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Cardiovascular Research Advance Access first published online on May 15, 2009
This version [Corrected Proof] published online on June 10, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp154
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2009. For permissions please email: journals.permissions@oxfordjournals.org.

Shear flow increases S-nitrosylation of proteins in endothelial cells

Bin Huang1, Shih Chung Chen2 and Danny Ling Wang1,*

1 Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, 128 sec. 2 Academia Rd. NanKang, Taipei 11529, Taiwan
2 Division of Cardiology, Taipei Medical University, Wan Fang Hospital, Taipei 11696, Taiwan

* Corresponding author. Tel: +886 2 23699132; fax: +886 2 27899143. E-mail address: lingwang{at}ibms.sinica.edu.tw

Aims: Endothelial 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 results: Human 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.

Conclusion: Post-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.

KEYWORDS Shear flow; S-nitrosylation; 2-D DIGE; CyDye switch; Endothelial cells

Time for primary review: 24 days


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