Josephs TM et al. (2015), Interspecies Variation in the Functional Conseq...

XB-ART-50895

PLoS One 2015 Jun 18;106:e0130292. doi: 10.1371/journal.pone.0130292.

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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 .

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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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Species referenced: Xenopus
Genes referenced: apaf1 casp3.2 hdac1

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	Fig 2. 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).
	Fig 3. Interaction between cytochrome c and Apaf-1 in a human apoptosome model.Apaf-1 in grey (NBD, HD1, HD2 and WHD), pale blue (8 blade ß-propeller), lemon (7 blade ß-propeller) with the ß7 loop in orange. Cytochrome c in salmon-pink with the 40-to-57 Ω loop in magenta. Inset shows expansion of the cytochrome c 40-to-57 Ω loop with Gly-41 as a sphere and Apaf-1 as an electrostatic surface. Drawn from Protein Data Bank entry 3J2T.
	Fig 4. Variation at residue 44 does not alter the functional impact of the G41S mutation in mouse somatic cytochrome c.A and B. Human cytochrome c and mouse somatic cytochrome c differ in sequence at surface exposed residues. A. Human ferricytochrome c with side chains of residues differing between human and mouse somatic shown as sticks with carbon in pale blue, oxygen in red and nitrogen in blue. The backbone of the 40-to-57 Ω loop is in cyan. Drawn from Protein Data Bank entry 3NWV. B. Clustal Omega sequence alignment of human (P99999) and mouse somatic (P62897) cytochromes c with the initiating Met removed. The residues comprising the 40-to-57 Ω loop are in cyan and Gly41 is in bold. C. Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in mouse WEHI164 (100 μg) cytosols with the addition of 50 nM, 100 nM or 150 nM of mouse somatic WT (white bars), G41S (light grey bars), A44P (dark grey bars) or G41S/A44P (black bars) cytochrome c. The rate in the absence of cytochrome c was 2.74 ± 0.24 pmol/min AMC. Data are presented as mean ± SD (n = 3). *P < 0.001 compared to WT. Progress curves are shown in S4 Fig.
	Fig 5. Cytochromes c from different specifies have different abilities to activate caspases.Cleavage of the caspase 3 substrate Ac-DEVD-AMC was monitored in human U937 (100 μg, A) and mouse WEHI164 (100 μg, B) cytosols with the addition of 0 nM, 25 nM, 50 nM or 100 nM human (white bars), mouse somatic (grey bars) or horse (black bars) cytochrome c. Data are presented as mean ± SD (n = 3). P < 0.05, *P < 0.001 compared to human (A) or mouse (B).
	Fig 1. Platelet counts and formation are normal in Cycs G41S/G41S mice.A. Platelet counts of 8 week old Cycs +/+, Cycs +/G41S and Cycs G41S/G41S mice. B. Anti-platelet serum was injected into Cycs +/+ mice (□, n = 7–11) and Cycs G41S/G41S mice (△, n = 8–13) to deplete platelets and platelet count was monitored over 10 days. Data are presented as mean ± SEM.

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