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.signaling complex assembly | TLR4 targeting | TIR domain recognition site | decoy peptides T oll-like receptors (TLRs) initiate innate immune responses by recognizing specific pathogen-associated molecules; for example, TLR4 recognizes lipopolysaccharides (LPSs) of Gramnegative bacteria (1, 2). Ligand recognition induces dimerization of cytoplasmic Toll/IL-1R resistance (TIR) domains of two receptor molecules and causes recruitment of intracellular TIR domain-containing adapters. Four adapter proteins participate in TLR4 signaling: myeloid differentiation factor 88 (MyD88) (3), TIR domain-containing adapter protein, also known as MyD88-adapterlike (TIRAP-Mal) (4, 5), TIR domain-containing adapter-inducing IFN-β, also known as TLR adaptor molecule 1 (TRIF-TICAM-1) (6, 7), and TRIF-related adaptor molecule also known as TLR adaptor molecule 2 (TRAM-TICAM-2) (8, 9). TIRAP-Mal is important for MyD88 recruitment to the signaling complex located at the plasma membrane to initiate early NF-κB and mitogenactivated protein kinase (MAPK) activation and induce "MyD88-dependent" proinflammatory cytokines, such as TNF-α and IL-1β (4, 5, 10). TRAM is important for TRIF recruitment to the endosomally located TLR4...

Toll-like Receptors in the Vascular System: Sensing the Dangers Within

“…Furthermore, these studies provided novel insights into the structural interactions and function of TIR domains that could potentially augment the efficacy of TLR agonists and antagonists. Fekonja et al (2012b) showed that the addition of a strong-coiled coil dimerization domain determined the degree of inhibition against various TLRs and prevented activation of the dimeric TIR platform and Piao et al (2013a) identified TRIF sites that are important for interaction with TLR4 and TRAM. Intense interest in the BB loop of TIR domain has guided research efforts in the development of inhibitory peptides (Toshchakov et al, 2007(Toshchakov et al, , 2011Piao et al, 2013a).…”

“…Fekonja et al (2012b) showed that the addition of a strong-coiled coil dimerization domain determined the degree of inhibition against various TLRs and prevented activation of the dimeric TIR platform and Piao et al (2013a) identified TRIF sites that are important for interaction with TLR4 and TRAM. Intense interest in the BB loop of TIR domain has guided research efforts in the development of inhibitory peptides (Toshchakov et al, 2007(Toshchakov et al, , 2011Piao et al, 2013a). The BB loop peptides, however, lack specificity for a specific TLR or TLR-adapter complex because they bind to proteins containing TIR domains with variable affinity (Toshchakov et al, 2007(Toshchakov et al, , 2011Fekonja et al, 2012a;Piao et al, 2013a).…”

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

“…The intracellular region of TLR4 also contains clusters of missense mutations within the Toll/IL-1 receptor (TIR) domain (Fig. 1A), which are essential for the recruitment of intracellular effectors of TLR4 signaling, including myeloid differentiation primary response 88 and TIR domain-containing adaptor protein (2,3). This mutational profile suggests that TLR4 gene function is disrupted in some breast tumors in which TLR4 may function as a tumor suppressor gene.…”

“…TLR4 is activated by a variety of ligands: DNA, RNA, and viral particles; chemotherapeutic agents; and lipopolysaccharides (LPS). TLR4 induction in immune cells can activate numerous cancer-associated signaling cascades, including the MAP kinase and NFkB pathways (2,3). These pathways transcriptionally activate the secretion of either proinflammatory cytokines, such as IL-6 and IL-8, or anti-inflammatory type I IFNs, including IFN-γ.…”

Characterization of SIGIRR/IL-1R8 Homolog from Zebrafish Provides New Insights into Its Inhibitory Role in Hepatic Inflammation

“…For antagonist inhibition assay, zebrafish were pretreated 1 h via i.p. with two TRIF antagonist peptides (with a penetration sequence at the N terminus) derived from a mice model (41,42). The sequences of the peptides for DrTRIF (DrT1 and DrT2) and control are shown as follows: RQIKIWFQNRRMKWKK-CIEDAI DNSAFV-NH2 (DrT1), RQIKIWFQNRRMKWKK-FSEDFAQAGRSTLR-NH2 (DrT2), and RQIKIWFQNRRMKWKK-SLHGRGDPMEAFII-NH2 (DrControl).…”