APOPTOSIS SIGNAl--BINDING OF CYTOCHROME C TO APAF 1

细胞凋亡信号--细胞色素C与APAF 1的结合

基本信息

批准号：
6498692
负责人：
George L McLendon
金额：
$ 18.43万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-01 至 2003-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6498692
关键词：
apoptosis biological signal transduction cofactor cysteine endopeptidases cytochrome c fluorescence resonance energy transfer hydrogen transport mass spectrometry mathematical model method development nuclear magnetic resonance spectroscopy point mutation protein binding protein protein interaction stoichiometry transcription factor ultraviolet spectrometry

项目摘要

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.

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）通常会导致癌症细胞生长失控。最近的工作已确定经过充分研究的蛋白质细胞色素 c (Cc) 是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 caspase-9，进而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发挥着关键作用。因此，该复合物为细胞凋亡发挥关键作用的癌症（和其他疾病）提供了有吸引力的治疗靶点。我们建议对 Apaf-1: Cc 相互作用进行详细研究。研究包括结合化学计量、结合自由能、环境影响和调节作用（pH、离子强度、dATP）。点突变对识别和结合的影响将使用大型 Cc 变体库进行评估。最后，提出了质谱酰胺氢交换实验来定位 Cc: Apaf-1 复合物中 Cc 识别 Apaf-1 的结合界面。