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。细胞凋亡途径的失灵(如P53突变)通常会导致肿瘤细胞生长失控。最近的研究发现，细胞色素c(CC)是细胞凋亡途径中的一个重要中间体。当CC从线粒体中释放出来时，它会与一种凋亡调节蛋白APAF-1结合。CC：APAF-1复合体激活蛋白水解酶caspase-9，caspase-9进而激活其他的蛋白水解酶和核酸酶。通过这种方式，细胞色素c：APAF-1的相互作用在促使细胞发生凋亡方面发挥了关键作用。因此，这种复合体为癌症(和其他疾病)提供了一个有吸引力的治疗靶点，在这些疾病中，细胞凋亡起着关键作用。我们建议对APAF-1：CC相互作用进行详细研究。研究包括结合化学计量、结合自由能、环境效应和调节效应(pH、离子强度、dATP)。点突变对识别和结合的影响将使用CC变异体的大型文库进行评估。最后，利用质谱酰胺氢交换实验对CC识别APAF-1的结合界面进行了定位。

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.