Inhibition of Cytochrome c-induced Caspase Activation

描述（由申请人提供）：细胞凋亡是一种细胞自杀程序，其中单个细胞从活组织中移除，而不会破坏整个组织结构。作为对凋亡信号的反应，细胞激活了一个被称为半胱天冬酶的细胞内蛋白酶家族，它参与了死亡细胞的有序分解和包装。在许多情况下（例如，在用化疗药物治疗细胞后），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从线粒体转运到细胞质。一旦细胞质，细胞色素c与凋亡调节因子Apaf-1相互作用，促进atp依赖性的特定caspase - 9的激活。这种caspase 9/Apaf-1/细胞色素c的复合体被称为凋亡体。已经证明，许多促进细胞存活的信号影响线粒体途径，阻止线粒体细胞色素c向细胞质的外溢。这可以有效地阻止caspase的激活，防止细胞死亡。然而，在这个提议中，我们关注的是细胞存活的另一种模式，通过细胞色素c后抑制凋亡。具体来说，我们已经发现激活的Erk激酶和癌蛋白Bcr-Abl都可以阻止caspase 9的激活，即使在存在胞浆细胞色素c的情况下也是如此。本文的目标是阐明这种抑制作用的机制。为此，我们建议在这些活化激酶存在和不存在的情况下仔细表征凋亡细胞（组成，翻译后修饰，结合伙伴）。此外，我们建议使用一种新的方法来鉴定和表征这些激酶在无细胞凋亡重建系统中的直接底物。最后，我们提出了两种筛选方法来鉴定新的凋亡调节因子。总的来说，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见解。

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.