Cytochrome c and Apoptosis

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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.