title: Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain.
abstract: Toll/IL-1R resistance (TIR) domain-containing adapter-inducing IFN-β (TRIF) is a Toll-like receptor (TLR) adapter that mediates MyD88-independent induction of type I interferons through activation of IFN regulatory factor 3 and NFκB. We have examined peptides derived from the TRIF TIR domain for ability to inhibit TLR4. In addition to a previously identified BB loop peptide (TF4), a peptide derived from putative helix B of TRIF TIR (TF5) strongly inhibits LPS-induced cytokine and MAPK activation in wild-type cells. TF5 failed to inhibit LPS-induced cytokine and kinase activation in TRIF-deficient immortalized bone-marrow-derived macrophage, but was fully inhibitory in MyD88 knockout cells. TF5 does not block macrophage activation induced by TLR2, TLR3, TLR9, or retinoic acid-inducible gene 1/melanoma differentiation-associated protein 5 agonists. Immunoprecipitation assays demonstrated that TF4 binds to TLR4 but not TRIF-related adaptor molecule (TRAM), whereas TF5 binds to TRAM strongly and TLR4 to a lesser extent. Although TF5 prevented coimmunoprecipitation of TRIF with both TRAM and TLR4, site-directed mutagenesis of the TRIF B helix residues affected TRIF-TRAM coimmunoprecipitation selectively, as these mutations did not block TRIF-TLR4 association. These results suggest that the folded TRIF TIR domain associates with TRAM through the TRIF B helix region, but uses a different region for TRIF-TLR4 association. The B helix peptide TF5, however, can associate with either TRAM or TLR4. In a mouse model of TLR4-driven inflammation, TF5 decreased plasma cytokine levels and protected mice from a lethal LPS challenge. Our data identify TRIF sites that are important for interaction with TLR4 and TRAM, and demonstrate that TF5 is a potent TLR4 inhibitor with significant potential as a candidate therapeutic for human sepsis.
authors.affiliation: Departments of Microbiology and Immunology, Medicine, and Microbiology and Immunology, and Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD 21201.
authors.forename: Wenji
authors.initials: W
authors.lastname: Piao
authors.forename: Lisa W
authors.initials: LW
authors.lastname: Ru
authors.forename: Kurt H
authors.initials: KH
authors.lastname: Piepenbrink
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sundberg
authors.forename: Stefanie N
authors.initials: SN
authors.lastname: Vogel
authors.forename: Vladimir Y
authors.initials: VY
authors.lastname: Toshchakov
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D004926:Escherichia coli; D015151:Immunoblotting; D047468:Immunoprecipitation; D016899:Interferon-beta / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D018123:Receptors, Interleukin / Q000378:metabolism*; D015398:Signal Transduction / Q000276:immunology*; D051197:Toll-Like Receptor 4 / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D016899:Interferon-beta; D018123:Receptors, Interleukin; D051197:Toll-Like Receptor 4; C476286:Ticam1 protein, mouse; C476416:Ticam2 protein, mouse
keywords: TIR domain recognition site; TLR4 targeting; decoy peptides; signaling complex assembly