Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c
Tracy M Josephs1, Moira E Hibbs1, Lily Ong1
1Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.
Insights
The human cytochrome c G41S variant causes thrombocytopenia, but this effect isn't seen in mice. This is because the G41S mutation impacts caspase activation differently across species, revealing species-specific interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- A human cytochrome c variant (G41S) causes mild thrombocytopenia (Thrombocytopenia Cargeeg) due to platelet production defects.
- The molecular mechanisms underlying this platelet defect remain unclear.
- Cytochrome c is highly conserved between humans and mice, yet a G41S knockin mouse model did not replicate the thrombocytopenia phenotype.
Purpose of the Study:
- To investigate the species-specific functional impact of the cytochrome c G41S mutation on caspase activation.
- To understand the disparity between human thrombocytopenia and the lack of a similar phenotype in the mouse model.
- To elucidate the species-specific interactions between cytochrome c and Apaf-1.
Main Methods:
- Generation of a mouse model with the G41S mutation in mouse cytochrome c (CycsG41S/G41S).
- Assays to measure caspase activation by wild-type and G41S cytochrome c from different species (human, mouse).
- Testing cytochrome c activity in Xenopus embryo extracts.
Main Results:
- The G41S mutation decreased caspase activation in mouse cytochrome c, contrasting with its effect in human cytochrome c.
- G41S mouse cytochrome c failed to activate caspases in Xenopus embryo extracts, unlike wild-type human cytochrome c.
- These findings highlight a species-specific interaction between cytochrome c and Apaf-1.
Conclusions:
- The functional consequences of cytochrome c mutations, specifically G41S, are species-dependent.
- Interactions between cytochrome c and Apaf-1 are not solely determined by electrostatic interactions, indicating other specificity factors.
- These results are crucial for interpreting studies on cytochrome c mutations and the intrinsic apoptosis pathway.
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 (CycsG41S/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.
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