Proteomic analysis of S-nitrosylated proteins in mesangial cells.
NO participates in numerous biological events in a variety of cell types including activated glomerular mesangial cells. Many of these events appear to be independent of the known effects of NO on soluble guanylyl cyclase. NO derived from all major isoforms of NO synthase can S-nitrosylate cysteine residues in target proteins, potentially altering their functional activities. Recent evidence suggests that S-nitrosylation is specific, is regulated, and may play an important regulatory role akin to phosphorylation. In the present study, the "biotin-switch" method of isolating S-nitrosylated proteins was coupled with two-dimensional PAGE protein separation followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and peptide mass fingerprinting to identify target proteins for S-nitrosylation in murine mesangial cells treated with NO donors or appropriate controls. This approach resolved 790 protein spots. We analyzed the most abundant spots and identified 34 known proteins. Of these, 31 are unique S-nitrosylated proteins not previously identified, including signaling proteins, receptors and membrane proteins, cytoskeletal or cell matrix proteins, and cytoplasmic proteins. Prominent among these were peroxisome proliferator activated receptor gamma, uroguanylin, GTP-binding protein alpha, protein 14-3-3, NADPH-cytochrome P450 oxidoreductase, transcription factor IIA, melusin, mitosin, phospholipase A2-activating protein, and protein-tyrosine phosphatase. The in vivo induction of S-nitrosylation was assayed by treating mesangial cells with interleukin-1beta followed by the biotin-switch and Western blot of selected targets. These results broaden our knowledge of potential signal transduction pathways and other cell functions mediated by NO S-nitrosylation.
Kuncewicz
Teresa
T
Department of Internal Medicine, The University of Texas Medical School at Houston, Houston, Texas 77030, USA.
Sheta
Essam A
EA
Goldknopf
Ira L
IL
Kone
Bruce C
BC
D000818:Animals; D001710:Biotin / Q000737:chemistry; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005920:Glomerular Mesangium / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000737:chemistry; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D026422:S-Nitrosoglutathione / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D020030:Nitric Oxide Donors; D020543:Proteome; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D001710:Biotin; D052247:Nitric Oxide Synthase Type II; D003545:Cysteine