Cysteine 203 of Cyclophilin D Is Critical for Cyclophilin D Activation of the Mitochondrial Permeability Transition Pore

Systems Biology Center, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2011; 286(46):40184-92. DOI: 10.1074/jbc.M111.243469
Source: PubMed


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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    • "Adenine nucleotide translocator (ANT), which is required for ATP/ADP exchange, is also basally S-glutathionylated[53]. Decreased PGlu of ANT is associated with mitochondrial permeability transition pore (MPTP) opening and apoptosis in neurological tissue[53]. Similarly cyclophilin D is S-glutathionylated which prevents induction of mi- toptosis[54]. To complicate matters further GSH is also able to S-glutathionylate target proteins. "
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    ABSTRACT: At its core mitochondrial function relies on redox reactions. Electrons stripped from nutrients are used to form NADH and NADPH, electron carriers that are similar in structure but support different functions. NADH supports ATP production but also generates reactive oxygen species (ROS), superoxide (O2·-) and hydrogen peroxide (H2O2). NADH-driven ROS production is counterbalanced by NADPH which maintains antioxidants in an active state. Mitochondria rely on a redox buffering network composed of reduced glutathione (GSH) and peroxiredoxins (Prx) to quench ROS generated by nutrient metabolism. As H2O2 is quenched, NADPH is expended to reactivate antioxidant networks and reset the redox environment. Thus, the mitochondrial redox environment is in a constant state of flux reflecting changes in nutrient and ROS metabolism. Changes in redox environment can modulate protein function through oxidation of protein cysteine thiols. Typically cysteine oxidation is considered to be mediated by H2O2 which oxidizes protein thiols (SH) forming sulfenic acid (SOH). However, problems begin to emerge when one critically evaluates the regulatory function of SOH. Indeed SOH formation is slow, non-specific, and once formed SOH reacts rapidly with a variety of molecules. By contrast, protein S-glutathionylation (PGlu) reactions involve the conjugation and removal of glutathione moieties from modifiable cysteine residues. PGlu reactions are driven by fluctuations in the availability of GSH and oxidized glutathione (GSSG) and thus should be exquisitely sensitive to changes ROS flux due to shifts in the glutathione pool in response to varying H2O2 availability. Here, we propose that energy metabolism-linked redox signals originating from mitochondria are mediated indirectly by H2O2 through the GSH redox buffering network in and outside mitochondria. This proposal is based on several observations that have shown that unlike other redox modifications PGlu reactions fulfill the requisite criteria to serve as an effective posttranslational modification that controls protein function.
    Full-text · Article · Aug 2016
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    • "Previous studies have demonstrated that post-translational modifications of CyPD by phosphorylation and nitrosylation regulate PTP opening [12] [13] [14]. Other studies showed that CyPD can also be acetylated on lysine 166 [15] [16]. "
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    ABSTRACT: How ischemic postconditioning can inhibit opening of the mitochondrial permeability transition pore (PTP) and subsequent cardiac myocytes death at reperfusion remains unknown. Recent studies have suggested that de-acetylation of cyclophilin D (CyPD) by sirtuin 3 (SIRT3) can modulate its binding to the PTP. The aim of the present study was to examine whether ischemic postconditioning (PostC) might activate SIRT3 and consequently prevent lethal myocardial reperfusion injury through a deacetylation of CyPD. Using hypoxia-reoxygenation (H/R) in H9C2 cells, we showed that SIRT3 overexpression prevented CyPD acetylation, limited PTP opening and reduced cell death by 24%. In vitro modification of the CyPD acetylation status in MEFs by site-directed mutagenesis altered capacity of PTP opening by calcium. Calcium Retention Capacity (CRC) was significantly decreased with CyPD-KQ that mimics acetylated protein compared with CyPD WT (871± 266 vs 1193 ± 263 nmoles Ca(2+)/mg protein respectively). Cells expressing non-acetylable CyPD mutant (CyPD-KR) displayed 20% decrease in cell death compare to cells expressing CyPD WT after H/R. Correspondingly, in mice we showed that cardiac ischemic postconditioning could not reduce infarct size and CyPD acetylation in SIRT3 KO mice, and was unable to restore CRC in mitochondria as it is observed in WT mice. Our study suggests that the increased acetylation of CyPD following myocardial ischemia-reperfusion facilitates PTP opening and subsequent cell death. Therefore ischemic postconditioning might prevent lethal reperfusion injury through an increased SIRT3 activity and subsequent attenuation of CyPD acetylation at reperfusion. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Apr 2015 · Journal of Molecular and Cellular Cardiology
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    • "Indeed, ablation of CyP-D renders the PTP insensitive to CsA and doubles the threshold Ca 2+ load required to open the PTP in the presence of phosphate anion (Pi) [41] [42] [43] [44]. Notably, CyP-D can undergo several post-translation modifications, including phosphorylation [45], acetylation [46], and nitrosylation [47] [48]; CyP-D can also interact with a variety of proteins, such as Bcl-2 [49], the Ser/Thr kinase GSK-3 [45], the chaperones Hsp90, TRAP1 [50] and Hsp60 [51], and the FOF1 ATP synthase [52]. These dynamic changes of CyP-D affect the PTP, providing a remarkable level of flexibility in its modes of regulation. "
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    Full-text · Article · Oct 2014 · Cell Calcium
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