Abstract
Cytochrome c is a small haem-containing protein that as an essential role in the electron transport chain as an electron carrier. Cytochrome c also plays a fundamental role in the initiation of the intrinsic apoptotic pathway. Intrinsic apoptosis is a process by which cytochrome c is released from the mitochondria into the cytosol where it is activates Apaf-1 upon binding to the Apaf-1 WD40 domains. Seven activated Apaf-1 proteins then come together to form the apoptosome. The apoptosome functions as a platform to recruit and activate caspases for cellular degradation.
Four naturally occurring mutations in human cytochrome c result in the disease of mild autosomal dominant thrombocytopenia, characterised by low blood platelet counts. Cytochrome c mutants known to cause this disease are G41S, Y48H, A51V, and H26Y. Three of the four mutations have been found to increase in vitro cytochrome c-induced caspase activity, except for the H26Y mutation whose effect on caspase activity is currently unknown. The mechanistic link between increased caspase activation and decreased blood platelet count is also unknown.
Mouse and human cytochrome c share a 91.4% sequence identity so it was expected that when a G41S knock-in mouse was made, it would have the thrombocytopenia phenotype. However, the G41S knock-in mouse had no change in phenotype, highlighting a potential species-specific effect between the mutation in mouse and human cytochrome c. Furthermore, in vitro analysis of mouse G41S cytochrome c found that it causes a decrease in caspase activity, thereby suggesting that the species-specific effects are due to differences within the cytochrome c-Apaf-1 interaction.
The source of species-specific changes between the mouse and human cytochrome c-Apaf-1 interaction is unknown. However, it is known that all the mutations occur within or are associated with the 40-57 Ω loop of cytochrome c, forming the hypothesis that the loop may play an important role in the affinity of binding between cytochrome c and Apaf-1. Moreover, out of eight residues that are differential between mouse and human cytochrome c, three of them are within the 40-57 Ω loop. This suggests there may be some evolutionary influence on the changes within the cytochrome c-Apaf-1 interaction.
This thesis aimed to determine the impact of the Y48H, A51V, and H26Y mutations on the ability of mouse cytochrome c to activate caspases in vitro and to use bioinformatic analysis to identify co-evolving residues in cytochrome c and Apaf-1 that may explain the differences of the cytochrome c-Apaf-1 interaction between Metazoan species. The results conclude that the thrombocytopenia mutations in mouse cytochrome c do cause species-specific effects on caspase activation, as there were no changes in caspase activity in mouse cell-free lysate. The bioinformatic analyses on Metazoan cytochrome c and Apaf-1 sequences did not detect any co-evolving residues within the sequences that could have explained the potential species-specific effects on cytochrome c-Apaf-1 binding.