APOPTOSIS SIGNAl--BINDING OF CYTOCHROME C TO APAF 1

细胞凋亡提供了破坏受损细胞的关键机制。细胞凋亡途径的失败（例如p53突变）通常导致癌症细胞生长不受调控。 最近的工作已经确定了充分研究的蛋白质，细胞色素c，（Cc）作为细胞凋亡途径中的关键中间体。 当从线粒体释放时，Cc与凋亡调节蛋白Apaf-1结合。 Cc：Apaf-1复合物激活蛋白酶半胱天冬酶-9，其进而激活另外的蛋白酶和核酸酶。 以这种方式，细胞色素c：Apaf-1相互作用在使细胞凋亡中起关键作用。 因此，这种复合物为细胞凋亡起关键作用的癌症（和其他疾病）提供了有吸引力的治疗靶标。 我们建议对Apaf-1：Cc相互作用进行详细的研究。研究包括结合化学计量、结合自由能、环境效应和调节剂效应（pH、离子强度、dATP）。 将使用大的Cc变体文库评估点突变对识别和结合的影响。 最后，质谱酰胺氢交换实验提出本地化的结合界面，其中Cc承认Apaf-1内的Cc：Apaf-1复合物。

Apoptosis provides a critical mechanism to destroy damaged cells. Failures in the apoptosis pathway (e.g. p53 mutations) commonly lead to the unregulated cell growth of cancer. Recent work has identified the well studied protein, cytochrome c, (Cc) as a critical intermediate in the apoptosis pathway. When released from mitochondria, Cc binds to an apoptotic regulator protein, Apaf-1. The Cc: Apaf-1 complex activates the protease caspase-9, which, in turn, activates additional proteases and nucleases. In this way the cytochrome c: Apaf-1 interaction plays a critical role in committing the cell to apoptosis. This complex thereby provides an attractive therapeutic target for cancer (and other diseases) where apoptosis plays a critical role. We propose to carry out detailed studies of the Apaf-1: Cc interaction. Studies include binding stoichiometry, binding free energies, environmental effects and regulator effects (pH, ionic strength, dATP). Effects of point mutations on recognition and binding will be assessed using a large library of Cc variants. Finally, mass spectrometric amide hydrogen exchange experiments are proposed to localize the binding interface by which Cc recognizes Apaf-1 within the Cc: Apaf-1 complex.