Shear flow increases S-nitrosylation of proteins in endothelial cells

Bin Huang, Shih Chung Chen, Danny Ling Wang

Research output: Contribution to journalArticle

59 Citations (Scopus)

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 languageEnglish
Pages (from-to)536-546
Number of pages11
JournalCardiovascular Research
Volume83
Issue number3
DOIs
Publication statusPublished - Aug 2009

Fingerprint

Protein S
Endothelial Cells
Proteins
Nitric Oxide
Tropomyosin
Umbilical Veins
Amino Acid Motifs
Nitric Oxide Synthase Type III
Electrophoresis, Gel, Two-Dimensional
Alkylation
Vimentin
Post Translational Protein Processing
Sulfhydryl Compounds

Keywords

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

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)
  • Physiology

Cite this

Shear flow increases S-nitrosylation of proteins in endothelial cells. / Huang, Bin; Chen, Shih Chung; Wang, Danny Ling.

In: Cardiovascular Research, Vol. 83, No. 3, 08.2009, p. 536-546.

Research output: Contribution to journalArticle

Huang, Bin ; Chen, Shih Chung ; Wang, Danny Ling. / Shear flow increases S-nitrosylation of proteins in endothelial cells. In: Cardiovascular Research. 2009 ; Vol. 83, No. 3. pp. 536-546.
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AB - 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.

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