Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c

Abstract

The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (Cycs G41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.

Multifunctional Cytochrome c: Learning New Tricks from an Old Dog

“…Noteworthy, while G41 is a highly conserved residue in all eukaryotic cyt c , the effect of H-cyt c G41S in binding Apaf-1 appears to be species-specific. Substitution of G41 in mouse or in Xenopus led to a marked decrease in caspase activation in embryo extracts, an effect not observed in the human counterpart …”

“…Substitution of G41 in mouse or in Xenopus led to a marked decrease in caspase activation in embryo extracts, an effect not observed in the human counterpart. 759 5.3.2. Cytochrome c Tyr48His.…”

Increased dynamics in the 40–57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

“…This has been highlighted recently through the discovery of a species-dependent interaction between cyt and Apaf-1 in cytosolic extracts, which is proposed to arise due to sequence variation between mitochondrial species in the 40–57 Ω-loop 45 . Further to this discovery, residues flanking Pro44 have for some time been associated with a conformational change in cells undergoing apoptosis 46 and recent modelling studies suggest H-cyt interacts with Apaf-1 through contact via the 40–57 Ω-loop 45 47 . Thus based on the present work we propose that the dynamics of 40–57 Ω-loop may be species-specific and tuned to regulate interaction with the cognate Apaf-1.…”

Altered structure and dynamics of pathogenic cytochrome c variants correlate with increased apoptotic activity

“…Furthermore, peripheral blood mononuclear cells from subjects with G41S cytochrome c do not show altered susceptibility to apoptotic stimuli (12). Interestingly, in the context of mouse cytochrome c, the G41S mutation causes increased peroxidase activity, decreased apoptosome activation, and is not associated with thrombocytopenia (13,17). This suggests that it may be the combination of impact on apoptosome activation and peroxidase activity that triggers abnormal platelet formation.…”

“…The Y48H mutation increases the apoptotic activity of mouse cytochrome c (9). However cytochrome c-induced apoptosis is species specific (17) and the impact of the A51V and Y48H mutation on apoptosome activation by human cytochrome c has not previously been determined. We now show that the Y48H and A51V mutations both caused an increase in cytochrome c-induced apoptosome activation compared to WT (Table 1), using a cell-free human caspase activity assay.…”