An Inter-Species Investigation into the Role of Cytochrome c in Apoptosis
Cytochrome c is a protein that has important roles in electron transport and cell death (apoptosis). In its role during the intrinsic apoptosis pathway, cytochrome c is released from the mitochondria for it to interact with the cytosolic protein Apaf-1 at its WD40 domain. The binding event between cytochrome c and Apaf-1 triggers the formation of the heptameric apoptosome which activates caspase enzymes for cell death. The first naturally occurring mutation in cytochrome c, characterised as a substitution of glycine to serine at position 41 (G41S), was identified in a New Zealand family with inherited thrombocytopenia. This mutation was found to cause an increase in the ability of human cytochrome c to activate caspases. The same G41S mutation when introduced into mouse cytochrome c exhibited the opposite effect with a decrease in caspase activation compared to mouse wildtype. The molecular mechanisms that underly these species-specific effects of the G41S mutation are unknown, though the dynamics of the 40-57 Ω-loop has been suggested to play a role. This thesis reports analysis of molecular dynamics simulations of mouse WT and G41S cytochrome c compared against human WT and G41S cytochrome c simulation data. Characterisation of the global stabilities of mouse and human cytochrome c variants and trialled expression of a Apaf-1 WD40 domain construct are also reported. Expression trials of the Apaf-1 WD40 domain construct ∆N594 in insect cell lines showed high expression but little solubility upon purification. Future work involving the redesign of a WD40 domain construct will be required to achieve a useable construct for investigating the interaction of Apaf-1 with cytochrome c. Molecular dynamics simulations of mouse WT and G41S cytochrome c showed no clear difference in mobility to human WT cytochrome c with these three variants not exhibiting any large conformational shifts. Human G41S shows a large increase in mobility, especially in the 40-57 Ω-loop region, over that of human WT cytochrome c. This indicates species-specific effects of the G41S mutation on mobility. Analysis of the global stabilities of WT and G41S cytochrome c of each species showed there is no difference in Tm. Human WT, mouse WT and mouse G41S exhibited the same thermodynamic parameters. Human G41S however showed a decrease in enthalpy and increase in entropy, much lower than the other three variants, indicating hG41S has a more disordered native structure. This aligns with the observations from the molecular dynamics simulations. It is likely that the increased mobility of human G41S may explain the increase in caspase activation seen with this variant. However, the dynamics do not explain the decrease in caspase activation seen with mouse G41S. Species variation in the WD40 domain of Apaf-1 may play a role in determining this effect, where mouse and human Apaf-1 may allow different ranges of mobility for interacting residues on cytochrome c to achieve tight binding.
Advisor: Ledgerwood , Elizabeth; Wilbanks, Sigurd
Degree Name: Master of Science
Degree Discipline: Biochemistry
Publisher: University of Otago
Keywords: Cytochrome c; Apoptosis; Molecular Dynamics; Apaf-1
Research Type: Thesis