家族将 CYP、GST 和许多其他信号分子的双模式靶向引导至多个亚细胞区室。线粒体靶向 CYP2E1 和 HO-1 诱导氧化应激，降低细胞色素 C 氧化酶和其他复合物的活性，并诱导酵母中的 rho 零表型。人类肝库的药物遗传学筛查显示 mt-CYP2E1 水平存在个体间差异，这可能与影响蛋白质靶向效率的突变有关。本申请的目的是使用多管齐下的方法启动关于增加线粒体输入 CYP2E1 和 HO-1 的遗传基础以及这些蛋白质在酒精介导的肝和心脏损伤中的作用的新研究：1) 研究线粒体 CYP2E1 和 HO-1 在 ROS 产生中的机制作用，调节细胞色素 c 氧化酶以及其他电子传递复合物的结构/功能。将在主要表达 mt 或主要 mc 靶向酶的稳定细胞系中研究乙醇介导的 ROS 产生和 mt 功能障碍，并将测试 CYP2E1 和 HO-1 的特定抑制剂在减轻损伤方面的作用。我们将使用新开发的线粒体特异性 mito-DEPMPO EPR 探针以及用于 ROS 测量的荧光探针。将研究酒精处理细胞中的线粒体氧化应激、线粒体 DNA 损伤和功能参数。同时酵母表达系统将用于评估细胞毒性。 2) 检测酒精和非酒精患者组织库肝脏和心脏中mt-CYP2E1和mt-HO-1的水平。我们将研究这些患者样本中 CcO 的结构/功能、mtDNA 完整性和 mt 功能。我们还将分析编码这两种蛋白信号区域的 mRNA 的 5' 或 3' 末端，以识别 mt 靶向效率改变的人类变体，了解肝脏和心脏组织中这些蛋白的 mt 含量个体间变异的遗传基础，并评估 mt 蛋白水平和酒精介导的组织损伤之间的相关性。 3) 评估线粒体靶向抗氧化剂 Mito-Q 以及 CYP2E1 和 HO-1 抑制剂在减轻两个代谢不同组织（肝脏和心脏）中酒精介导的病变的能力。我们将研究酒精治疗或酒精 + LPS 治疗的大鼠的肝脏病变、mt 功能障碍、心肌缺血/再灌注损伤。另一个目标是开发 mt 靶向肽形式的分子干预措施，旨在干扰 mt-CYP2E1、HO-1 的催化活性，以减少或预防酒精组织损伤。将使用Langendorff灌注系统进行心肌缺血/再灌注。这些结果应该为酒精引起的线粒体毒性和组织损伤的遗传基础提供有价值的新见解，此外还有助于确定开发治疗干预措施的重要分子靶点。本申请的目的是利用细胞和动物模型启动双峰靶向 CYP2E1 和 HO-1 在酒精诱导的肝脏和心脏毒性中的作用的研究。将研究具有显着增强的线粒体靶向性的 CYP2E1 和 HO-1 的人类遗传变异形式，以了解酒精引起的毒性的个体间变异的遗传基础。拟议的研究项目基于这样的假设：线粒体靶向 CYP2E1 和 HO-1 有助于 ROS 产生、氧化应激和线粒体功能障碍，是导致组织损伤的重要因素。