Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore.

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.

The CypD cDNA plasmid used in our original manuscript from Ori-Gene included a 62-base pair intronic fragment (Fig. 1). Unfortunately, we did not recognize the problem with this commercial plasmid until after publication of the paper. We evaluated whether this residual intron was cleaved during processing following transfection; however, this sequence was retained in the RNA isolated from the transfected mouse embryonic fibroblast (MEF) cells as assessed by RT-PCR with primers targeting the region spanning the 62-base pair insert. This finding is validated where we find that two protein products are generated whether we expressed the WT CypD or mutated plasmids in MEFs from mice lacking CypD (Fig 2). As shown in Fig. 2, there are no bands in MEFs from the CypD-KO mice (lane 2) and a single band for the WT MEFs (lane 1), but two bands were observed when we expressed either the mutant CypD (lanes 3 and 6) or WT CypD (lane 5). The two bands are at the correct molecular weight for the protein with and without the extra 62 bases, and we usually see a ratio of ϳ7:3 of the lower molecular weight (without the residual intronic sequence) to the higher molecular weight product.
To validate the results of our published work, we obtained a new plasmid for the WT CypD, where we confirmed the absence of the intronic region. As done in our article, we then mutated cysteine 203 to a serine residue (C203S). We then sequenced both plasmids to confirm their fidelity (Fig. 1). With the new plasmids, we repeated the key experiments of our paper to validate the results. We expressed the WT CypD and C203S-CypD in MEFs from the CypD Ϫ/Ϫ mice. As shown in Fig. 3, typically similar levels of WT CypD and C203S-CypD were expressed in CypD Ϫ/Ϫ MEFs. The corrected "Results" and figures are shown below.

Cysteine 203 of CypD Is Required for mPTP Opening-We
repeated key experiments to confirm the role of cysteine 203 of CypD in the inhibition of mPTP opening using the calcein AM-cobalt chloride quenching method and also by measuring calcium retention capacity (CRC). Using the calcein-cobalt method, we found that mutation of Cys-203 to a serine protected MEFs from H 2 O 2 (46 ϩ 13% versus 16 ϩ 5%, Fig.  4A) and ionomycin-induced (79 ϩ 5% versus 45 ϩ 7%, Fig. 4B) opening of the mPTP. Inhibition of mPTP in MEFs overexpressing mutant CypD was similar to that observed in the CypD null MEFs.
Consistent with the calcien-cobalt results, we found that CypD Ϫ/Ϫ MEFs or CypD Ϫ/Ϫ MEFs overexpressing C203S-CypD sequester more calcium than MEFs overexpressing WT CypD (Fig. 4, red or purple versus blue lines). Fig. 4D shows that mPTP opening is sensitive to CsA in KO MEFs transfected with WT CypD (turquoise line), but CsA offers no additional inhibition on Ca 2ϩ uptake in KO MEFs transfected with the mutated CypD (orange line). Taken altogether, these data confirm that Cys-203 is necessary for CypD to facilitate mPTP opening.  The mutated C203S-CypD plasmid sample was sequenced to confirm the site-directed mutagenesis. The replaced serine corresponding codon TCT is colored red.

C203S-CypD Reduces H 2 O 2 -induced Cell Death-To
FIGURE 2. Western blot indicating two product bands from the unedited plasmids. CypD Ϫ/Ϫ MEFs (KO) were transfected with pCMV-XL6 (control plasmids), old WT CypD, or old mutated C203S-CypD plasmids for 48 h. After 48 h of transfection, proteins were extracted, and samples were analyzed by Western blot analysis using anti-CypD or anti-actin (as a loading control). A representative blot is shown.