Mechanisms and Functions of Bimodally Targeted Cytochrome P450s to Mitochondria

双峰靶向细胞色素 P450 线粒体的机制和功能

• 批准号: 7934368
• 负责人: NARAYAN G AVADHANI
• 金额: $ 25.43万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934368
• 关键词: A549; Affinity; All-Trans-Retinol; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animal Model; Arts; Binding; Biogenesis; Biological Assay; CYP1B1 gene; CYP2D6 gene; CYP2E1 gene; Cardiac Myocytes; Cells; Chemicals; Chlorpromazine; Cyclic AMP-Dependent Protein Kinases; Cytochromes; Cytosol; DNA Damage; Data; Destinations; Drug or Chemical; Endoplasmic Reticulum; Epithelial Cells; Estrogen Receptors; Family; Ferredoxin-NADP Reductase; Funding; Genes; Genetic Transcription; Glucocorticoid Receptor; Glutathione S-Transferase; Grant; Heart; Hormone Receptor; Hydrogen Peroxide; Hypoxia; Injury; Ions; Ischemia; Knock-out; Knockout Mice; Laboratories; Lung; Measures; Mediating; Metabolism; Mitochondria; Mitochondrial DNA; Molecular Conformation; Monoamine Oxidase; Mus; Myocardial Ischemia; N-terminal; NADH dehydrogenase (ubiquinone); Neurotoxins; Nuclear Hormone Receptors; Oxidative Stress; Oxidoreductase; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Pharmaceutical Preparations; Phosphorylation; Physiological; Play; Process; Production; Proteins; Publishing; Regulation; Reperfusion Injury; Research; Role; Serine; Signal Transduction; Site; Stimulus; Stress; Stress-Induced Protein; Superoxides; System; TP53 gene; Tamoxifen; Tissues; Toxic effect; Transcript; Transcription Initiation; Work; Xenobiotics; base; biological adaptation to stress; cholanthrene; drug metabolism; electron donor; heme oxygenase-1; human TFRC protein; mitochondrial dysfunction; mouse model; novel; numb protein; preference; public health relevance; receptor; response; stable cell line; toxicant

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.

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-CYP）。 PI 实验室的工作发现了一种新的“嵌合”信号家族，该信号将 CYP 和许多核激素受体双模式靶向多个亚细胞区室。已经描述了激活神秘线粒体靶向信号的两种主要机制：1）胞质内切蛋白酶的 N 末端加工，2）PKA 或 PKC 介导的磷酸化信号激活。当前资助期间的工作已经对细胞质内切蛋白酶进行了表征，该酶可激活大量蛋白质的 mt 靶向信号。结果还表明mt-CYP2D6可能具有神经毒素MPTP和其他药物的代谢活性。此外，缺氧和心肌缺血会在心肌细胞中诱导 mt-CYP1B1 和 CYP2C6。本提案的目的是启动关于 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) 定义异生素诱导的胞质内切蛋白酶在调节 GR、RxR1、ER2 和其他蛋白质等 mt 生物合成和 mt 功能蛋白的 mt 靶向中的作用。将研究这些应激诱导蛋白对 mt 转录、mtDNA 包装、DNA 损伤和 ROS 产生的作用。研究BNF、Dex和缺氧诱导p90（多聚酶1）基因的机制。 2) 研究mt-CYP2D6在布呋洛尔、他莫昔芬、氯丙嗪等药物和MPTP等毒物代谢中的作用。主要表达 mt-CYP2D6 或 mc-CYP2D6 的稳定细胞系，结合选择性沉默 CYP 还原酶、肾上腺氧还蛋白 (Adx)、MAO 敲除和 CYPR 敲除小鼠模型，将用于研究 mt-CYP2D6 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mt靶向的CYP1B1、CYP2C6和其他CYP在缺氧的H9C2心肌细胞和缺血的小鼠心脏中的作用，以及这些CYP在ROS产生和mt毒性中的作用。新型线粒体靶向 EPR 探针 Mito-DEPMPO（DEPMPO 与三苯基鏻离子缀合）和 Mito-DHE 将用于测量线粒体产生的 ROS，并将生成 mt 靶向 Adx 肽，用于选择性抑制整个细胞和组织中的 mt-CYP。公共健康相关性：该提案的重点是阐明异生物质诱导细胞毒性和氧化或化学损伤的机制。该提案旨在利用最先进的检测系统阐明靶向 CYP2D6、2C6 和 1B1 的线粒体以及许多外源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。最后，将使用细胞和动物模型研究线粒体靶向 CYP 在缺血再灌注损伤中的作用。