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: 7900513
• 负责人: NARAYAN G AVADHANI
• 金额: $ 35.08万
• 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/7900513
• 关键词: Affect; Alcohols; Animal Model; Antioxidants; Attenuated; Biogenesis; COS Cells; 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; 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; 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 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）和可诱导的血红素氧酶1（HO-1）和许多其他蛋白质除了其建立良好的ER或细胞质目的地外，还针对线粒体。 PI实验室的工作导致发现了一个新的“嵌合”信号家族，该家族指导CYP，GST和许多其他信号分子的双峰靶向到一个以上的亚细胞隔室。线粒体靶向的CYP2E1和HO-1诱导氧化应激，细胞色素C氧化酶和其他复合物的活性降低，以及酵母中诱导的Rho零表型。人肝库的药物遗传学筛查显示MT-CYP2E1水平的个体间变化，这可能与影响蛋白质靶向效率的突变有关。 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.将在稳定的细胞系中研究乙醇介导的ROS产生和MT功能，主要表达MT或主要是MC靶向的酶，并且将测试CYP2E1和HO-1的特定抑制剂在减弱损伤中的特定抑制剂。我们将使用新开发的线粒体特异性线粒体 - 脱离EPR探针以及荧光探针进行ROS测量。将研究用酒精治疗的细胞中的线粒体氧化应激，mtDNA损伤和功能参数。在平行的酵母表达系统中，将用于评估细胞毒性。 2）研究来自酒精和非酒精性患者的人体组织库中MT-CYP2E1和MT-HO-1的水平。我们将研究这些患者样本中CCO，mtDNA完整性和MT功能的结构/功能。我们还将分析编码两种蛋白质信号区域的mRNA的5'或3端，以鉴定具有MT靶向效率改变的人类变体，以了解这些蛋白质蛋白在肝脏和心脏组织中这些蛋白质中个体含量的遗传基础的遗传基础，并评估MT菌株损伤和酒精培养基培养基的任何腐蚀性。 3）评估CYP2E1的线粒体靶向抗氧化剂，MITO-Q和抑制剂的能力，以及HO-1在两个代谢上不同的组织（肝脏和心脏）中衰减酒精介导的病变的能力。我们将研究酒精治疗或酒精 + LPS治疗的大鼠的肝病变，MT功能，心肌缺血/再灌注损伤。另一个目的是以靶向MT靶向的肽的形式开发分子干预措施，这些肽旨在干扰MT-CYP2E1，HO-1的催化活性，以减少或预防酒精组织损伤。心肌缺血/再灌注将使用Langendorff灌注系统进行。这些结果应提供有价值的新见解，除了酒精诱导的线粒体毒性和组织损伤之外，还可以帮助确定发展治疗干预措施的重要分子靶标。该应用的目的是使用细胞和动物模型对双峰靶向CYP2E1和HO-1在酒精诱导肝脏和心脏的毒性中的作用进行研究。将研究CYP2E1和HO-1的人类遗传变异形式，其线粒体靶向大量增加，以了解酒精诱导毒性的个体间变异的遗传基础。拟议的研究项目基于以下假设：线粒体靶向的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。