Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages.
Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identification of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identification of the "universe" of S-nitrsoylated proteins. In this study using endotoxin-stimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identified as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins specific to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not sufficient.
Gao
Chengjiang
C
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Guo
Hongtao
H
Wei
Junping
J
Mi
Zhiyong
Z
Wai
Philip Y
PY
Kuo
Paul C
PC
D000578:Amidines / Q000494:pharmacology; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation / Q000379:methods; D015153:Blotting, Western; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000378:metabolism
D000578:Amidines; D001596:Benzylamines; D008070:Lipopolysaccharides; C104820:N-(3-(aminomethyl)benzyl)acetamidine; D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D001710:Biotin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse