Molecular Structure and Regulation of the Permeability Transition Pore

渗透率转变孔的分子结构和调控

• 批准号: 7729756
• 负责人: MICHAEL A FORTE
• 金额: $ 34.15万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7729756
• 关键词: Affinity; Affinity Chromatography; Alzheimer' s Disease; Apoptosis; Apoptotic; Applied Genetics; Benzodiazepine Receptor; Binding; Biochemical; Biochemical Genetics; Bioenergetics; Brain; Cell Membrane Permeability; Cell Respiration; Cell Survival; Cell physiology; Cells; Collagen; Cyclosporine; DBL Oncoprotein; Data; Development; Electrons; Energy Metabolism; Event; Funding; Genetic screening method; Goals; Heart; Homeostasis; Human; Hyperactive behavior; In Vitro; Inner mitochondrial membrane; Ions; Knockout Mice; Laboratories; Lead; Link; Membrane; Metabolism; Mitochondria; Modeling; Molecular; Molecular Genetics; Molecular Structure; Mus; Muscular Dystrophies; Nerve Degeneration; Neurons; Organism; Oxidoreductase; Oxygen; Pathway interactions; Peripheral; Permeability; Physiology; Play; Property; Protein Isoforms; Proteins; Protons; Reactive Oxygen Species; Reagent; Regulation; Reperfusion Injury; Role; Scheme; Series; Signal Transduction; Site; System; Testing; Tissues; base; cell growth regulation; experience; extracellular; genetic analysis; human disease; in vivo; mitochondrial dysfunction; mitochondrial membrane; mitochondrial permeability transition pore; molecular mass; novel strategies; protein complex; public health relevance; pyridine nucleotide; receptor expression; respiratory; response; solute; tool; uptake; voltage

DESCRIPTION (provided by applicant): Mitochondria play a pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2+ homeostasis and apoptosis. Given this multifactorial role, these aspects of cellular function must operate as an integrated system. Consequently, mitochondrial Ca2+ homeostasis must be tightly regulated and is based in a series of specific uptake and release systems. Yet, mitochondria can easily undergo an inner mitochondrial membrane (IMM) permeability increase to relatively large solutes called the permeability transition (PT), through the regulated opening of an IMM pore, the mitochondrial permeability transition pore (PTP). A great deal of information is available about the functional properties of the PTP and pathological activation of the PTP can have dramatic consequences on mitochondrial function. As a result, the PTP has long been known to play a key role in mitochondrial dysfunction associated with human pathological events such as ischemia-reperfusion injury and neurodegeneration. However, despite detailed functional characterization over the last 30 years, none of the candidate pore components in traditional models of the PTP has withstood critical genetic tests. In essence then, we have a remarkably poor understanding of the molecular components forming and regulating the PTP. The overall goal of this proposal is to use the pharmacological, biochemical and genetic tools that we have developed for the unbiased identification of proteins involved in the formation of the PTP, followed by a variety of tests to confirm their roles in PTP activity. Since the PTP has been demonstrated to play a critical role in variety of human diseases, we anticipate that the rigorous identification of proteins forming or regulating the formation of the PTP increases our ability to define therapies targeting this important complex of proteins. Our specific plans include: 1) The peripheral benzodiazepine receptor (PBR), which remains the only biochemically identified component included in traditional models of the PTP that has not been subjected to rigorous genetic testing. The goal of this aim is to apply genetic tests of the involvement of the PBR in the formation or regulation of the PTP through conditional elimination of PBR expression. 2) Biochemical data indicate that CyPD binds with high affinity to a limited set of IMM sites and genetic analysis has demonstrated that it is a key regulator of the PTP. Consequently, the goal of this aim is to employ the CyPD molecule in tandem affinity purification strategies for the identification of components of the PTP, followed by rigorous testing of their roles. 3) In higher organisms, a significant fraction of the p66 isoform of ShcA is localized to mitochondria where it binds cyt. c and acts as an oxidoreductase, shuttling electrons from cyt c to molecular oxygen in the creation of reactive oxygen species (ROS). Since ROS are potent inducers of the PTP, the PTP has been proposed to constitute the immediate downstream target of mitochondrial p66 action in the activation of apoptotic pathways. The goal of this aim will be to use of the genetic and molecular tools at our disposal to define the exact relationship between p66-dependent pathways and the PTP, a link that has yet to be critically established. PUBLIC HEALTH RELEVANCE: The mitochondrial permeability transition pore has been studied for over 50 years and has been implicated, for example, in ischemia-reperfusion injury of the heart and brain, muscular dystrophy caused by collagen VI deficiency, and in the axonal damage occurring during MS among many other pathological conditions. Since little is known of the molecular composition of the PTP, our goals in this application are to use the pharmacological, biochemical and genetic tools we have established for the unbiased identification of proteins involved in the formation of the PTP and to use a variety of in vitro and in vivo tests to confirm their roles, either as core components of the pore itself, or regulators of pore activity. Since the PTP is of direct relevance to variety of human pathological conditions, we anticipate that the rigorous and careful identification of proteins forming or regulating the formation of the PTP will increase our ability to define therapies targeting these proteins as treatments for a wide variety of human diseases.

