Key Event Title
Key Event Component
|protein glutathionylation||nitric oxide synthase, endothelial||increased|
|protein glutathionylation||cysteine residue||increased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Peptide Oxidation Leading to Hypertension||KeyEvent|
Level of Biological Organization
|endothelial cell of vascular tree|
|Homo sapiens||Homo sapiens||Moderate||NCBI|
|Bos taurus||Bos taurus||Moderate||NCBI|
|Mus musculus||Mus musculus||Weak||NCBI|
|Rattus norvegicus||Rattus norvegicus||Weak||NCBI|
How This Key Event Works
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). 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. This modification of eNOS also alters endothelium-dependent vasodilation, which can lead to hypertension. S-glutathionylation of eNOS can be reversed by treatment with reducing agents 2-mercaptoethanol or dithiothreitol, which restores eNOS activity.
How It Is Measured or Detected
There are four general approaches to detect protein S-glutathionylation (Pastore and Piemonte, 2013).
- 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.
- Labeling of Glutathione: Use 35S-cysteine radiolabeling or biotin labeling to detect glutathione adducts on S-thiolated proteins.
- 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.
- 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
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
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.
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.