Molecular Structure and Regulation of the Permeability Transition Pore

描述（由申请人提供）：线粒体在细胞存活和组织发育中发挥关键作用，其作用包括能量代谢、细胞Ca2+稳态调节和细胞凋亡。考虑到这种多因素作用，细胞功能的这些方面必须作为一个综合系统来运作。因此，线粒体Ca2+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，线粒体可以很容易地经历一个线粒体内膜（IMM）通透性增加到相对较大的溶质，称为通透性过渡（PT），通过调节IMM孔的开放，线粒体通透性过渡孔（PTP）。关于PTP的功能特性有大量的信息，PTP的病理激活可以对线粒体功能产生巨大的影响。因此，人们早就知道PTP在与人类病理事件（如缺血再灌注损伤和神经退行性变）相关的线粒体功能障碍中发挥关键作用。然而，尽管在过去的30年里详细的功能表征，在PTP的传统模型中没有一个候选孔成分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的分子成分的理解非常贫乏。本提案的总体目标是利用我们开发的药理学，生化和遗传学工具来公正地鉴定参与PTP形成的蛋白质，然后进行各种测试来确认它们在PTP活性中的作用。由于PTP已被证明在多种人类疾病中发挥着关键作用，我们预计，对形成或调节PTP形成的蛋白质的严格鉴定将增加我们确定针对这一重要蛋白质复合物的治疗方法的能力。我们的具体计划包括：1)外周苯二氮卓受体（PBR），它仍然是传统PTP模型中唯一未经严格基因检测的生化鉴定成分。这一目的的目标是通过条件消除PBR表达，应用PBR参与PTP形成或调节的基因测试。2)生化数据表明CyPD与有限的IMM位点具有高亲和力结合，遗传分析表明它是PTP的关键调节因子。因此，本研究的目的是采用CyPD分子的串联亲和纯化策略来鉴定PTP的成分，然后对其作用进行严格的测试。3)在高等生物体中，ShcA的p66异构体的很大一部分定位于线粒体，并与细胞结合。它作为一种氧化还原酶，在生成活性氧（ROS）的过程中，将电子从cyt c传送到分子氧中。由于ROS是PTP的有效诱导剂，PTP被认为是线粒体p66激活凋亡通路的直接下游靶点。这一目标的目标将是利用我们掌握的遗传和分子工具来定义p66依赖途径和PTP之间的确切关系，这一联系尚未得到严格的建立。公共卫生相关性：线粒体通透性过渡孔已经被研究了50多年，并与心脏和大脑的缺血再灌注损伤、胶原VI缺乏引起的肌肉萎缩、多发性硬化症期间发生的轴突损伤以及许多其他病理状况有关。由于对PTP的分子组成知之甚少，我们在本应用中的目标是使用我们已经建立的药理学，生化和遗传学工具来公正地鉴定参与PTP形成的蛋白质，并使用各种体外和体内测试来确认它们的作用，无论是作为孔本身的核心成分，还是作为孔活性的调节剂。由于PTP与多种人类病理状况直接相关，我们预计对形成或调节PTP形成的蛋白质的严格和仔细的鉴定将增加我们确定针对这些蛋白质的治疗方法的能力，作为治疗多种人类疾病的方法。

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.