Acute hypoxia enhances proteins' S-nitrosylation in endothelial cells.
Hypoxia-induced responses are frequently encountered during cardiovascular injuries. Hypoxia triggers intracellular reactive oxygen species/nitric oxide (NO) imbalance. Recent studies indicate that NO-mediated S-nitrosylation (S-NO) of cysteine residue is a key posttranslational modification of proteins. We demonstrated that acute hypoxia to endothelial cells (ECs) transiently increased the NO levels via endothelial NO synthase (eNOS) activation. A modified biotin-switch method coupled with Western blot on 2-dimensional electrophoresis (2-DE) demonstrated that at least 11 major proteins have significant increase in S-NO after acute hypoxia. Mass analysis by CapLC/Q-TOF identified those as Ras-GTPase-activating protein, protein disulfide-isomerase, human elongation factor-1-delta, tyrosine 3/tryptophan 5-monooxygenase activating protein, and several cytoskeleton proteins. The S-nitrosylated cysteine residue on tropomyosin (Cys 170) and beta-actin (Cys 285) was further verified with the trypsic peptides analyzed by MASCOT search program. Further understanding of the functional relevance of these S-nitrosylated proteins may provide a molecular basis for treating ischemia-induced vascular disorders.
Chen
Shih Chung
SC
Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan.
Huang
Bin
B
Liu
Yu Chi
YC
Shyu
Kou Gi
KG
Lin
Pen Y
PY
Wang
Danny Ling
DL
D000595:Amino Acid Sequence; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D015153:Blotting, Western; D015687:Cell Hypoxia; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D007511:Ischemia / Q000378:metabolism; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D012694:Serine / Q000378:metabolism
D011506:Proteins; D009569:Nitric Oxide; D012694:Serine; D001710:Biotin; D052250:Nitric Oxide Synthase Type III