APOPTOSIS SIGNAl--BINDING OF CYTOCHROME C TO APAF 1

细胞凋亡是破坏受损细胞的重要机制。凋亡途径的失败（如p53突变）通常导致癌症细胞生长不受调节。最近的工作已经确定了一种被充分研究的蛋白质，细胞色素c （Cc）是凋亡途径的关键中间体。当从线粒体中释放出来时，Cc与凋亡调节蛋白Apaf-1结合。Cc: Apaf-1复合物激活蛋白酶caspase-9， caspase-9反过来激活其他蛋白酶和核酸酶。通过这种方式，细胞色素c: Apaf-1相互作用在细胞凋亡中起着关键作用。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）提供了一个有吸引力的治疗靶点。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学、结合自由能、环境效应和调节剂效应（pH、离子强度、dATP）。点突变对识别和结合的影响将使用大型Cc变异库进行评估。最后，提出了质谱酰胺氢交换实验来定位Cc: Apaf-1配合物中Cc识别Apaf-1的结合界面。

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.