title: Proteomic analysis of the NOS2 interactome in human airway epithelial cells.
abstract: The cytokine-inducible isoform of nitric oxide synthase (NOS2) is constitutively expressed in human respiratory epithelia and is upregulated in inflammatory lung disease. Here, we sought to better define the protein interactions that may be important for NOS2 activity and stability, as well as to identify potential targets of NOS2-derived NO, in the respiratory epithelium. We overexpressed Flag-tagged, catalytically-inactive NOS2 in A549 cells and used mass spectrometry to qualitatively identify NOS2 co-immunoprecipitating proteins. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify the coordinate effects of cytokine stimulation on NOS2-protein interactions. Multi-protein networks dominated the NOS2 interactome, and cytokine-inducible interactions with allosteric activators and with the ubiquitin-proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination. The ubiquitin ligase scaffolding protein, FBXO45, was identified as a novel, direct NOS2 interactor. Similar to the SPRY domain-containing SOCS box (SPSB) proteins, FBXO45 requires Asn27 in the (23)DINNN(27) motif of NOS2 for its interaction. However, FBXO45 is unique from the SPSBs in that it recruits a distinct E3 ligase complex containing MYCBP2 and SKP1. Collectively, these findings demonstrate the general utility of interaction proteomics for defining new aspects of NOS2 physiology.
authors: Division of Pulmonary, Allergy and Critical Care Medicine, Duke University Medical Centers, Durham, NC 27710, United States. Electronic address: mwfoster@duke.edu.
authors: Matthew W
authors: MW
authors: Foster
authors: J Will
authors: JW
authors: Thompson
authors: Michael T
authors: MT
authors: Forrester
authors: Yonggang
authors: Y
authors: Sha
authors: Timothy J
authors: TJ
authors: McMahon
authors: Dawn E
authors: DE
authors: Bowles
authors: M Arthur
authors: MA
authors: Moseley
authors: Harvey E
authors: HE
authors: Marshall
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D020545:Respiratory Mucosa / Q000166:cytology*; D016415:Sequence Alignment; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D016207:Cytokines; D020543:Proteome; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D044767:Ubiquitin-Protein Ligases
keywords: B30.2/SPRY domain; Nitric oxide; Nitric oxide synthase; Proteomics; Ubiquitination