Analysis of the molecular determinants of Apaf-1 function

Apaf-1功能的分子决定因素分析

• 批准号: 231043840
• 负责人: Dr. Susanne Eschenburg
• 金额: --
• 依托单位: Institut für Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/231043840?language=en
• 关键词: Analysis; molecular; determinants; Apaf; function

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.

Apaf-1（凋亡蛋白酶激活因子1）是线粒体凋亡途径的关键参与者。在这一途径中，决定性的死亡信号是细胞色素c从线粒体膜间空间释放到细胞质中。在与细胞色素c结合后，Apaf-1寡聚成七聚体凋亡复合物，引发下游的caspase级联反应。我们的目标是阐明这一过程的分子决定因素，并为凋亡细胞组装制定一个完整和连贯的模型。我们将通过结合突变研究、核苷酸结合研究、小角度散射实验和Apaf-1 ATP状态的x射线晶体学分析的结果来确定核苷酸交换在释放Apaf-1自抑制中的作用。为了提供凋亡的准原子模型，我们将确定凋亡体亚复合物的x射线结构，并将其与低温电镜研究相结合。结合x射线晶体学和生化分析，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。借助易于结晶的小鼠Apaf-1同源物，我们将确定Apaf-1中的配体结合位点。这些研究将为利用Apaf-1作为药物靶点，针对细胞凋亡机制的治疗控制奠定基础。