Molecular Structure and Regulation of the Permeability Transition Pore

描述（由申请人提供）：线粒体凭借其在能量代谢、细胞 Ca2 稳态调节和细胞凋亡中的作用，在细胞存活和组织发育中发挥着关键作用。鉴于这种多因素作用，细胞功能的这些方面必须作为一个集成系统运行。因此，线粒体 Ca2 稳态必须受到严格调节，并且基于一系列特定的摄取和释放系统。然而，通过调节 IMM 孔（线粒体通透性转变孔 (PTP)）的开放，线粒体可以轻松地对相对较大的溶质进行线粒体内膜 (IMM) 通透性增加，称为通透性转变 (PT)。关于 PTP 的功能特性有大量信息，PTP 的病理激活可能对线粒体功能产生巨大的影响。因此，长期以来人们都知道 PTP 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发挥着关键作用。然而，尽管过去 30 年进行了详细的功能表征，传统 PTP 模型中的候选孔隙成分都没有经受住关键的基因测试。从本质上讲，我们对形成和调节 PTP 的分子成分知之甚少。该提案的总体目标是利用我们开发的药理学、生化和遗传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提高我们定义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 模型中唯一经过生化鉴定且尚未经过严格基因测试的成分。该目的的目的是通过有条件消除 PBR 表达来应用基因测试来检测 PBR 参与 PTP 的形成或调节。 2) 生化数据表明，CyPD 以高亲和力与一组有限的 IMM 位点结合，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采用 CyPD 分子来鉴定 PTP 的成分，然后对其作用进行严格测试。 3) 在高等生物中，ShcA 的 p66 同工型的很大一部分定位于线粒体，在那里它与细胞色素结合。 c 并充当氧化还原酶，在产生活性氧 (ROS) 的过程中将电子从细胞色素 c 穿梭至分子氧。由于 ROS 是 PTP 的有效诱导剂，因此有人提出 PTP 构成细胞凋亡途径激活中线粒体 p66 作用的直接下游靶标。这一目标的目标是利用我们掌握的遗传和分子工具来定义 p66 依赖性途径和 PTP 之间的确切关系，这一联系尚未得到严格确立。公共健康相关性：线粒体通透性转变孔的研究已有 50 多年的历史，并且与心脏和大脑的缺血再灌注损伤、VI 型胶原蛋白缺乏引起的肌营养不良以及 MS 期间发生的轴突损伤等许多其他病理状况有关。由于对 PTP 的分子组成知之甚少，我们在此应用中的目标是使用我们建立的药理学、生化和遗传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孔活性的调节剂。由于 PTP 与各种人类病理状况直接相关，因此我们预计，对形成或调节 PTP 形成的蛋白质进行严格和仔细的鉴定将提高我们定义针对这些蛋白质的治疗方法的能力，以治疗多种人类疾病。

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.