Analysis of the molecular determinants of Apaf-1 function

APAF-1(凋亡性蛋白水解酶激活因子1)是线粒体细胞凋亡途径中的关键分子。在这一途径中，决定性的死亡信号是细胞色素c从线粒体膜间间隙释放到胞浆中。当与细胞色素c结合时，APAF-1寡聚形成七聚体凋亡体复合体，从而触发下游的caspase级联反应。我们的目标是阐明这一过程的分子决定因素，并建立一个完整和一致的凋亡体组装模型。我们将结合突变研究、核苷酸结合研究、小角散射实验和对APAF-1的ATP状态的X射线晶体分析的结果，确定核苷酸交换在释放APAF-1的自身抑制作用中的作用。为了提供凋亡体的准原子模型，我们将测定凋亡体亚复合体的X射线结构，并将其与低温电子显微镜研究相结合。结合X射线结晶学和生化分析，我们试图阐明APAF-1的选定蛋白质和小分子相互作用伙伴的作用模式。借助易结晶的小鼠APAF-1同源基因，我们将确定APAF-1中的配体结合位点。这些研究将为APAF-1作为药物靶点用于细胞凋亡机制的治疗控制奠定基础。

Apaf-1 (apoptotic protease activating factor 1) is a key player in the mitochondrial pathway of apoptosis. The decisive death signal in this pathway is the release of cytochrome c from the mitochondrial intermembrane space into the cytosol. Upon binding of cytochrome c Apaf-1 oligomerizes into the heptameric apoptosome complex, which triggers the downstream caspase cascade. We aim to elucidate the molecular determinants of this process and to formulate a complete and coherent model for apoptosome assembly. We will determine the role of nucleotide exchange for the release of the autoinhibition of Apaf-1 by combining the results from mutational studies, nucleotide binding studies, small angle scattering experiments, and X-ray crystallographic analysis of an ATP state of Apaf-1. To provide a quasi atomic model of the apoptosome, we will determine X-ray structures of apoptosomal subcomplexes and combine them with cryoEM studies. Combining X-ray crystallographic and biochemical analyses, we seek to clarify the modes of action of selected protein and small molecule interaction partners of Apaf-1. With the aid of the readily crystallizable murine Apaf-1 ortholog, we will determine the ligand binding sites in Apaf-1. These studies will lay the ground for the use of Apaf-1 as drug target aiming at the therapeutic control of the apoptosis machinery.