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除去而不破坏整个组织结构。响应于凋亡信号，细胞激活称为半胱天冬酶的细胞内蛋白酶家族，其参与垂死细胞的有序分解和包装。在许多情况下（例如，在用化学治疗剂处理细胞后），胱天蛋白酶活化通过线粒体依赖性途径进行，导致呼吸链组分细胞色素c从线粒体易位到细胞质。一旦细胞质，细胞色素c与凋亡调节因子Apaf-1相互作用，以促进特异性caspase-9的ATP依赖性活化。这种半胱天冬酶9/Apaf-1/细胞色素c的复合物被称为线粒体。已经证明，许多促进细胞存活的信号冲击线粒体途径，阻止线粒体细胞色素c流出到细胞质。这可以有效地防止半胱天冬酶激活和阻止细胞死亡。然而，在这个提议中，我们专注于细胞存活的另一种模式，通过后细胞色素c抑制的线粒体起作用。具体地说，我们已经发现，激活的Erk激酶和癌蛋白Bcr-Abl可以防止半胱天冬酶9的激活，即使在细胞溶质细胞色素c的存在下。本提案的目的是阐明这种抑制的潜在机制。为此，我们提出了一个仔细的表征的aptosome（组合物，翻译后修饰，结合伙伴）在这些激活的激酶的存在和不存在。此外，我们建议使用一种新的方法来确定和表征这些激酶在无细胞凋亡重建系统的直接底物。最后，我们提出了两个屏幕，以确定其他新的染色体调节。总的来说，这些实验应该提供深入了解细胞的生存机制后，线粒体释放细胞色素c。

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.