API

Event: 927

Key Event Title

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KE1 : S-Glutathionylation, eNOS

Short name

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S-Glutathionylation, eNOS

Key Event Component

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Process Object Action
protein glutathionylation nitric oxide synthase, endothelial increased
protein glutathionylation cysteine residue increased

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Peptide Oxidation Leading to Hypertension KeyEvent

Stressors

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Level of Biological Organization

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Biological Organization
Molecular

Cell term

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Cell term
endothelial cell of vascular tree


Organ term

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Taxonomic Applicability

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Term Scientific Term Evidence Link
Homo sapiens Homo sapiens Moderate NCBI
Bos taurus Bos taurus Moderate NCBI
Mus musculus Mus musculus Weak NCBI
Rattus norvegicus Rattus norvegicus Weak NCBI

Life Stage Applicability

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Life stage Evidence
All life stages Strong

Sex Applicability

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Term Evidence
Unspecific Strong

How This Key Event Works

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S-glutathionylation is a redox-dependent, reversible post-translational modification that is involved in the regulation of various regulatory, structural, and metabolic proteins (Pastore and Piemonte, 2013). Under oxidative stress, S-glutathionylation targets cysteine residues of a protein and adds glutathione through thiol-disulfide exchange with oxidized glutathione (GSSG) or reaction of oxidant-induced protein thiyl radicals with reduced glutathione (Chen et al., 2010, 2011, Schuppe et al. 1992). Endothelial nitric oxide synthase (eNOS) regulates vascular function by generating nitric oxide which is involved in endothelium-dependent relaxation, and control of blood pressure and vascular tone. It has been shown that cysteine residues are important for the maintenance of normal eNOS function. Under oxidative stress, S-glutathionylation of eNOS was induced by GSSG at residue sites Cys 689 and Cys 908, resulting in a decrease in eNOS activity and an increase in superoxide generation, also known as eNOS uncoupling. Furthermore, eNOS S-glutathionylation was shown to be abundant in the vessel walls of spontaneously hypertension rats (SHRs), in contrast to non-hypertensive rats.   SHRs demonstrated impaired endothelium-dependent vasodilation, which was reversible upon administration of the reducing agent, dithiothreitol (Chen et al. 2010).  Similarly in human aortic endothial cells, exposure to ultrafine particles caused a decrease in NO production in a dose-depedent manner.  This was shown to be prevented upon over-expression of glutaredoxin-1, which inhibits eNOS S-glutathionylation (Du et al. 2013).


How It Is Measured or Detected

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There are four general approaches to detect protein S-glutathionylation (Pastore and Piemonte, 2013).

  1. Quantification of Total S-Glutathionylated Proteins: Use sample lysis or homogenization in non-reducing buffer containing N-ethylmaleimide to eliminate thiols, followed by protein precipitation, reduction of gluthionyl-protein adducts, and derivatization of protein thiols or free glutathione with fluorescence probes. Fluorescence can be measured by fluorometric analysis with or without prior HPLC separation. This method allows for quantification of glutathionylated proteins but cannot detect glutathione adducts on specific proteins.
  2. Labeling of Glutathione: Use 35S-cysteine radiolabeling or biotin labeling to detect glutathione adducts on S-thiolated proteins.
  3. Use of Anti-Glutathione Antibodies: Use commercially available anti-glutathione to detect glutathionylated proteins by Western blots, immunoprecipitation or immunocytolocalization. This method is useful for analysis of individual proteins like eNOS but not for large-scale detection of glutathionylated proteins.
  4. Top-Down Proteomic Approach: Use liquid chromatography-coupled mass spectrometry to identify S-glutathionylated proteins on whole protein extract from cells without using labeling or anti-glutathione antibody.

Evidence Supporting Taxonomic Applicability

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S-glutathionylation of eNOS has been demonstrated in humans, cows, mice and rats (Chen et al., 2010; De Pascali et al., 2014; Du et al., 2013).


Evidence for Perturbation by Stressor



References

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Chen, C.-A., Wang, T.-Y., Varadharaj, S., Reyes, L.A., Hemann, C., Talukder, M.A.H., Chen, Y.-R., Druhan, L.J., and Zweier, J.L. (2010). S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118.

Chen CA, Lin CH, Druhan LJ, Wang TY, Chen YR, Zweier JL.  Superoxide induces endothelial nitric-oxide synthase protein thiyl radical formation, a novel mechanism regulating eNOS function and coupling.  J Biol Chem. 2011 286(33):29098-107.

De Pascali, F., Hemann, C., Samons, K., Chen, C.-A., and Zweier, J.L. (2014). Hypoxia and reoxygenation induce endothelial nitric oxide synthase uncoupling in endothelial cells through tetrahydrobiopterin depletion and S-glutathionylation. Biochemistry (Mosc.) 53, 3679–3688.

Du, Y., Navab, M., Shen, M., Hill, J., Pakbin, P., Sioutas, C., Hsiai, T.K., and Li, R. (2013). Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS. Biochem. Biophys. Res. Commun. 436, 462–466.

Pastore, A., and Piemonte, F. (2013). Protein glutathionylation in cardiovascular diseases. Int. J. Mol. Sci. 14, 20845–20876.

Schuppe I, Moldéus P, and Cotgreave IA. Protein-specific S-thiolation in human endothelial cells during oxidative stress. (1992) Biochem. Pharmacol. 44: 1757–1764.