Mechanisms and Functions of Bimodally Targeted Cytochrome P450s to Mitochondria

描述（由申请人提供）：P450（CYP）和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（mt-tR）。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和许多核激素受体的双峰靶向引导到一个以上的亚细胞区室。已经描述了两种主要机制用于激活隐蔽线粒体靶向信号：1）通过胞质内蛋白酶的N-末端加工，和2）通过PKA或PKC介导的磷酸化的信号激活。在目前的资助期间的工作已经导致了细胞质内切蛋白酶的特性，激活大量蛋白质的mt靶向信号。结果还表明，mt-CYP 2D 6可能对神经毒素MPTP和其他药物的代谢具有活性。此外，心肌细胞中的mt-CYP 1B 1和CYP 2C 6被缺氧和心肌缺血诱导。该提案的目的是启动对mt-CYP和HO-1在药物代谢、ROS产生和细胞毒性中的病理生理学作用的研究：1）确定外源性诱导的胞质内蛋白酶在调节蛋白质如GR、RxR 1、ER 2和其他蛋白质对mt-生物发生和mt-功能的mt-靶向中的作用。这些胁迫诱导蛋白在线粒体转录、线粒体DNA包装、DNA损伤和活性氧产生中的作用将被研究。本研究将探讨BNF、Dex和缺氧诱导p90基因表达的机制。2)将研究mt-CYP 2D 6在药物如丁呋洛尔、他莫昔芬、氯丙嗪和毒物如MPTP代谢中的作用。主要表达mt-CYP 2D 6或mc-CYP 2D 6的稳定细胞系与选择性沉默α还原酶、肾上腺素还蛋白（Adx）和MAO敲除和CYPR敲除小鼠模型的组合将用于研究mt-CYP 2D 6在药物代谢、药物诱导的线粒体功能障碍中的作用。3)将研究线粒体靶向的CYP 1B 1、CYP 2C 6和其他CYP在缺氧的H9 C2心肌细胞和缺血的小鼠心脏中的作用，并研究这些CYP在ROS产生和线粒体毒性中的作用。新的线粒体靶向EPR探针Mito-DEPMPO（与三苯基鳞离子缀合的DEPMPO）和Mito-DHE将用于测量细胞内产生的ROS，并且将产生mt靶向Adx肽用于选择性抑制全细胞和组织中的mt-CYP。公共卫生相关性：该提案的重点是阐明机制，其中外源性物质诱导细胞毒性和氧化或化学损伤。该提案旨在阐明线粒体靶向CYP 2D 6、2C 6和1B 1以及许多外源性诱导激素受体在诱导线粒体功能障碍、mtDNA损伤和ROS产生中的作用，使用最先进的测定系统。最后，线粒体靶向CYP在缺血再灌注损伤中的作用将使用细胞和动物模型进行研究。

DESCRIPTION (provided by applicant): P450s (CYPs) and a number of other proteins are bimodally targeted to mitochondria (mt-CYP) 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 and a number of nuclear hormone receptors to more than one subcellular compartment. Two major mechanisms have been described for the activation of cryptic mitochondria targeting signals: 1) N-terminal processing by a cytosolic endoprotease, and 2) signal activation by PKA or PKC-mediated phosphorylation. Work during the current grant period has resulted in the characterization of cytoplasmic endoprotease which activates mt-targeting signals of a large number of proteins. Results also show that mt-CYP2D6 may have activity for the metabolism of neurotoxin MPTP and other drugs. Additionally mt-CYP1B1 and CYP2C6 are induced in cardiomyocytes by hypoxia and myocardial ischemia. The objective of this proposal is to initiate studies on pathophysiological roles of mt- CYPs and HO-1 in drug metabolism, ROS production, and cellular toxicity: 1) Define the role of xenobiotic-induced cytosolic endoprotease in modulating mt- targeting of proteins such as GR, RxR1, ER2, and other proteins on mt-biogenesis, and mt- function. Role of these stress induced proteins on mt transcription, mtDNA packaging, DNA damage and ROS production will be studied. Mechanism of induction of p90 (polyserase 1) gene by BNF, Dex and hypoxia will be studied. 2) The role of mt-CYP2D6 in the metabolism of drugs such as bufuralol, tamoxifen, chlorpromazine and toxicants such as MPTP will be investigated. Stable cell lines predominantly expressing either the mt-CYP2D6 or mc-CYP2D6 in combination with selective silencing of CYP reductase, adrenodoxin (Adx), and MAO knock out and CYPR knock out mouse models will be used to investigate the role of mt-CYP2D6 in drug metabolism, drug-induced mitochondrial dysfunction. 3) The role of mt-targeted CYP1B1, CYP2C6, and other CYPs in H9C2 cardiomyocytes subjected to hypoxia and mouse hearts subjected to ischemia will be investigated and the role of these CYPs in ROS production, and mt-toxicity will be studied. Novel mitochondria targeted EPR probe Mito-DEPMPO (DEPMPO conjugated to triphenylphosphonium ion) and Mito-DHE will be used for measuring mitochondrially produced ROS and mt-targeted Adx peptides will be generated for selective inhibition of mt- CYPs in whole cells and tissues. PUBLIC HEALTH RELEVANCE: The proposal is focused to elucidate mechanisms by which xenobiotic agents induce cellular toxicity and oxidative or chemical damage. The proposal is aimed to elucidate the role of mitochondria targeted CYP2D6, 2C6 and 1B1 and also a number of xenobiotic inducible hormone receptors in inducing mitochondrial dysfunction, mtDNA damage and ROS production using state of the art assay systems. Finally, the role of mitochondria targeted CYPs in ischemia reperfusion injury will be studied using cell and animal models.