Molecular Dissection of the Permeability Transition Pore

渗透率转变孔的分子解剖

• 批准号: 6872901
• 负责人: MICHAEL A FORTE
• 金额: $ 31.31万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6872901
• 关键词: cyclosporines; inhibitor /antagonist; intermolecular interaction; laboratory mouse; membrane activity; membrane permeability; membrane structure; mitochondria; mitochondrial membrane; peptidylprolyl isomerase; protein structure function

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.

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，Ca 2+稳态，代谢和生物能量学作为一个综合系统发挥作用，因为能量守恒用于驱动每个过程。线粒体能量守恒（ATP产生）需要呼吸驱动的跨线粒体内膜（IMM）的质子电化学电势差（Δ mu H）的形成，其由呼吸复合物的质子泵送产生。梯度的维持需要IMM对质子、带电物质和溶质的低渗透性。然而，线粒体在体外可以容易地经历IMM渗透性增加到分子量为约1，500 Da或更低的溶质。这种渗透性变化，称为渗透性转换（PT），是由膜孔，线粒体渗透性转换孔（PTP）的开放调节。体外PTP开放对线粒体功能具有显著影响（例如，Δ μ H的崩溃和吡啶核苷酸的耗尽）和结构（细胞色素c的释放），其导致呼吸抑制。长期以来，该过程一直被研究为体内线粒体功能障碍的潜在靶点，以及通过释放细胞色素c和其他对凋亡机制有活性的膜间蛋白作为程序性细胞死亡（PCD）的介体。然而，尽管在过去30年中进行了详细的功能表征，但形成PTP的分子组分尚未明确确定，也没有定义PTP在体内的精确作用。这项建议是基于可能的协同作用，通过结合我们两个实验室的新方法。我们的具体计划包括以下目标：目标1：在PTP的化学抑制剂的筛选中，我们已经通过共价修饰哺乳动物VDAC 1（VDAC 1）的亚型1，在功能测定中将Ro 68-3400鉴定为PTP的高亲和力（nM）阻断剂。类似的实验也表明，酵母VDAC 1是该化合物的特异性靶向。我们计划利用我们在哺乳动物和酵母VDAC方面的经验来确定VDAC与该化合物的高亲和力缔合的结构要求，检查其他哺乳动物VDAC亚型被Ro 68-3400修饰的能力，并测试用这种新型PTP阻断剂处理的线粒体对BCL-2家族蛋白的敏感性。目标二：传统上，PTP被认为是通过IMM中的腺嘌呤核苷酸转运子（ANT）、OMM中的VDAC和基质调节蛋白线粒体亲环素D（CyP-D）的相互作用在内/外膜接触位点形成的动态多蛋白复合物。然而，最近的数据并不支持ANT参与PTP复合体的证据。因此，在这一目标中，我们计划利用Ro 68-3400作为一个特定的工具，以进一步确定形成PTP的核心组分，特别关注孔复合物中VDAC的IMM伴侣的识别。目标三：环孢菌素A（CsA）和非免疫抑制类似物的抑制作用已成为标准的诊断工具，在分离的线粒体，活细胞和体内的PTP的特性。在这些研究中，CsA的靶点CyP-D是PTP中唯一作用已明确的组分。该目的的目标是明确地解决与CyP-D对PTP的影响、PTP在凋亡程序的特定方面的参与以及其在对人类疾病具有重要意义的特定病理过程中的作用有关的基本问题，通过使用小鼠，其中CyP-D和MVDAC 1的表达已经通过“敲除”策略消除。