Abstract
AimsEndothelial cells (ECs) constantly exposed to shear flow increase nitric oxide production via the activation of endothelial nitric oxide synthase. Nitric oxide-mediated S-nitrosylation has recently been identified as an important post-translational modification that may alter signalling and/or protein function. S-nitrosylation of endothelial proteins after shear flow treatment has not been fully explored. In this study, the CyDye switch method was utilized to examine S-nitrosylated proteins in ECs after exposure to shear flow.Methods and resultsHuman umbilical vein ECs were subjected to shear flow for 30 min, and S-nitrosylated proteins were detected by the CyDye switch method. In principle, free thiols in proteins become blocked by alkylation, the S-nitrosylated bond is reduced by ascorbate, and then CyDye labels proteins. Proteins that separately labelled with Cy3 or Cy5 were mixed and subjected to two-dimensional gel electrophoresis for further analysis. More than 100 S-nitrosoproteins were detected in static and shear-treated ECs. Among these, 12 major proteins of heterogeneous function showed a significant increase in S-nitrosylation following shear flow. The S-nitrosylated residues in tropomyosin and vimentin, which were localized in the hydrophobic motif of each protein, were identified as Cys170 and Cys328, respectively.ConclusionPost-translational S-nitrosylation of proteins in ECs can be detected by a reliable CyDye switch method. This flow-induced S-nitrosylation of endothelial proteins may be essential for the adaptation and remodelling of ECs under flow conditions.
Original language | English |
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Pages (from-to) | 536-546 |
Number of pages | 11 |
Journal | Cardiovascular Research |
Volume | 83 |
Issue number | 3 |
DOIs | |
Publication status | Published - Aug 2009 |
Keywords
- 2-D DIGE
- CyDye switch
- Endothelial cells
- S-nitrosylation
- Shear flow
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine
- Physiology (medical)
- Physiology