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Updated: Aug 6, 2026
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Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
S-nitrosoprotein formation and localization in endothelial cells
1Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, MA 02118, USA.
Summary
Researchers developed a new method to detect protein S-nitrosation in endothelial cells. This technique identified key S-nitrosated proteins, like GAPDH, revealing nitric oxide
Area of Science:
- Biochemistry
- Cell Biology
- Proteomics
Background:
- Protein S-nitrosation is a crucial, yet challenging to study, post-translational modification.
- Existing methods lack specificity and are hindered by the instability of cellular S-nitrosothiols.
Purpose of the Study:
- To develop a rapid and specific method for detecting endothelial S-nitrosoproteins in situ.
- To identify specific S-nitrosated proteins within endothelial cells and understand their cellular localization and dynamics.
Main Methods:
- Adaptation of the biotin switch method for endothelial cells, involving thiol alkylation and reductive thiol generation.
- Labeling of newly formed thiols with biotin or Texas red derivatives.
- Confocal microscopy for subcellular localization and half-life determination.
- Proteomic analysis using 2D gel electrophoresis and mass spectrometry.
Main Results:
- Successfully detected S-nitrosated proteins formed from exogenous donors or endogenous nitric oxide (NO) production.
- S-nitrosoproteins were primarily localized in mitochondria and peri-mitochondrial compartments with a half-life of approximately 1 hour.
- Identified key S-nitrosated proteins including GAPDH, vimentin, beta-galactosidase, peroxiredoxin 1, beta-actin, and ubiquitin-conjugating enzyme E2.
- GAPDH was the most abundant S-nitrosated protein in resting endothelial cells, suggesting a role in NO-mediated glycolysis regulation.
Conclusions:
- The developed method enables specific detection and identification of endothelial S-nitrosoproteins.
- S-nitrosation is a dynamic process in endothelial cells, with specific protein targets and subcellular localization.
- Findings suggest a regulatory role for NO via S-nitrosation in endothelial cell function, particularly in glycolysis.
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