Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore.
Year: 2012
Type: article
Abstract: VOLUME 286 (2011) PAGES 40184–40192 The CypD cDNA plasmid used in our original manuscript from OriGene 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 ∼7:3 of the lower molecular weight (without the residual intronic sequence) to the higher molecular weight product.FIGURE 2Western blot indicating two product bands from the unedited plasmids. CypD−/− 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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−/− mice. As shown in Fig. 3, typically similar levels of WT CypD and C203S-CypD were expressed in CypD−/− MEFs. The corrected “Results” and figures are shown below. 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 H2O2 (46 + 13% versus 16 + 5%, Fig. 4A) and ionomycin-induced (79 + 5% versus 45 + 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−/− MEFs or CypD−/− 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 Ca2+ 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. To determine whether C203S-CypD would protect MEFs from H2O2-induced cell death, MEFs overexpressing WT CypD or C203S-CypD were treated with H2O2 for 6 h to induce cell death. After treatment, we determined the percent of cell death using the LIVE/DEAD viability kit (Molecular Probes). The KO MEFs overexpressing C203S-CypD showed reduced cell death as compared with KO MEFs expressing WT CypD (39.6 + 8.9% versus 21.7 + 3.4%, Fig. 5). Our results indicate the importance of cysteine 203 in modulating mPTP opening in response to oxidative stress-induced cell death and reconfirm the results of the publication.
Source: Journal of Biological Chemistry
Authors: Tiffany T. Nguyen, Mark V. Stevens, Mark Kohr, Charles Steenbergen, Michael N. Sack, Elizabeth Murphy
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W2586687464