Inhibition of Cytochrome c-induced Caspase Activation

描述(申请人提供)：细胞凋亡是一种细胞自杀程序，在不破坏整个组织结构的情况下，将单个细胞从活组织中移除。作为对凋亡信号的反应，细胞激活被称为caspase的细胞内蛋白水解酶家族，该家族参与了死亡细胞的有序分解和包装。在许多情况下(例如，在用化疗药物处理细胞后)，caspase的激活通过线粒体依赖的途径进行，导致呼吸链成分细胞色素c从线粒体转移到细胞质。一旦细胞质内，细胞色素c与凋亡调节因子APAF-1相互作用，促进特定caspase-9依赖于ATP的激活。这种caspase 9/APAF-1/细胞色素c的复合体被称为凋亡体。已有研究表明，许多促进细胞存活的信号作用于线粒体途径，阻止线粒体细胞色素c外流至细胞质。这可以有效地阻止caspase的激活和阻止细胞死亡。然而，在这项提案中，我们关注的是另一种细胞生存模式，即通过细胞色素c后抑制凋亡体发挥作用。具体地说，我们发现激活的Erk激酶和癌蛋白bcr-abl都可以阻止caspase 9的激活，即使在胞浆细胞色素c存在的情况下也是如此。这项提议的目的是阐明这种抑制的机制(S)。为此，我们建议在存在和不存在这些激活的激酶的情况下对凋亡体(组成、翻译后修饰、结合伙伴)进行仔细的表征。此外，我们建议使用一种新的方法来识别和表征无细胞凋亡重建系统中这些激酶的直接底物。最后，我们提出了两个筛选来识别其他新的凋亡体调控因子。总而言之，这些实验应该为线粒体释放细胞色素c后的细胞存活机制提供洞察力。

DESCRIPTION (provided by applicant): Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without destroying overall tissue architecture. In response to apoptotic signals, cells activate a family of intracellular proteases known as caspases, which participate in the orderly dismantling and packaging of the dying cell. Under many circumstances (e.g. following treatment of cells with chemotherapeutic agents), caspase activation proceeds through a mitochondrial-dependent pathway, leading to translocation of the respiratory chain component, cytochrome c, from the mitochondria to the cytoplasm. Once cytoplasmic, cytochrome c interacts with an apoptotic regulator, Apaf-1 to promote ATP-dependent activation of a specific caspase, caspase 9. This complex of caspase 9/Apaf-1/cytochrome c is referred to as the apoptosome. It has been demonstrated that many signals promoting cell survival impinge upon the mitochondrial pathway, preventing the efflux of mitochondrial cytochrome c to the cytoplasm. This can effectively prevent caspase activation and forestall cell death. In this proposal, however, we focus on an alternative mode of cell survival, acting through post-cytochrome c inhibition of the apoptosome. Specifically, we have found that both activated Erk kinases and the oncoprotein Bcr-Abl can prevent activation of caspase 9, even in the presence of cytosolic cytochrome c. It is the goal of this proposal to elucidate the mechanism(s) underlying this inhibition. Towards this end we propose a careful characterization of the apoptosome (composition, post-translational modifications, binding partners) in the presence and absence of these activated kinases. Moreover, we propose to use a new methodology to identify and characterize direct substrates of these kinases in a cell-free apoptotic reconstitution system. Finally, we propose two screens to identify additional novel apoptosomal regulators. Collectively, these experiments should provide insight into cellular survival mechanisms acting after mitochondrial release of cytochrome c.