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

描述(申请人提供)：细胞色素P4502E1(细胞色素P4502E1)和诱导型血红素加氧酶1(HO-1)和其他一些蛋白质除了其已确定的内质网或细胞质目的地外，还以双峰方式靶向线粒体。PI实验室的工作导致了一系列新的“嵌合”信号的发现，这种信号将Cyps、GST和许多其他信号分子的双峰靶向指向不止一个亚细胞室。线粒体靶向的CYP2E1和HO-1在酵母中诱导氧化应激，降低细胞色素C氧化酶等复合体的活性，并诱导Rho零表型。人类肝库的药物遗传学筛查显示，mt-CYP2E1水平在个体之间存在差异，这可能与影响蛋白质靶向效率的突变有关。本研究的目的是通过多管齐下的方法，对线粒体中增加的细胞色素C氧化酶和HO-1的输入以及这些蛋白在酒精所致的肝脏和心脏损伤中的作用展开新的研究：1)研究线粒体中的细胞色素C氧化酶和其他电子传递复合体的结构/功能在ROS产生中的作用。乙醇介导的ROS产生和mt功能障碍将在稳定表达mt或mc靶向酶的细胞系中进行研究，并将测试特定的CYP2E1和HO-1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的mito-DEPMPO EPR探针和荧光探针来测量ROS。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)检测酒精中毒者和非酒精性中毒者肝脏和心脏组织中mt-CYP2E1和mt-HO-1的表达水平。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质信号区的mRNAs的5‘或3’端，以确定具有mt靶向效率改变的人类变异，了解这些蛋白质在肝脏和心脏组织中mt含量个体间差异的遗传基础，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)评估线粒体靶向抗氧化剂Mito-Q以及CYP2E1和HO-1抑制剂在减轻肝脏和心脏两种不同代谢组织中酒精介导的损伤的能力。我们将研究酒精处理或酒精+脂多糖处理的大鼠的肝脏损伤、线粒体功能障碍、心肌缺血/再灌注损伤。另一个目标是开发mt靶向多肽形式的分子干预，旨在干扰mt-CYP2E1，HO-1的催化活性，以期减少或防止酒精组织损伤。心肌缺血/再灌注将使用朗宁多夫灌流系统进行。这些结果应该为酒精诱导的线粒体毒性和组织损伤的遗传学基础提供有价值的新见解，此外还有助于确定重要的分子靶点，以开发治疗干预措施。本应用的目的是利用细胞和动物模型研究双峰靶向的细胞色素P450-2和环氧合酶-1在酒精对肝脏和心脏毒性中的作用。将对线粒体靶向性大大增加的人类CYP2E1和HO-1的遗传变异形式进行研究，以了解酒精诱导的毒性个体间差异的遗传基础。提出的研究项目是基于这样的假设，即线粒体靶向的CYP2E1和HO-1参与了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.