Cytochrome c and Apoptosis

细胞色素 c 和细胞凋亡

• 批准号: 0109366
• 负责人: Gary Pielak
• 金额: $ 10万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0109366&HistoricalAwards=false
• 关键词: Cytochrome; Apoptosis

Cytochrome c and apoptosis activating factor-1 (apaf-1) are the judge andjury of mitochondrially-mediated programmed cell death (apoptosis). Theirinteraction seals a cell's fate by launching a tightly controlled program that endsin cellular disintegration, and imbalances in this regulation are associated withcancer, neurological diseases, and heart disease. Given the biological andmedical relevance of this protein-protein reaction, it is surprising that itsmolecular basis is unknown. The broad long-term goal of this project is todetermine the molecular basis for the interaction between cytochrome c andapaf-1. The main hypotheses are: a) Cytochrome c uses the same cationicpatch to interact with apaf-1 and its redox partners. b) Apaf-1 uses specificanionic patches to interact with cytochrome c.The specific aims are 1) Quantify apaf-1 / cytochrome c binding, 2) Identifyprobable binding sites on apaf-1 and cytochrome c, 3) Test the binding sites.Sedimentation equilibrium methods will be used to quantify binding, butalternative methods are also discussed. Probable binding sites on the WD repeatdomain of apaf-1 have been identified in modeling studies, and the redox-partnerbinding site on cytochrome c has been known for many years. The siteswill be tested by using site directed mutagenesis. To ensure that biologicallysignificant data are obtained, the variants will first be assessed by using a quickand biologically relevant caspase activation assay. Once the biologicallyrelevant residues have been identified, the biophysics of the variants will becharacterized. Circular dichroism spectropolarimetry will be used to assess theirintegrity. Sedimentation velocity experiments on apaf-1 variants will be used toassess their effects on intraprotein interactions. Sedimentation equilibriumtechniques from aim 1 will be used to quantify their effects on the free energyand stoichiometry of complex formation.

细胞色素c和凋亡激活因子-1(APAF-1)是线粒体介导的细胞程序性死亡的判断和标志。它们的相互作用通过启动一种严格受控的程序来封存细胞的命运，这种程序最终导致细胞解体，而这种调节的失衡与癌症、神经疾病和心脏病有关。考虑到这种蛋白质-蛋白质反应的生物学和医学相关性，令人惊讶的是它的分子基础尚不清楚。该项目的长期目标是确定细胞色素c和apaf-1相互作用的分子基础。主要假设是：a)细胞色素c使用相同的阳离子补丁与APAF-1及其氧化还原伙伴相互作用。2)APAF-1利用特定的阴离子斑块与细胞色素C相互作用，其具体目的是1)定量APAF-1/细胞色素C的结合，2)确定APAF-1和细胞色素C上可能的结合部位，3)检测结合部位。在模型研究中已经确定了APAF-1 WD重复结构域上可能的结合部位，而细胞色素c上的氧化还原伙伴结合部位已经知道很多年了。这些网站将通过定点突变进行测试。为了确保获得具有生物学意义的数据，首先将使用快速和生物相关的半胱氨酸天冬氨酸酶激活试验对变异进行评估。一旦确定了与生物相关的残基，变异体的生物物理特性就会被表征。将使用圆二色光谱旋光法来评估它们的完整性。APAF-1变异体的沉降速度实验将被用来评估它们对蛋白质内相互作用的影响。来自目标1的沉积平衡技术将被用来量化它们对络合物形成的自由能和化学计量的影响。