Shear flow increases S-nitrosylation of proteins in endothelial cells.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Huang
Bin
B
Cardiovascular Division, Institute of Biomedical Sciences, Academia Sinica, 128 sec. 2 Academia Rd. NanKang, Taipei 11529, Taiwan.
Chen
Shih Chung
SC
Wang
Danny Ling
DL
D000222:Adaptation, Physiological; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002232:Carbocyanines; D002478:Cells, Cultured; D003545:Cysteine; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D013314:Stress, Mechanical; D014335:Tropomyosin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D011506:Proteins; D014335:Tropomyosin; D014746:Vimentin; C089187:cyanine dye 3; C085321:cyanine dye 5; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D003545:Cysteine