A second binding site for double-stranded RNA in TLR3 and consequences for interferon activation

Pirher, Nina; Ivičak, Karolina; Pohar, Jelka; Benčina, Mojca; Jerala, Roman

doi:10.1038/nsmb.1453

Cited by 67 publications

(69 citation statements)

References 20 publications

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“…In addition, TLR3 has two distinct binding sites for dsRNA binding, one located from the N terminus to LRR3 and the other in LRR19 through 21 of the ectodomain. Both are required for binding to poly(I:C) (43)(44)(45)(46)(47)(48), in comparison to TLR9 and TLR7, which recognize ligands primarily through one binding site (49). Both cleaved and uncleaved TLR3 appear to be competent for signaling, but proteolytic cleavage may confer additional activity to the recognition of some ligands by TLR3, as we have observed for the recognition of Reovirus dsRNA and poly(A:U) RNA in RAW267.4 cells.…”

Section: Discussionmentioning

confidence: 71%

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Singh

Kao

2012

Journal of Biological Chemistry

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Background: Whether TLR3 requires proteolytic processing is not clear. Results: TLR3 was found to be processed by cathepsins within Loop1 of leucine-rich repeat 12. Proteolytic processing is not required for TLR3 signaling but modulated its response to ligands and affected TLR3 localization in endosomes. Conclusion: Cathepsin processing can alter TLR3 signaling. Significance: This work contributes to understanding of TLR3 structure and function.

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Section: Discussionmentioning

confidence: 71%

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Singh

Kao

2012

Journal of Biological Chemistry

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“…In contrast, some structural studies have reported that efficient binding of dsRNA to TLR3 requires a minimum of 40-50 nt (16,37). The recent identification of a second binding site for dsRNA in TLR3 confirms the ability of 21-nt siRNA to bind this immune receptor (38) and helps to resolve some of the conflicting biological and structural data. Our data that 21-nt siRNA phosphorylates TLR3 demonstrate that dsRNAs of this minimum length directly activate this immune sensor.…”

Section: Discussionmentioning

confidence: 84%

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth

Cho

Albuquerque

Kleinman

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

132

120

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Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.angiogenesis ͉ innate immunity ͉ lymphangiogenesis ͉ ischemia ͉ wound healing

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“…Because H60A bound dsRNA at pH 5.5, the inability of H60A-transfected reporter cells to respond to dsRNA suggests that the pH of the intracellular compartment in which TLR3 encounters dsRNA is higher than 5.5, consistent with binding occurring in early, but not late endosomes. Interestingly, Pirher et al (12) found that H39R, a mutation that replaces a positively charged imidazole side chain with a positively charged guanidinium group, retained almost full signaling capacity. This was also reflected in binding capacity because the H39R mutant bound dsRNA equivalently to WT TLR3 at pH below 6.5 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

Wang

Liu

Davies

et al. 2010

Journal of Biological Chemistry

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TLR3 (Toll-like receptor 3) recognizes dsRNA, a potent indicator of viral infection. The extracellular domain of TLR3 dimerizes when it binds dsRNA, and the crystal structure of the dimeric complex reveals three sites of interaction on each extracellular domain, two that bind dsRNA and one that is responsible for dimer formation. The goal of this study was to determine which amino acid residues are essential for forming a stable receptor⅐ligand complex and whether dimerization of TLR3 is required for dsRNA binding. Using a novel ELISA to analyze dsRNA binding by mutant TLR3 constructs, we identified the essential interacting residues and determined that the simultaneous interaction of all three sites is required for ligand binding. In addition, we show that TLR3 is unable to bind dsRNA when dimerization is prevented by mutating residues in the dimerization site or by immobilizing TLR3 at low density. We conclude that dimerization of TLR3 is essential for ligand binding and that the three TLR3 contact sites individually interact weakly with their binding partners but together form a high affinity receptor⅐ligand complex.Many viruses produce dsRNA at some stage in their replication cycle. Although short stretches of dsRNA are normally found in the microRNA, tRNA, and rRNA of most cells, only viruses synthesize long dsRNA molecules. Therefore, dsRNA longer than ϳ30 bp can serve as a potent molecular signature of viral infection in higher organisms, including man. Consequently, dsRNA is recognized by a number of pattern recognition receptors of the innate immune system, including TLR3 (Toll-like receptor 3) (1-3). TLR3 is a type I transmembrane receptor with an N-terminal extracellular domain (ECD), 2 a single transmembrane helix, and a C-terminal cytoplasmic signaling domain of the TIR (Toll/IL-1 receptor) family. The ECD of TLR3 is located in the interior of endosomes, where it encounters and binds dsRNA and transduces signals that initiate inflammatory and adaptive antiviral responses. dsRNA enters the endosomes either by direct uptake from the medium or by phagocytosis of virally infected cells (4), and as the dsRNA transits from early to late endosomes, the pH decreases progressively from pH ϳ6.2 to 5.5 (5).Recent crystallographic studies have shown how TLR3 recognizes dsRNA at the molecular level (for a review, see Ref. 6). The TLR3 ECD can be described as a coil comprising 23 tandem leucine-rich repeat (LRR) motifs bent into the shape of a horseshoe and capped at the N-and C-terminal ends by specialized structures known as the LRR-NT and LRR-CT domains, respectively (7,8). The TLR3 ECD is decorated with 15 N-linked glycans and has one surface that is devoid of glycan and free to interact with dsRNA. Recombinant TLR3 ECD protein binds dsRNA under mildly acidic conditions, at pH values similar to those found in early and late endosomes (9). Although the TLR3 ECD is monomeric in solution, it binds as dimers to 45-bp segments of dsRNA, and several dimers can bind to long dsRNA strands (9). The x-ray structure of ...

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A second binding site for double-stranded RNA in TLR3 and consequences for interferon activation

Cited by 67 publications

References 20 publications

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth

Dimerization of Toll-like Receptor 3 (TLR3) Is Required for Ligand Binding

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