Abstract
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.
Highlights
NO participates in numerous biological events in a variety of cell types including activated glomerular mesangial cells
Since S-nitrosylated proteins often serve as major effectors of NOrelated bioactivity in various cell types and tissues, identification of the full complement of proteins that can be modified in this manner has been an emerging area of investigation
NO and derived species could potentially exert important regulatory control on mesangial cell function via S-nitrosylation of proteins involved in a variety of cellular processes
Summary
Reagents—S-Nitrosoglutathione (GSNO), S-nitroso-N-acetylpenicillamine, GSSG, and GSH were from Sigma. Sample Preparation for Proteomic Analysis—To prepare extracts of cytosolic and membrane-associated proteins, cells were harvested by scraping, resuspended in 20 volumes of HEN buffer (25 mM HEPES, pH 7.9, 1 mM EDTA, 0.1 mM neocuproine) containing 0.4% CHAPS, and allowed to swell for 15 min at 4 °C. After resuspending the proteins in HEN buffer containing 1% SDS, sodium ascorbate solution (1 mM final concentration) and biotin-HPDP The pellet was resuspended in 0.1 ml HEN buffer containing 1% SDS/mg of protein in the initial protein sample. Western Analysis—Proteins isolated by the biotin-switch method were resolved by SDS-PAGE, electroblotted to Hybond-P membranes, and immunoblotted with primary antibodies as indicated in the text and figure legends. Bound antibody was visualized by the ECL chemiluminescence detection system (Amersham Biosciences) using peroxidase-conjugated sheep anti-mouse or goat anti-rabbit IgG as appropriate
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