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Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c.

Josephs TM, Hibbs ME, Ong L, Morison IM, Ledgerwood EC - PLoS ONE (2015)

Bottom Line: 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.Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1.These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.

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.

No MeSH data available.


Related in: MedlinePlus

Mutations at residue 41 have species-specific effects on caspase activation.A. and B. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in A. mouse WEHI164 (100 μg) and B. human U937 (100 μg) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM mouse somatic WT (white bars) or G41S (black bars) cytochrome c at pH 7.25. All reactions contained 1 mM dATP. Data are presented as mean ± SD (n = 3). *P < 0.01, **P < 0.001 compared to WT. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in Xenopus cytosolic extracts (2 μg) with the addition of 100 nM human cytochrome c without (white bars) or with (black bars) the addition of human Apaf-1. Data are presented as mean ± SD (n = 3). *p < 0.01, **p < 0.001 compared to without human Apaf-1. Progress curves are shown in S2B–S2D Fig).
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pone.0130292.g002: Mutations at residue 41 have species-specific effects on caspase activation.A. and B. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in A. mouse WEHI164 (100 μg) and B. human U937 (100 μg) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM mouse somatic WT (white bars) or G41S (black bars) cytochrome c at pH 7.25. All reactions contained 1 mM dATP. Data are presented as mean ± SD (n = 3). *P < 0.01, **P < 0.001 compared to WT. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in Xenopus cytosolic extracts (2 μg) with the addition of 100 nM human cytochrome c without (white bars) or with (black bars) the addition of human Apaf-1. Data are presented as mean ± SD (n = 3). *p < 0.01, **p < 0.001 compared to without human Apaf-1. Progress curves are shown in S2B–S2D Fig).

Mentions: There were two possible explanations for the difference in phenotype between Thrombocytopenia Cargeeg subjects and the CycsG41S/G41S knockin mice. There are differences in platelet formation between humans and mice, with mice producing a higher number of smaller platelets [22]. Thus the role of cytochrome c in platelet formation may not be conserved between humans and mice. In keeping with this there is substantial evidence that neither the intrinsic nor extrinsic apoptosis pathway is necessary for platelet formation in mice [12,21]. Alternatively the functional effect of the G41S mutation may not be conserved between human and mouse cytochrome c. To investigate the latter explanation, the activities of WT and G41S somatic mouse cytochromes c were assayed using cytosolic extracts obtained from either mouse or human cell lines (Fig 2). We have previously reported that human G41S cytochrome c has up to 2-fold increased caspase-inducing activity compared to human WT cytochrome c [1,5] (S2A Fig). In contrast here we observed that mouse G41S cytochrome c had 2-fold reduced caspase-inducing activity compared to mouse WT cytochrome c in mouse cytosolic extracts (Fig 2A) although interestingly the same activity as mouse WT cytochrome c in human cytosolic extracts (Fig 2B). In contrast de Rocco et al. reported that Cycs-/-/Cyct-/- mouse lung fibroblasts expressing mouse G41S cytochrome c were significantly more sensitive to activation of the intrinsic apoptosis pathway compared to Cycs-/-/Cyct-/- mouse lung fibroblasts expressing WT mouse cytochrome c [2]. The reason for the discrepancy between the results with recombinant protein and transduced cell lines is unclear. However the absence of thrombocytopenia in the CYCG41S/G41S mice supports our finding of lack of functional conservation of the proapoptotic effect of the G41S mutation from human to mouse cytochrome c.


Interspecies Variation in the Functional Consequences of Mutation of Cytochrome c.

Josephs TM, Hibbs ME, Ong L, Morison IM, Ledgerwood EC - PLoS ONE (2015)

Mutations at residue 41 have species-specific effects on caspase activation.A. and B. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in A. mouse WEHI164 (100 μg) and B. human U937 (100 μg) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM mouse somatic WT (white bars) or G41S (black bars) cytochrome c at pH 7.25. All reactions contained 1 mM dATP. Data are presented as mean ± SD (n = 3). *P < 0.01, **P < 0.001 compared to WT. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in Xenopus cytosolic extracts (2 μg) with the addition of 100 nM human cytochrome c without (white bars) or with (black bars) the addition of human Apaf-1. Data are presented as mean ± SD (n = 3). *p < 0.01, **p < 0.001 compared to without human Apaf-1. Progress curves are shown in S2B–S2D Fig).
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pone.0130292.g002: Mutations at residue 41 have species-specific effects on caspase activation.A. and B. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in A. mouse WEHI164 (100 μg) and B. human U937 (100 μg) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM mouse somatic WT (white bars) or G41S (black bars) cytochrome c at pH 7.25. All reactions contained 1 mM dATP. Data are presented as mean ± SD (n = 3). *P < 0.01, **P < 0.001 compared to WT. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in Xenopus cytosolic extracts (2 μg) with the addition of 100 nM human cytochrome c without (white bars) or with (black bars) the addition of human Apaf-1. Data are presented as mean ± SD (n = 3). *p < 0.01, **p < 0.001 compared to without human Apaf-1. Progress curves are shown in S2B–S2D Fig).
Mentions: There were two possible explanations for the difference in phenotype between Thrombocytopenia Cargeeg subjects and the CycsG41S/G41S knockin mice. There are differences in platelet formation between humans and mice, with mice producing a higher number of smaller platelets [22]. Thus the role of cytochrome c in platelet formation may not be conserved between humans and mice. In keeping with this there is substantial evidence that neither the intrinsic nor extrinsic apoptosis pathway is necessary for platelet formation in mice [12,21]. Alternatively the functional effect of the G41S mutation may not be conserved between human and mouse cytochrome c. To investigate the latter explanation, the activities of WT and G41S somatic mouse cytochromes c were assayed using cytosolic extracts obtained from either mouse or human cell lines (Fig 2). We have previously reported that human G41S cytochrome c has up to 2-fold increased caspase-inducing activity compared to human WT cytochrome c [1,5] (S2A Fig). In contrast here we observed that mouse G41S cytochrome c had 2-fold reduced caspase-inducing activity compared to mouse WT cytochrome c in mouse cytosolic extracts (Fig 2A) although interestingly the same activity as mouse WT cytochrome c in human cytosolic extracts (Fig 2B). In contrast de Rocco et al. reported that Cycs-/-/Cyct-/- mouse lung fibroblasts expressing mouse G41S cytochrome c were significantly more sensitive to activation of the intrinsic apoptosis pathway compared to Cycs-/-/Cyct-/- mouse lung fibroblasts expressing WT mouse cytochrome c [2]. The reason for the discrepancy between the results with recombinant protein and transduced cell lines is unclear. However the absence of thrombocytopenia in the CYCG41S/G41S mice supports our finding of lack of functional conservation of the proapoptotic effect of the G41S mutation from human to mouse cytochrome c.

Bottom Line: 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.Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1.These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Otago School of Medical Sciences, University of Otago, Dunedin, New Zealand.

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.

No MeSH data available.


Related in: MedlinePlus