Cytochrome c and Apoptosis

细胞色素c和凋亡激活因子-1（apoptosis activating factor-1，apaf-1）是细胞介导的程序性细胞死亡（programmed cell death，apoptosis）的法官和陪审员。它们的相互作用通过启动一个严格控制的程序来决定细胞的命运，该程序最终导致细胞解体，而这种调节的不平衡与癌症、神经系统疾病和心脏病有关。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，它的分子基础是未知的。该项目的广泛长期目标是确定细胞色素c和apaf-1之间相互作用的分子基础。主要的假设是：a）细胞色素c使用相同的阳离子补丁与apaf-1及其氧化还原配偶体相互作用。B）Apaf-1与细胞色素c的相互作用是通过特定的阴离子斑来实现的，具体的目的是：1）定量分析apaf-1与细胞色素c的结合，2）确定apaf-1与细胞色素c可能的结合位点，3）检测结合位点。在模型研究中已经确定了apaf-1的WD重复结构域上可能的结合位点，并且细胞色素c上的氧化还原伴侣结合位点已经知道很多年了。将使用定点突变对这些位点进行检测。为了确保获得具有生物学意义的数据，将首先通过使用快速和生物学相关的半胱天冬酶活化测定来评估变体。一旦生物学相关的残基已经确定，变异的生物物理学将被表征。圆二色谱偏振光谱法将用于评估其完整性。对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。沉积平衡技术从目标1将被用来量化其对自由能和化学计量复杂的形成。

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.