title: Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore.
abstract: The mitochondrial permeability transition pore (mPTP) opening plays a critical role in mediating cell death during ischemia/reperfusion (I/R) injury. Our previous studies have shown that cysteine 203 of cyclophilin D (CypD), a critical mPTP mediator, undergoes protein S-nitrosylation (SNO). To investigate the role of cysteine 203 in mPTP activation, we mutated cysteine 203 of CypD to a serine residue (C203S) and determined its effect on mPTP opening. Treatment of WT mouse embryonic fibroblasts (MEFs) with H(2)O(2) resulted in an 50% loss of the mitochondrial calcein fluorescence, suggesting substantial activation of the mPTP. Consistent with the reported role of CypD in mPTP activation, CypD null (CypD(-/-)) MEFs exhibited significantly less mPTP opening. Addition of a nitric oxide donor, GSNO, to WT but not CypD(-/-) MEFs prior to H(2)O(2) attenuated mPTP opening. To test whether Cys-203 is required for this protection, we infected CypD(-/-) MEFs with a C203S-CypD vector. Surprisingly, C203S-CypD reconstituted MEFs were resistant to mPTP opening in the presence or absence of GSNO, suggesting a crucial role for Cys-203 in mPTP activation. To determine whether mutation of C203S-CypD would alter mPTP in vivo, we injected a recombinant adenovirus encoding C203S-CypD or WT CypD into CypD(-/-) mice via tail vein. Mitochondria isolated from livers of CypD(-/-) mice or mice expressing C203S-CypD were resistant to Ca(2+)-induced swelling as compared with WT CypD-reconstituted mice. Our results indicate that the Cys-203 residue of CypD is necessary for redox stress-induced activation of mPTP.
authors.affiliation: Systems Biology Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Tiffany T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Mark V
authors.initials: MV
authors.lastname: Stevens
authors.forename: Mark
authors.initials: M
authors.lastname: Kohr
authors.forename: Charles
authors.initials: C
authors.lastname: Steenbergen
authors.forename: Michael N
authors.initials: MN
authors.lastname: Sack
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D020125:Mutation, Missense; D020030:Nitric Oxide Donors; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects
chemical_list: D000081406:Peptidyl-Prolyl Isomerase F; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D020030:Nitric Oxide Donors; D016877:Oxidants; C000631446:PPIF protein, mouse; D006861:Hydrogen Peroxide; D021983:Cyclophilins; D003545:Cysteine; D002118:Calcium