TY - JOUR
T1 - Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore
AU - Nguyen, Tiffany T.
AU - Stevens, Mark V.
AU - Kohr, Mark
AU - Steenbergen, Charles
AU - Sack, Michael N.
AU - Murphy, Elizabeth
PY - 2011/11/18
Y1 - 2011/11/18
N2 - 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 2O 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 2O 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 altermPTP 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 stressinduced activation of mPTP.
AB - 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 2O 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 2O 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 altermPTP 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 stressinduced activation of mPTP.
UR - http://www.scopus.com/inward/record.url?scp=81155123702&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=81155123702&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.243469
DO - 10.1074/jbc.M111.243469
M3 - Article
C2 - 21930693
AN - SCOPUS:81155123702
SN - 0021-9258
VL - 286
SP - 40184
EP - 40192
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -