Cyclosporine A-induced nitration of tyrosine 34 MnSOD in endothelial cells: Role of mitochondrial superoxide

Departamento Medicina Celular y Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Cardiovascular Research (Impact Factor: 5.94). 07/2010; 87(2):356-65. DOI: 10.1093/cvr/cvq028
Source: PubMed

ABSTRACT AimsCyclosporine A (CsA) has represented a fundamental therapeutic weapon in immunosupression for the past three decades. However, its clinical use is not devoid of side effects, among which hypertension and vascular injury represent a major drawback. Endothelial cells are able to generate reactive oxygen and nitrogen species upon exposure to CsA, including formation of peroxynitrite. This may result in endothelial cell toxicity and increased tyrosine nitration. We have now studied the subcellular origin of superoxide formation in endothelial cells treated with CsA and the biochemical consequences for the function of mitochondrial enzymes.Methods and resultsBy using electron spin resonance and endothelial cells lacking functional mitochondria, we showed that superoxide anion is generated in mitochondria. This was associated with an effect of CsA on bioenergetic parameters: increased mitochondrial membrane potential and inhibition of cellular respiration. In addition, CsA inhibited the activity of the mitochondrial enzymes aconitase and manganese superoxide dismutase (MnSOD). The use of murine lung endothelial cells deficient in endothelial nitric oxide synthase (eNOS) and NOS/peroxynitrite inhibitors allowed us to establish that the presence of eNOS and concomitant NO synthesis and peroxynitrite formation were essential for CsA induced nitration and inhibition of MnSOD activity. As the latter has been shown to become inactivated by nitration, we sought to identify this modification by mass spectrometry analysis. We found that CsA induced specific MnSOD tyrosine 34 nitration both in the recombinant protein and in endothelial cells overexpressing MnSOD.Conclusion
We propose that CsA induced endothelial damage may be related to increased mitochondrial superoxide formation and subsequent peroxynitrite-dependent nitroxidative damage, specifically targeting MnSOD. The inactivation of this key antioxidant enzyme by tyrosine nitration represents a pathophysiological cellular mechanism contributing to self-perpetuation and amplification of CsA-related vascular toxicity.

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Available from: Eduardo Rial, Sep 28, 2015
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    • "The nature of each peak was determined by selected MS/MS ion monitoring (SMIM) using a high-performance liquid chromatography (HPLC)-linear ion trap mass spectrometer. [31] [32] The detector was programmed to perform multiple fragmentations on doubly charged precursor ions [M + 2H] 2+ corresponding to the YCVQQLK peptide modified with IAM, NEM, DMD, DMBA or CPD (m/z 453.8, 503.8, 510.3, 489.2 and 518.1, respectively). "
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    ABSTRACT: Owing to its labile nature, a new role for cysteine sulfenic acid (–SOH) modification has emerged. This oxidative modification modulates protein function by acting as a redox switch during cellular signaling. The identification of proteins that undergo this modification represents a methodological challenge, and its resolution remains a matter of current interest. The development of strategies to chemically modify cysteinyl-containing peptides for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis has increased significantly within the past decade. The method of choice to selectively label sulfenic acid is based on the use of dimedone or its derivatives. For these chemical probes to be effective on a proteome-wide level, their reactivity toward –SOH must be high to ensure reaction completion. In addition, the presence of an adduct should not interfere with electrospray ionization, the efficiency of induced dissociation in MS/MS experiments or with the identification of Cys-modified peptides by automated database searching algorithms. Herein, we employ a targeted proteomics approach to study the electrospray ionization and fragmentation effects of different –SOH specific probes and compared them to commonly used alkylating agents. We then extend our study to a whole proteome extract using shotgun proteomic approaches. These experiments enable us to demonstrate that dimedone adducts do not interfere with electrospray by suppressing the ionization nor impede product ion assignment by automated search engines, which detect a + 138 Da increase from unmodified peptides. Collectively, these results suggest that dimedone can be a powerful tool to identify sulfenic acid modifications by high-throughput shotgun proteomics of a whole proteome. Copyright © 2014 John Wiley & Sons, Ltd.
    Journal of Mass Spectrometry 04/2014; 49(4). DOI:10.1002/jms.3336 · 2.38 Impact Factor
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    • "The drugs themselves could promote nitration, but we know of no evidence supporting this. Cyclosporin can promote tyrosine nitration in endothelial cells [34], but only two samples in this study were from patients taking cyclosporin and both were negative for NN. "
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    Arthritis research & therapy 02/2014; 16(1):R48. DOI:10.1186/ar4477 · 3.75 Impact Factor
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    • "Even though pretreatment of tempol or co-enzyme Q 10 significantly reduced superoxide overproduction and normalized the activities of mitochondrial complex enzymes, tempol or co-enzyme Q 10 did not increase MnSOD activity. Although the present study cannot explain this phenomenon, one possible explanation is that tempol and co-enzyme Q 10 did not stop the ischemia–reperfusioninduced MnSOD nitration because some studies have shown that MnSOD nitration decreases the MnSOD activity (Filipovic et al., 2007; Redondo-Horcajo et al., 2010; Tangpong et al., 2008), which needs further study. Nevertheless, this lack of effect by tempol or co-enzyme Q 10 on MnSOD activity may explain the partial improvement in the size muscle infarction by tempol or co-enzyme Q 10 in ischemia– reperfusion, and it is possible that other factors (such as calcium overload) also contribute to the ischemia–reperfusion injuries besides superoxide. "
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    European journal of pharmacology 10/2010; 650(1):328-34. DOI:10.1016/j.ejphar.2010.10.037 · 2.53 Impact Factor
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