Molecular Dissection of the Permeability Transition Pore

描述(申请人提供)：线粒体通过其在能量代谢、调节细胞内钙稳态和细胞凋亡中的作用，在细胞生存和组织发育中发挥关键作用。鉴于这一多因素的作用，由于能量守恒被用来驱动每个过程，所以钙稳态、新陈代谢和生物能量学作为一个综合系统发挥作用。线粒体能量守恒(ATP生产)需要呼吸驱动跨线粒体内膜(IMM)形成质子电化学电位差(Delta MuH)，这是由呼吸复合体的质子泵作用产生的。保持梯度要求IMM对质子、带电物种和溶质的低渗透性。然而，在体外，线粒体很容易对分子质量在1,500 Da或更低的溶质发生IMM通透性增加。这种通透性变化被称为通透性转换(PT)，由膜孔即线粒体通透性转换孔(PTP)的打开来调节。PTP的体外开放对线粒体的功能(例如，mU H的崩溃和吡啶核苷酸的耗尽)和结构(细胞色素c的释放)具有显著的影响，从而导致呼吸抑制。长期以来，这一过程被认为是体内线粒体功能障碍的潜在靶点，也是细胞程序性死亡(PCD)的媒介，它通过释放细胞色素c和其他活跃在细胞凋亡机制上的膜间蛋白来实现。然而，尽管在过去的30年里进行了详细的功能鉴定，但形成PTP的分子组成还没有被确定，PTP在体内的确切作用也没有被定义。这项建议的基础是通过我们两个实验室现有的新方法的组合而可能产生的协同效应。我们的具体计划包括以下目标：目的1：在PTP化学抑制剂的筛选中，我们通过对哺乳动物VDAC(VDAC1)亚型1的共价修饰，在功能分析中鉴定了Ro 68-3400是PTP的高亲和力(NM)阻断剂。类似的实验也证明了酵母VDAC1是这种化合物的特异性靶点。我们计划利用我们在哺乳动物和酵母VDAC上的经验来确定VDAC与这种化合物高亲和力结合的结构要求，检查其他哺乳动物VDAC亚型被RO68-3400修饰的能力，并测试用这种新型PTP阻断剂处理的线粒体对bcl2家族蛋白质的敏感性。目的2：传统上，PTP被认为是一种动态的多蛋白复合体，通过IMM的腺嘌呤核苷酸转运体(ANT)、OMM中的VDAC和基质调节蛋白线粒体亲环素D(CYP-D)的相互作用而在内外膜接触部位形成。然而，最近的数据并没有支持蚂蚁与PTP复合体有关的证据。因此，为了实现这一目标，我们计划利用RO68-3400作为一种特定的工具来进一步确定形成PTP的核心成分，特别是在孔道复合体中识别VDAC的IMM伙伴。目的：环孢菌素A(CsA)和非免疫抑制类似物对PTP的抑制作用已成为鉴定分离线粒体、活细胞和体内PTP的标准诊断工具。在这些研究中，CsA的靶标CYP-D是PTP中唯一被明确确定其作用的成分。这一目标的目的是明确地解决与CYP-D对PTP的影响，PTP参与细胞凋亡计划的特定方面，以及它在对人类疾病有重要意义的特定病理过程中的作用相关的基本问题，通过使用已通过“敲除”策略消除了CYP-D和MVDAC1表达的小鼠。

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 Ca 2+ homeostasis and apoptosis. Given this multifactorial role, Ca 2+ homeostasis, metabolism, and bioenergetics function as an integrated system since energy conservation is used to drive each process. Mitochondrial energy conservation (ATP production) requires the respiration-driven formation of a proton electrochemical potential difference (delta mu H) across the inner mitochondrial membrane (IMM), which is created by proton pumping by the respiratory complexes. Maintenance of the gradient demands a low permeability of the IMM to protons, charged species and solutes. Yet, mitochondria in vitro can easily undergo an IMM permeability increase to solutes with molecular masses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), is regulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). PTP opening in vitro has dramatic consequences on mitochondrial function (e.g., collapse of the delta mu H and depletion of pyridine nucleotides) and structure (release of cytochrome c) that lead to respiratory inhibition. This process has long been studied as a potential target for mitochondrial dysfunction in vivo and as a mediator of programmed cell death (PCD) through the release of cytochrome c and other intermembrane proteins active on the apoptotic machinery. However, despite detailed functional characterization over the last 30 years, the molecular components forming the PTP have been not been definitively established nor has the precise role of the PTP in vivo been defined. This proposal is based in the synergy possible through the combination of novel approaches available in our two laboratories. Our specific plans include the following aims: Aim 1: In screens for chemical inhibitors of the PTP, we have identified Ro 68-3400 in functional assays as a high affinity (nM) blocker of the PTP through covalent modification of isoform 1 of mammalian VDAC (VDAC1). Similar experiments have also demonstrated that yeast VDAC1 is specifically targeted by this compound. We plan to use our experience with both mammalian and yeast VDAC to pin-point the structural requirements for high affinity association of VDAC with this compound, examine other mammalian VDAC isoforms for their ability to be modified by Ro 68-3400 and test the sensitivity of mitochondria treated with this novel PTP blocker to proteins in the BCL-2 family. Aim 2: Traditionally, the PTP has been considered to be a dynamic multiprotein complex formed at inner/outer membrane contact sites through the interaction of the adenine nucleotide translocator (ANT) of the IMM, VDAC in the OMM and a matrix regulatory protein, mitochondrial cyclophilin D (CyP-D). However, evidence implicating the ANT in the PTP complex has not been supported by recent data. Therefore, in this aim we plan to take advantage of Ro 68-3400 as a specific tool to further define the core components forming the PTP, with a specific focus on the identification of the IMM partner for VDAC in the pore complex. Aim 3: Inhibition by cyclosporin A (CsA) and non-immunosuppressive analogs has become the standard diagnostic tool for the characterization of the PTP in isolated mitochondria, in living cells, and in vivo. The target of CsA in these studies, CyP-D, is the only component of the PTP whose role has been definitively established. The goal of this aim is to unambiguously resolve basic questions related to the influence of CyP-D on the PTP, the participation of the PTP in specific aspects of the apoptotic program, and its role in specific pathological processes of significance to human, disease through the use of mice in which the expression of CyP-D and MVDAC1 have been eliminated by "knock-out" strategies.