Release of Apoptogenic Proteins from Brain Mitochondria

描述(由申请人提供)：我们对钙升高和促凋亡蛋白诱导线粒体促凋亡因子释放的机制的理解存在根本差距。我们的长期目标是确定线粒体在神经细胞凋亡中的作用。本研究的目的是阐明由钙升高或促凋亡蛋白启动的脑线粒体促凋亡蛋白释放的机制。这项研究的中心假设是，脑线粒体中活性氧的产生增加、脂质过氧化增强、磷脂酶A2的激活和K内流是导致钙升高或促凋亡蛋白TBID和Bax诱导的促凋亡蛋白释放的主要过程。在特定的目标1中，我们将建立钙诱导的脑线粒体肿胀和凋亡蛋白释放的钾依赖机制。线粒体K通道和腺嘌呤核苷酸转位酶的抑制剂将应用于分离的脑线粒体或培养的神经元，以确定它们在钙诱导的肿胀和凋亡蛋白释放中的作用。在特定的目标2中，我们将确定线粒体K通道的激活和通透性转变在多大程度上有助于促凋亡蛋白TBID和Bax诱导的促凋亡因子的释放。通透性转换的抑制剂和K通道的阻断剂将被用来确定它们在诱导凋亡的蛋白释放中的作用。在具体目标3中，我们将确定活性氧物种、脂质过氧化和磷脂酶A2在TBID和Bax诱导的促凋亡蛋白释放中的作用。各种抗氧化剂和磷脂酶A2抑制剂将被用来抑制促凋亡蛋白的释放。在特定的目标4中，我们将确定caspase在暴露于TBID和Bax的脑线粒体中释放促凋亡蛋白的作用。暴露于TBID和Bax并用重组caspase处理的分离的脑线粒体将被用来检验这一假说。这项研究为更好地理解钙升高或促凋亡蛋白TBID和Bax诱导线粒体外膜通透性的分子机制奠定了基础，并有助于填补我们对这些现象的认识空白。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of the mechanisms of the release of mitochondrial apoptogenic factors induced by elevated Ca2+ and by pro-apoptotic proteins. Our long-term goal is to establish the role of mitochondria in neuronal apoptosis. The objective of this study is to delineate the mechanisms of release of apoptogenic proteins from brain mitochondria initiated by elevated Ca2+ or by pro-apoptotic proteins. The central hypothesis of the proposed research is that an increased generation of reactive oxygen species, augmentation of lipid peroxidation, activation of phospholipase A2, and K+ influx in brain mitochondria are the major processes leading to the release of apoptogenic proteins induced by elevated Ca2+ or pro-apoptotic proteins tBID and BAX. In Specific Aim 1 we will establish K+-dependent mechanisms of the Ca2+-induced swelling of brain mitochondria and release of apoptogenic proteins. Inhibitors of mitochondrial K+ channels and the adenine nucleotide translocase will be applied to isolated brain mitochondria or to cultured neurons to establish their role in the Ca2+-induced swelling, and release of apoptogenic proteins. In Specific Aim 2 we will determine the extent to which an activation of mitochondrial K+ channels and the permeability transition contributes to the release of apoptogenic factors induced by pro-apoptotic proteins tBID and BAX. Inhibitors of the permeability transition and blockers of K+ channels will be used to identify their role in the release of the apoptogenic proteins. In Specific Aim 3 we will establish the role of reactive oxygen species, lipid peroxidation and phospholipase A2 in the release of apoptogenic proteins induced by tBID and BAX. Various antioxidants and inhibitors of phospholipase A2 will be used to inhibit the release of apoptogenic proteins. In Specific Aim 4 we will determine the role of caspases in the release of apoptogenic proteins from brain mitochondria exposed to tBID and BAX. Isolated brain mitochondria exposed to tBID and BAX and treated with recombinant caspases will be used to test this hypothesis. The proposed research lays the foundation for a better understanding of the molecular mechanisms of the permeabilization of the outer mitochondrial membrane induced by elevated Ca 2+ or pro-apoptotic proteins tBID and BAX and contributes to filling in a gap in our knowledge of these phenomena.