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. 2011 Nov 18;286(46):40184-92.
doi: 10.1074/jbc.M111.243469. Epub 2011 Sep 19.

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

Tiffany T Nguyen  1 , Mark V Stevens, Mark Kohr, Charles Steenbergen, Michael N Sack, Elizabeth Murphy
Affiliations

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

Tiffany T Nguyen et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
MS/MS spectra for CypD. Representative MS/MS spectra showing fragmentation of the KIVITDC*QLS peptide (IonScore, 38). Peaks in the spectrum that are marked red correspond to matched b ions and peaks that are marked blue correspond to matched y ions. The number paired with each ion identification (i.e. b2, y4, etc.) indicates the number of amino acids present on N-terminal fragments for b ions and C-terminal fragments for y ions. This peptide identification was observed with ischemic preconditioning in two of five biological replicates; this modification was not observed in any of the hearts with control perfusion.
FIGURE 2.
FIGURE 2.
H2O2-induced mPTP opening is blocked by GSNO. In A, CypD WT MEFs were transfected with pCMV-XL6 (control (Con) plasmids). CypD−/− MEFs (KO) were transfected either with pCMV-XL6 (control plasmids) or WT CypD for 48 h as labeled in each panel. After transfections, mPTP opening was assessed using H2O2 in the presence and absence of a NO donor, GSNO, using the calcein-cobalt quenching technique. *, p < 0.05 versus WT+empty or KO+CypD (n = 5). In B, PPIase activity was measured using recombinant CypD in the presence of 0, 0.25, 0.5, and 1 mm GSNO. The reaction was initiated with PPIase substrate and chymotrypsin as described under “Experimental Procedures.” The kobs for PPIase activity was calculated using non-linear regression analysis as described under “Experimental Procedures.” The k0 is the PPIase activity in the absence of CypD (the non-enzymatic rate) was also calculated using non-linear regression and was subtracted from the kobs. The results represent average measurements from three independent assays.
FIGURE 3.
FIGURE 3.
Confirmation of C203S-CypD mutation by restriction digestion and DNA sequencing. In A, 1 μg of mutated CypD (mCypD) plasmid was digested with 2 units of restriction enzyme, NotI, at 37 °C for 1 h. The resulting reaction and undigested samples were analyzed on a 1% agarose gel. A representative ethidium bromide stained gel is shown. In B, a mutated C203S-CypD plasmid sample was sequenced to confirm the site-directed mutagenesis. The replaced serine corresponding codon TCT is in boldface.
FIGURE 4.
FIGURE 4.
Expression of mutated C203S-CypD in CypD−/− MEFs. CypD−/− MEFs (KO) were transfected with pCMV-XL6 (control plasmids), WT CypD, or 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-β-actin (as a loading control). Representative autoradiographs are shown (n = 4). mCypD, mutant CypD.
FIGURE 5.
FIGURE 5.
mPTP opening is blocked by C203S-CypD. CypD−/− MEFs (KO) were transfected either with WT CypD or C203S-CypD plasmids for 48 h as labeled in each panel. After transfections, mPTP opening was assessed using H2O2 (A and B) or ionomycin (C) in the presence and absence of an NO donor, GSNO, or CsA using the calcein-cobalt quenching technique. In D, after transfections, CRC was measured using calcium-sensitive probe calcium green-5N in the presence of 5 μm Ca2+ pulses. A representative figure is shown from three independent assays. *, p < 0.05 versus WT+empty or KO+CypD (n = 5). mCypD, mutant CypD.
FIGURE 6.
FIGURE 6.
C203S-CypD reduces H2O2-induced cell death. CypD−/− MEFs (KO) were transfected with WT CypD or mutated C203S-CypD plasmids for 48 h. After transfections, MEFs were treated with 1 mm H2O2 for 6 h to induce cell death. Cell death was assessed using the LIVE/DEAD viability kit (Molecular Probes) according to the manufacturer's instructions. The results represent average measurements from three independent assays. mCypD, mutant CypD. *, p < 0.05, versus KO + WT CypD in the presence of H2O2.
FIGURE 7.
FIGURE 7.
Induction of mutated C203S-CypD expression in the liver mitochondria of CypD−/− mice abolished Ca2+-induced mPTP opening. Liver samples were obtained 72 h after adenovirus injections with an adenoviral vector driving WT CypD, mutated C203S-CypD, or AdCMV-GFP (used as a control) expressions. Immunoblot analysis of 40 μg of mitochondrial protein extracted from mouse livers, and the resulting blots were probed with anti-CypD, GFP, and β-ATP synthase (ATPsyn) antibodies. Representative blots are shown (n = 6). mCypD, mutant CypD.
FIGURE 8.
FIGURE 8.
C203S-CypD expression in the liver mitochondria of CypD−/− mice does not alter mitochondrial oxygen consumption and membrane potential. Liver mitochondrial samples were obtained as described in Fig. 7. Respiratory control ratio (RCR) (A) was determined using glutamate and malate as substrates by calculating state 3/state 2 using a Oxygraph electrode. In B, membrane potential was monitored using the fluorescent dye TMRE in coupled respiring mitochondria and then in the presence of 1 μm protonophore carbonyl cyanide p-chlorophenylhydrazone. In C, mitochondrial swelling assay was measured by absolute absorbance at baseline and after Ca2+ (250 μm) addition in the presence and absence of CsA in isolated liver mitochondria from each of the indicated groups as described in A. The data show the profile of absorbance at 540 nm of mitochondrial suspensions after exposure to 250 μm CaCl2. The results represent average measurements from four independent preparations of liver mitochondria from separate mice. In D, CRC was measured using the calcium-sensitive probe calcium green-5N in the presence of 10 μm and 50 μm Ca2+ pulses. mCypD, mutant CypD.
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