Investigating the Molecular Basis of Cytochrome c Functions

The only known naturally occurring mutation in cytochrome c, identified in a New Zealand family, is associated with mild thrombocytopenia due to dysregulation of platelet formation in the bone marrow. The mutation is from a glycine to serine at residue 41 (G41S) spanning seven generations in the family. Cytochrome c has crucial roles in both cellular energy production and as a stimulus for programmed cell death. The energy producing function of G41S cytochrome c is unaffected, however interestingly the protein is more efficient at triggering cell death (apoptosis). During apoptosis, cytochrome c is released from mitochondria and binds to the apoptotic protease activating factor-1 (Apaf-1) in the cytosol. Seven cytochrome c-Apaf-1 complexes form the apoptosome that acts as a caspase activating platform to facilitate cell death. The G41S cytochrome c mutant is the only known variant with an increased ability to trigger caspase activation. Therefore, this variant provides a new opportunity to further understand the role of cytochrome c in platelet formation and in apoptosis. This thesis aimed to investigate the role of G41S cytochrome c in the apoptotic pathway by addressing two broad aims. Firstly by characterising a knock-in mouse model expression G41S cytochrome c. Secondly by determining the structural, biophysical and functional consequences of mutations to residue 41 of cytochrome c. The cytochrome c mediated apoptotic pathway has been implicated in platelet formation. However, controversy exists on whether the apoptotic pathway is required for platelet formation or rather needs to be suppressed for platelet production to occur in mice. Knock-in G41S cytochrome c mice were purchased to further analyse the role of cytochrome c in platelet formation. As the mouse and human cytochrome c sequence is 91% conserved, it was assumed that the mouse G41S cytochrome c would have the same caspase-inducing activity as human G41S cytochrome c. However, the G41S knock-in mice had normal platelet counts and no other discernible phenotype. The consequences of the null phenotype in mice provided a previously unexpected insight into the species specificity of the interaction between cytochrome c and Apaf-1. The difference in caspase activation when cytochrome c from one species is used with cytosolic extracts from another needs to be taken into consideration when the data is analysed and interpreted. A mutational and biophysical analysis of cytochrome c was undertaken to investigate the molecular basis of the increased caspase activation of human G41S cytochrome c. Additional Gly41 variants (G41A and G41T) were made to understand the role of residue 41 of cytochrome c in caspase activation. Residue changes at this position were shown to both increase and decrease induction of caspase activation. A correlation between the enhanced caspase-inducing activation of human G41S with enhanced Apaf- 1 binding affinity was found. Directed mutations of Gly41 did not alter the global structure of cytochrome c, but consistently altered the haem environment. The change to the haem environment of cytochrome c not only affects the interaction with Apaf-1, but a correlation between increased peroxidase activity of G41S cytochrome c and cytochrome c release from mitochondria resulted. The species dependent interaction of cytochrome c with Apaf-1 highlighted the importance of maintaining species in apoptosis assays, as sequence similarity did not correlate with conserved function. Additionally, the global structural similarity of cytochrome c did not indicate invariance of function. Mutation of Gly41 to Ser not only enhanced the in vivo apoptotic activity of cytochrome c via increased caspase activation in the cytosol, but also by a more efficient release of cytochrome c from mitochondria. These two mechanisms are independent and the combination of both mechanisms in a single variant is unprecedented