The first naturally occurring mutation identified in the highly conserved cytochrome c protein was discovered in a New Zealand family. The missense mutation,\results in the substitution of glycine for serine at residue 41 (with the initiator methionine omitted). The mutation causes only mild autosomal dominant thrombocytopenia in family members heterozygous for the mutation.
Two additional naturally occurring missense mutations in the cytochrome c gene, Y48H and A51V (c.145T>C; p.Y49H, and c.155C>T; p.A52V respectively), which result in thrombocytopenia have subsequently been identified. The naturally occurring mutants are all clustered on the same loop of cytochrome c, suggesting a role for cytochrome c and the loop in platelet production.
Cytochrome c is a small haem protein bound to the inner mitochondrial membrane with two main functions. Firstly, the protein plays a key role in energy production, shuttling electrons from complex III to complex IV in the electron transport chain. Secondly, cytochrome c is a crucial protein in the initiation of apoptosis. Cytochrome c is released from the mitochondria, binds to and forms the apoptosome and activates the caspase cascade, and subsequently cell death. Apoptosis has been implicated in platelet production, but its role remains controversial. It is therefore of great interest to study the biochemistry of these naturally occurring cytochrome c mutants which result in thrombocytopenia.
Platelets are produced from a mature megakaryocyte, a blood progenitor cell in the bone marrow. To determine the effect that the cytochrome c G41S mutation has on megakaryocyte differentiation we aimed to establish the megakaryoblastic cell line (MEG-01) overexpressing the G41S mutation. However experiments to test the suitability of using the MEG-01 cell line showed that the differentiating agent PMA caused an increased endogenous cytochrome c expression. Overexpression of cytochrome c as also correlated with increased cell density. Thus we were unable use the MEG-01 cell line to determine the effects of the G41S mutation on megakaryocyte differentiation.
Secondly, we sought to functionally characterise the impact of two additional cytochrome c mutations, Y48H and A51V to determine which properties of cytochrome c contribute to the Thrombocytopenia phenotype. As previously reported for G41S cytochrome c A51V and Y48H mutations enhanced the ability of recombinant cytochrome c to activate caspases in vitro. In addition, the three mutants had normal redox potential, were equally as stable and had the correct haem incorporation. We also determined that platelets and lymphocytes from Thrombocytopenia Cargeeg subjects have no obvious defect in mitochondrial respiration, in the presence of the heterozygous G41S mutation.
