Proteomic analysis of the NOS2 interactome in human airway epithelial cells.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Foster, MW; Thompson, JW; Forrester, MT; Sha, Y; McMahon, TJ; Bowles, DE; Moseley, MA; Marshall, HE

Published Date

  • November 1, 2013

Published In

Volume / Issue

  • 34 /

Start / End Page

  • 37 - 46

PubMed ID

  • 23438482

Pubmed Central ID

  • 23438482

Electronic International Standard Serial Number (EISSN)

  • 1089-8611

Digital Object Identifier (DOI)

  • 10.1016/j.niox.2013.02.079

Language

  • eng

Conference Location

  • United States