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

描述（由申请人提供）：细胞色素P4502 E1（CYP 2 E1）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，还以双峰方式靶向线粒体。PI实验室的工作导致了一个新的“嵌合”信号家族的发现，该家族将CYP、GST和许多其他信号分子的双峰靶向引导到一个以上的亚细胞区室。线粒体靶向的CYP 2 E1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。人肝库的药物遗传学筛选显示mt-CYP 2 E1水平的个体间差异，这可能与影响蛋白质靶向效率的突变相关。本申请的目的是使用多管齐下的方法启动关于CYP 2 E1和HO-1的线粒体输入增加的遗传基础以及这些蛋白质在酒精介导的肝脏和心脏损伤中的作用的新研究：1）研究线粒体CYP 2 E1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转移复合物的结构/功能。将在主要表达mt靶向酶或主要表达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，并将检测CYP 2 E1和HO-1特异性抑制剂在减轻损伤中的作用。我们将使用新开发的线粒体特异性DEPMPO EPR探针和荧光探针进行ROS测量。将研究酒精处理细胞的线粒体氧化应激、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)检测酒精性和非酒精性患者肝脏和心脏组织中线粒体CYP 2 E1和线粒体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。我们还将分析编码两种蛋白质的信号区的mRNA的5'或3'末端，以鉴定具有改变的mt靶向效率的人类变体，以了解肝脏和心脏组织中这些蛋白质的mt含量的个体间变异的遗传基础，并评估mt蛋白水平与酒精介导的组织损伤之间的任何相关性。 3)评估靶向抗氧化剂Mito-Q和CYP 2 E1和HO-1抑制剂在两种代谢不同的组织（肝脏和心脏）中减弱酒精介导的病变的能力。我们将研究酒精处理或酒精+ LPS处理的大鼠的肝脏病变，线粒体功能障碍，心肌缺血/再灌注损伤。另一个目的是开发以mt靶向肽形式的分子干预，其被设计为干扰mt-CYP 2 E1，HO-1的催化活性，以减少或预防酒精性组织损伤。将使用Langendorff灌注系统进行心肌缺血/再灌注。这些结果将为酒精诱导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。本申请的目的是使用细胞和动物模型启动关于双峰靶向CYP 2 E1和HO-1在酒精诱导的肝脏和心脏毒性中的作用的研究。将研究线粒体靶向大幅增加的CYP 2 E1和HO-1的人类遗传变异形式，以了解酒精诱导毒性个体间变异的遗传基础。拟议的研究项目是基于这样的假设，即细胞靶向的CYP 2 E1和HO-1有助于ROS的产生，氧化应激和线粒体功能障碍是导致组织损伤的重要因素。

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.