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* Department of Biochemistry and Biophysics,
University of North Carolina-Duke Michael Hooker Proteomics Center, and
Department of Pediatrics, Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; and
Department of Chemistry and Institute of Biomedical Sciences, Fudan University, Shanghai, China
Emerging evidences suggest TLR-mediated signaling is tightly regulated by a specific chain of intracellular protein-protein interactions, some of which are yet to be identified. Previously we utilized a dual-tagging quantitative proteomics approach to uncover MyD88 interactions in LPS-stimulated cells and described the function of Fliih, a leucine-rich repeat (LRR) protein that negatively regulates NF-
B activity. Here we characterize two distinct LRR-binding MyD88 interactors, LRRFIP2 and Flap-1, and found that both are positive regulators of NF-B activity. Upon LPS stimulation, LRRFIP2 was also found to positively regulate cytokine production in macrophages, suggesting a functional role in TLR4-mediated inflammatory response. Furthermore, we observed that immediately following LPS stimulation both LRRFIP2 and Flap-1 compete with Fliih for interacting with MyD88 to activate the signaling. By using a novel multiplex quantitative proteomic approach, we found that at endogenous levels these positive and negative regulators interact with MyD88 in a timely and orderly manner to differentially mediate the NF-B activity through the course of signaling from initiation to prolongation, and to repression. Based on these data, we describe a mechanistic model in which selective modulation of TLR signaling is achieved by temporal and dynamic interactions of MyD88 with its regulators.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grant 1R01AI064806-01A2 and by the Office of Science, Biological and Environmental Research, U.S. Department of Energy Grant DE-FG02-07ER64422.
2 Address correspondence and reprint requests to Dr. Xian Chen, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. E-mail address: xian_chen{at}med.unc.edu
3 Abbreviations used in this paper: LRR, leucine-rich repeat; Flap-1, Fli-I LRR-associated protein 1, or LRRFIP1; HA, hemagglutinin; iTRAQ, isobaric tag for relative and absolute quantitation; LC-MS/MS, liquid chromatography-mass spectroscopy/mass spectroscopy; LRRFIP2, LRR Fli-I-interacting protein 2; shRNA, short hairpin RNA.
4 The online version of this article contains supplemental material.
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