描述（由申请人提供）：线粒体由于其在能量代谢、细胞Ca 2+稳态调节和细胞凋亡中的作用，在细胞存活和组织发育中起关键作用。鉴于这种多因素作用，细胞功能的这些方面必须作为一个综合系统运作。因此，线粒体Ca 2+稳态必须严格调节，并基于一系列特定的摄取和释放系统。然而，线粒体可以容易地经历线粒体内膜（IMM）的渗透性增加，通过IMM孔（线粒体渗透性转换孔（PTP））的调节打开，相对较大的溶质被称为渗透性转换（PT）。关于PTP的功能特性的大量信息是可用的，并且PTP的病理激活可以对线粒体功能产生显著的后果。因此，PTP长期以来被认为在与人类病理事件如缺血-再灌注损伤和神经变性相关的线粒体功能障碍中起关键作用。然而，尽管在过去的30年中详细的功能表征，没有一个候选孔组件在传统模型的PTP经受住了关键的基因测试。从本质上讲，我们对形成和调节PTP的分子组分的理解非常贫乏。该提案的总体目标是使用我们开发的药理学，生物化学和遗传学工具，用于无偏见地鉴定参与PTP形成的蛋白质，然后进行各种测试以确认它们在PTP活性中的作用。由于PTP已被证明在各种人类疾病中发挥关键作用，我们预计，对形成或调节PTP形成的蛋白质的严格鉴定将提高我们定义靶向这种重要蛋白质复合物的疗法的能力。我们的具体计划包括：1）外周苯二氮卓受体（PBR），它仍然是PTP传统模型中唯一未经严格基因检测的生化鉴定成分。该目的的目的是通过条件性消除PBR表达来应用PBR参与PTP形成或调节的遗传测试。2)生物化学数据表明，CyPD以高亲和力结合到一组有限的IMM位点，遗传分析表明它是PTP的关键调节因子。因此，该目标的目标是在串联亲和纯化策略中使用CyPD分子来鉴定PTP的组分，然后严格测试它们的作用。3)在高等生物体中，ShcA的p66亚型的显著部分定位于线粒体，在那里它结合cyt。作为一种氧化还原酶，在产生活性氧（ROS）的过程中将电子从细胞色素c穿梭到分子氧。由于ROS是PTP的有效诱导剂，因此PTP已被提出构成线粒体p66在细胞凋亡途径活化中作用的直接下游靶标。这一目标的目标将是利用我们掌握的遗传和分子工具来确定p66依赖性途径和PTP之间的确切关系，这一联系尚未得到关键性的建立。公共卫生相关性：线粒体渗透性转换孔已经被研究了超过50年，并且已经涉及例如心脏和脑的缺血-再灌注损伤、由胶原VI缺乏引起的肌营养不良症、以及在MS期间发生的轴突损伤以及许多其他病理状况。由于对PTP的分子组成知之甚少，我们在本申请中的目标是使用我们已经建立的药理学、生物化学和遗传学工具来无偏地鉴定参与PTP形成的蛋白质，并使用各种体外和体内试验来确认它们的作用，无论是作为孔本身的核心组分，还是孔活性的调节剂。由于PTP与各种人类病理状况直接相关，我们预期严格和仔细鉴定形成或调节PTP形成的蛋白质将提高我们将靶向这些蛋白质的疗法定义为多种人类疾病的治疗的能力。