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）是线粒体介导的程序性细胞死亡（凋亡）的法官和工作。他们的互动通过启动严格控制的程序结束细胞分解的程序来封印一个细胞的命运，并且该调节中的失衡与癌症，神经系统疾病和心脏病有关。鉴于该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。该项目的长期长期目标是确定细胞色素C和PAPAF-1之间相互作用的分子基础。主要的假设是：a）细胞色素C使用相同的阳离子与APAF-1及其氧化还原伴侣相互作用。 b）APAF-1使用特定的离子化斑块与细胞色素相互作用C。特定目的是1）量化APAF-1 /细胞色素c结合，2）在APAF-1和细胞色素c上识别可探测的结合位点，3）测试结合位点。结合平衡方法将用于量化结合方法，但也将讨论结合方法。在建模研究中已经确定了APAF-1的WD重复量上的可能结合位点，并且CytoChrome C上的氧化还原 - 合并位点已知多年。该站点将通过使用位点的诱变进行测试。为了确保获得生物学意​​义的数据，将首先使用Quick And和与生物学相关的caspase激活测定法评估这些变体。一旦确定了生物含量的残基，这些变体的生物物理学将呈现。圆形二色性光谱法将用于评估其整合性。 APAF-1变体上的沉积速度实验将用于弥补其对肾上蛋白相互作用的影响。 AIM 1的沉积平衡技术将用于量化其对复合形成的自由能和化学计量的影响。