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中的作用，细胞Ca 2+稳态的调节和细胞凋亡而在细胞存活和组织发育中起关键作用。鉴于这种多因素的作用，Ca 2+稳态，代谢和生物能力作为集成系统的作用，因为能源保护用于驱动每个过程。线粒体能量保存（ATP产生）需要呼吸驱动的形成质子电化学势差（Delta Mu H），跨线粒体膜（IMM），这是由质子通过呼吸复合物泵泵产生的。维持梯度要求IMM对质子，带电的物种和溶质的渗透性较低。然而，在体外线粒体可以轻松地将IMM渗透性增加到分子量约为1,500 da或更低的溶质中。这种渗透性变化称为渗透率跃迁（PT），受膜孔的打开，即线粒体渗透性过渡孔（PTP）。 PTP在体外开放对线粒体功能（例如，三角洲Mu H的崩溃和吡啶核苷酸的消耗）和结构（细胞色素C的释放）对线粒体功能产生巨大后果。长期以来，通过释放细胞色素C和其他活跃于凋亡机器上的膜间蛋白，这一过程一直是体内线粒体功能障碍的潜在靶标，并作为程序性细胞死亡（PCD）的介体。但是，尽管在过去30年中详细的功能表征，但尚未确定形成PTP的分子成分，也没有确定PTP在体内的确切作用。该提议通过我们的两个实验室中可用的新方法结合起来基于协同作用。我们的具体计划包括以下目的：目标1：在PTP化学抑制剂的筛选中，我们通过功能测定法中的RO 68-3400确定为PTP的高亲和力（NM）阻滞剂，通过共价修改哺乳动物VDAC（VDAC1）的同工型1。类似的实验也表明，该化合物专门针对酵母VDAC1。我们计划利用哺乳动物和酵母VDAC的经验来确定VDAC与该化合物的高亲和力协会的结构要求，检查其他哺乳动物VDAC同工型，以通过RO 68-3400修改它们的能力，并测试与这种新颖的PTP Blocker对Blcl-nemples in bcl-2 proteins nebles the ptp blocker the bcl-smpl-2的敏感性。 AIM 2：传统上，PTP被认为是通过IMM的腺嘌呤核苷酸易位剂（ANT）的相互作用，在OMM中，OMM中的VDAC和基质调节蛋白，线粒体环蛋白D（CYP-D）形成的动态多蛋白复合物。但是，最近的数据尚未支持牵涉到PTP复合物中ANT的证据。因此，在此目标中，我们计划利用RO 68-3400作为进一步定义PTP的核心组件的特定工具，并特别关注孔复合体中VDAC的IMM伴侣的识别。 AIM 3：环孢菌素A（CSA）和非免疫抑制类似物的抑制已成为孤立线粒体，活细胞和体内PTP表征PTP的标准诊断工具。 CSA在这些研究中的靶标CYP-D是PTP的唯一组成部分，其作用已被确定。此目的的目的是明确解决与CYP-D对PTP的影响，PTP参与凋亡程序的特定方面的影响，以及其在使用CYP-DAC1和MVDAC1的表达中，其在对人类意义的特定病理学过程中的作用已被淘汰。