Molecular Structure and Regulation of the Permeability Transition Pore

描述(由申请人提供)：线粒体通透性转换孔(PTP)的激活显然在一些最广泛传播和治疗挑战的人类疾病中起着关键作用。我们的研究已经证实，PTP有两种工作模式：1)短暂的，PTP作为线粒体的钙释放通道；2)持久的，最终导致细胞死亡和疾病。虽然在功能水平上有很好的特征，但我们对形成PTP的分子或它是如何调控的知之甚少。我们迫切需要基本问题的答案，这些问题涉及哪些蛋白质实际上形成了PTP通道，以及是什么调节了体内PTP的开放。因此，我们的目标是确定在正常和疾病状态下对PTP的结构和调控做出贡献的分子。如果我们能够有效地识别和/或设计针对PTP从正常到病理转变的有价值的治疗方法，这些信息是至关重要的。在这里，我们将使用生化和遗传工具来确定PTP的结构成分，以及如何在体内动态调节PTP活性。这一应用的具体目标是通过Forte和Bernardi实验室提供的新方法的独特组合而可能产生的协同作用。这项应用的具体目标是：目的1-OMM蛋白在PTP活性调节中的测试作用：虽然PTP主要是一种IMM事件，但长期以来人们一直认为OMM中的蛋白质应该显著地调节PTP活性。我们首先将重点放在一个特定的OMM蛋白，TSPO，它在PTP调节中的作用已经被强烈建议。我们在这里的研究将使我们更深入地了解胞质元素如何影响PTP活性。目的2-确定PTP的关键结构成分：尽管我们越来越多地认识到它在正常和病理细胞反应中的基本作用，但形成PTP的分子仍然是一个谜。在这里，我们将使用可用的线粒体蛋白质组中的信息来识别形成PTP孔的蛋白质。我们期望，对构成PTP的任何单一组件的识别将为我们提供缺失的“钩子”，为识别更多组件提供机会。目的3-线粒体p66ShcA和ROS对PTP的激活：很明显，孔道的开闭转换可以在多个水平上进行调节；这些上游通路的错误调节可以导致PTP的持续的、病理性的激活。这一目的的目的是研究假设，通过p66ShcA(P66)的作用产生的ROS在过度氧化应激条件下作用于PTP激活的上游。我们期望在这一目标完成后，能够清楚地了解一种新的PTP活性上游激活剂的作用。这些研究将为未来的审问奠定基础，旨在扩大我们对生理和病理环境中线粒体和PTP活性的理解。显然，这些结果将是开发新的治疗策略的基础，这些治疗策略专门针对PTP明显涉及的许多疾病过程中的毛孔。

DESCRIPTION (provided by applicant): Activation of the mitochondrial permeability transition pore (PTP) clearly plays a key role in some of the most wide-spread and therapeutically challenging human diseases. Our studies have established that the PTP operates in two modes 1) transiently, whereby the PTP acts as a mitochondrial Ca2+ release channel or 2) persistently, which ultimately results in cell death and disease. Although well characterized on a functional level, we know remarkably little about the molecules that form the PTP or how it is regulated. We urgently need answers to basic questions concerning what proteins actually form the PTP channel and what modulates the opening of the PTP in vivo. As a result, our goal is to identify the molecules that contribute to the structure and regulation of the PTP in both normal and disease states. This information is critical if we are to be able to effectively identify and/or deign valuable therapeutics targeting the transition of the PTP from normal to pathological. Here, we will use biochemical and genetic tools to identify structural components of the PTP and how PTP activity can be dynamically regulated in vivo. The specific objectives of this application are based in the synergy possible through the unique combination of novel approaches available in the Forte and Bernardi laboratories. The specific objectives of this application are: Aim 1 - Test role of OMM proteins in the regulation of PTP activity: While the PTP is primarily an IMM event, it has long been appreciated that proteins in the OMM should prominently regulate PTP activity. We will initially focus on a specific OMM protein, Tspo, whose role in PTP regulation has been strongly suggested. Our studies here will allow us to gain a deeper understanding of how cytosolic elements can impact PTP activity. Aim 2 - Identify key structural components of the PTP: Despite our increasing appreciation of its fundamental role in normal and pathological cellular responses, the molecules that form the PTP have remained a mystery. Here, we will use information in available mitochondrial proteomes to identify proteins forming the pore of the PTP. It is our expectation that the identification of any single component forming the PTP will supply us with the missing "hook", providing the opportunity to identify additional components. Aim 3 - Mitochondrial p66ShcA and ROS activation of the PTP: It is clear that pore open-closed transitions can be regulated at many levels and, by extension; misregulation of these upstream pathways can lead to persistent, pathological activation of the PTP. The goal of this aim is to investigate the hypothesis that ROS generated through the action of p66ShcA (p66) functions upstream of the activation of the PTP in conditions of excess oxidative stress. We anticipate on the completion of this aim to have clear understanding of the role of one novel upstream activator of PTP activity. These studies will set the stage for future interrogation aimed at extending our understanding of mitochondria and PTP activity in physiological and pathological settings. Clearly, these outcomes will be fundamental to developing novel therapeutic strategies specifically targeting the pore in the many disease processes in which the PTP has been clearly implicated.