Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain

Ranjith-Kumar, C. T.; Miller, William R.; Xiong, Jin; Russell, William K.; Lamb, Roberta J.; Santos, J.-P. Dos; Duffy, Karen E.; Cleveland, Larissa; Park, Mary; Bhardwaj, Kanchan; Wu, Zhaoxiang; Russell, David H.; Sarisky, Robert T.; Mbow, Moustapha; Kao, C. Cheng

doi:10.1074/jbc.m610946200

Cited by 58 publications

(62 citation statements)

References 28 publications

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“…5), suggest that at least 21 nucleotides are required to activate TLR3. To understand the structural basis of the divergence between 19 and 21 nucleotides, we performed docking simulations between dimeric TLR3, which is required for signalling 17,41,42 , and dsRNA using crystal structures of TLR3 and 19-nucleotide dsRNA, the latter being shortened or extended as necessary. Docking of dsRNA of various lengths on a TLR3 ectodomain (ECD) monomer did not clarify the observed minimal length requirement for TLR3 activation because the ECD solenoid is approximately the same width as a single turn of an RNA helix.…”

Section: Targeted Sirnas Also Suppress Cnv Via Tlr3mentioning

confidence: 99%

“…Therefore, a TLR3 monomer cannot discriminate between dsRNAs greater than about 10 nucleotides in length (data not shown), indicating that TLR3 dimerization is required for the observed length needed for activation. We developed a model of a 2:1 TLR3-dsRNA complex, constrained by these observations or interpretations of known data: (1) active TLR3 involves a specific ECD carboxy terminus dimer, formation of which is stabilized by dsRNA binding 17,41,42 ; (2) TLR3 residues most critical for RNA binding and receptor activation, H539 and N541 (ref. 41), lie in a region 40-45 Å from the ECD C terminus; (3) dsRNA of 21 nucleotides or longer is required for receptor activation (Fig.…”

Section: Targeted Sirnas Also Suppress Cnv Via Tlr3mentioning

confidence: 99%

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Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Kleinman

Yamada

Takeda

et al. 2008

Nature

854

683

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Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro-and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-a/b activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-c and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3-RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world's population, and that siRNAs might induce unanticipated vascular or immune effects.Therapeutic application of long, double-stranded (ds)RNAmediated RNAi and sequence-specific gene silencing through RNAi by short synthetic RNA duplexes is challenging because mammalian cells do not uptake 'naked' siRNA (whether chemically modified or not) without cell-permeating entities [1][2][3][4] . To minimize systemic exposure, initial clinical trials of siRNA were launched using intraocular injection in patients with CNV. CNV, wherein the retina is invaded by choroidal vessels beneath the retinal pigmented epithelium (RPE), is a late stage of age-related macular degeneration that afflicts 30-50 million people globally. The preclinical bases for trials of naked VEGFA siRNA (Bevasiranib) or VEGFR1 siRNA (AGN211745/ siRNA-027) were single reports in mice 5,6 that such siRNAs suppressed laser-injury-induced CNV, a model predictive of efficacy in humans 7,8 . These findings were interpreted as anomalous examples of local delivery surmounting the impediment to intracellular entry 9-11 . Instead, we show in two animal models that suppression of neovascularization is a generic property of siRNAs independent of sequence, target and internalization.Sequence-independent angiogenesis suppression by siRNA Numerous synthetic non-targeted 21-nucleotide duplex siRNAs from multiple vendors, when injected into the vitreous humour of wild-type mice, uniformly and dose-dependently suppressed CNV (Fig. 1a, b and Supplementary Fig. 1). siRNAs targeting jellyfish green fluorescent ...

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Section: Targeted Sirnas Also Suppress Cnv Via Tlr3mentioning

confidence: 99%

Section: Targeted Sirnas Also Suppress Cnv Via Tlr3mentioning

confidence: 99%

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Kleinman

Yamada

Takeda

et al. 2008

Nature

854

683

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“…These results demonstrate that although N284I is almost completely inactive, L412F could have reduced affinity to the ligand. Furthermore, the ligand-binding surface of TLR3 was recently mapped to a charged surface of TLR3 (30,31) to leucine-rich repeats 17-20, which is C-terminal to L412F.…”

Section: Tlr3 Single Nucleotide Polymorphismsmentioning

confidence: 99%

“…7A). Ranjith-Kumar et al (31) have documented that the dominant negative activity is useful to genetically analyze TLR3-TLR3 interactions. We used a TLR3⌬TIR (missing residues 756 -904 of the TIR domain) as a control because it was previously shown to be a dominant negative of WT TLR3 (33).…”

Section: Tlr3 Single Nucleotide Polymorphismsmentioning

confidence: 99%

Effects of Single Nucleotide Polymorphisms on Toll-like Receptor 3 Activity and Expression in Cultured Cells

Ranjith-Kumar

Miller

Sun

et al. 2007

Journal of Biological Chemistry

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138

137

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Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-B protein.Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.

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“…TLRs comprise a family of innate immune receptors that recognize various pathogen-associated molecular patterns. TLR3 is a sensor of dsRNA (14), such as those found in viral genomes or replication intermediates, that undergoes dimerization (15)(16)(17)(18) and phosphorylation (19) to initiate a signaling cascade that can ultimately result in apoptotic cell death (20)(21)(22)(23). In this study, we sought to further explore this newly defined intersection between angiogenesis and innate immunity.…”

mentioning

confidence: 99%

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.

133

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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Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain

Cited by 58 publications

References 28 publications

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Effects of Single Nucleotide Polymorphisms on Toll-like Receptor 3 Activity and Expression in Cultured Cells

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

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