TIRP,a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling

The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-kappaB and potentiates IL-1 receptor-mediated NF-kappaB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-kappaB activation. Moreover, TIRP-mediated NF-kappaB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKbeta. Our findings suggest that TIRP is involved in IL-1-triggered NF-kappaB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKbeta     

Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain-containing proteins

Pathogenic microbes have evolved sophisticated molecular strategies to subvert host defenses. Here we show that virulent bacteria interfere directly with Toll-like receptor (TLR) function by secreting inhibitory homologs of the Toll/interleukin-1 receptor (TIR) domain. Genes encoding TIR domain containing-proteins (Tcps) were identified in Escherichia coli CFT073 (TcpC) and Brucella melitensis (TcpB). We found that TcpC is common in the most virulent uropathogenic E. coli strains and promotes bacterial survival and kidney pathology in vivo. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1, and we show that the Tcps impede TLR signaling through the myeloid differentiation factor 88 (MyD88) adaptor protein, owing to direct binding of Tcps to MyD88. Tcps represent a new class of virulence factors that act by inhibiting TLR- and MyD88-specific signaling, thus suppressing innate immunity and increasing virulence.     

Targeting Toll-like receptor (TLR) signaling by Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal)-derived decoy peptides

Toll/interleukin-1 receptor (TIR) domain-containing adapter protein/MyD88 adapter-like (TIRAP/Mal) is an adapter protein that facilitates recruitment of MyD88 to TLR4 and TLR2 signaling complexes. We previously generated a library of cell-permeating TLR4 TIR-derived decoy peptides fused to the translocating segment of the Drosophila Antennapedia homeodomain and examined each peptide for the ability to inhibit TLR4 signaling (Toshchakov, V. Y., Szmacinski, H., Couture, L. A., Lakowicz, J. R., and Vogel, S. N. (2011) J. Immunol. 186, 4819-4827). We have now expanded this study to test TIRAP decoy peptides. Five TIRAP peptides, TR3 (for TIRAP region 3), TR5, TR6, TR9, and TR11, inhibited LPS-induced cytokine mRNA expression and MAPK activation. Inhibition was confirmed at the protein level; select peptides abolished the LPS-induced cytokine production measured in cell culture 24 h after a single treatment. Two of the TLR4 inhibitory peptides, TR3 and TR6, also inhibited cytokine production induced by a TLR2/TLR1 agonist, S-(2,3-bis(palmitoyloxy)-(2R,2S)-propyl)-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH; however, a higher peptide concentration was required to achieve comparable inhibition of TLR2 versus TLR4 signaling. Two TLR4 inhibitory peptides, TR5 and TR6, were examined for the ability to inhibit TLR4-driven cytokine induction in mice. Pretreatment with either peptide significantly reduced circulating TNF-α and IL-6 in mice following LPS injection. This study has identified novel TLR inhibitory peptides that block cellular signaling at low micromolar concentrations in vitro and in vivo. Comparison of TLR4 inhibition by TLR4 and TIRAP TIR-derived peptides supports the view that structurally diverse regions mediate functional interactions of TIR domains.     

Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4

Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.     

The axon degeneration gene SARM1 is evolutionarily distinct from other TIR domain-containing proteins

Many forms of neurodegenerative disease are characterized by Wallerian degeneration, an active program of axonal destruction. Recently, the important player which enacts Wallerian degeneration was discovered, the multidomain protein SARM1. Since the SARM1 protein has classically been thought of as an innate immune molecule, its role in Wallerian degeneration has raised questions on the evolutionary forces acting on it. Here, we synthesize a picture of SARM1’s evolution through various organisms by examining the molecular and genetic changes of SARM1 and the genes around it. Using proteins that possess domains homologous to SARM1, we established distances and Ka/Ks values through 5671 pairwise species–species comparisons. We demonstrate that SARM1 diverged across species in a pattern similar to other SAM domain-containing proteins. This is surprising, because it was expected that SARM1 would behave more like its TIR domain relatives. Going along with this divorce from TIR, we also noted that SARM1’s TIR is under stronger purifying selection than the rest of the TIR domain-containing proteins (remaining highly conserved). In addition, SARM1’s synteny analysis reveals that the surrounding gene cluster is highly conserved, functioning as a potential nexus of gene functionality across species. Taken together, SARM1 demonstrates a unique evolutionary pattern, separate from the TIR domain protein family.     

Characterization of a novel Toll/interleukin-1 receptor (TIR)-TIR gene differentially expressed in common bean (Phaseolus vulgaris cv. Othello) undergoing a defence response to the geminivirus Bean dwarf mosaic virus

Common bean ( Phaseolus vulgaris L.) cultivar (cv.) Othello develops a hypersensitive response-associated vascular resistance to infection by Bean dwarf mosaic virus (BDMV), a single-stranded DNA virus (genus Begomovirus , family Geminiviridae ). A PCR-based cDNA subtraction approach was used to identify genes involved in this resistance response. Eighteen clones, potentially involved with BDMV resistance, were identified based upon being up-regulated in BDMV-infected tissues and/or having sequence similarity with known resistance-associated genes. Analysis of these clones revealed potential genes involved in pathogen defence, including pathogenesis-related protein genes and resistance gene analogues (RGAs). Further characterization of one RGA, F1-10 , revealed that it encodes a predicted protein with a double Toll/interleukin-1 receptor (TIR) motif. Full-length ( F1-10 ) and spliced ( F1-10sp ) forms of the RGA were strongly up-regulated in BDMV-infected cv. Othello hypocotyl tissues by 4 days post-inoculation, but not in equivalent mock-inoculated tissues. In agroinfiltration experiments, F1-10 , but not F1-10sp , mediated resistance to BDMV in the susceptible common bean cv. Topcrop. By contrast, transgenic Nicotiana benthamiana lines expressing F1-10 or F1-10sp were not resistant to BDMV. Interestingly, when these transgenic lines were inoculated with the potyvirus Bean yellow mosaic virus, some F1-10 lines showed a more severe symptom phenotype compared with non-transgenic control plants. Based on these findings, F1-10 was named: Phaseolus vulgaris VIRUS response TIR-TIR GENE 1 ( PvVTT1 ).     

Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling

MyD88 is a Toll/IL-1 receptor (TIR) domain-containing adapter common to signaling pathways via Toll-like receptor (TLR) family. However, accumulating evidence demonstrates the existence of a MyD88-independent pathway, which may explain unique biological responses of individual TLRs, particularly TLR3 and TLR4. TIR domain-containing adapter protein (TIRAP)/MyD88 adapter-like, a second adapter harboring the TIR domain, is essential for MyD88-dependent TLR2 and TLR4 signaling pathways, but not for MyD88-independent pathways. Here, we identified a novel TIR domain-containing molecule, named TIR domain-containing adapter inducing IFN-beta (TRIF). As is the case in MyD88 and TIRAP, overexpression of TRIF activated the NF-kappaB-dependent promoter. A dominant-negative form of TRIF inhibited TLR2-, TLR4-, and TLR7-dependent NF-kappaB activation. Furthermore, TRIF, but neither MyD88 nor TIRAP, activated the IFN-beta promoter. Dominant-negative TRIF inhibited TLR3-dependent activation of both the NF-kappaB-dependent and IFN-beta promoters. TRIF associated with TLR3 and IFN regulatory factor 3. These findings suggest that TRIF is involved in the TLR signaling, particularly in the MyD88-independent pathway.     

Structure of the Toll/interleukin 1 receptor (TIR) domain of the immunosuppressive Brucella effector BtpA/Btp1/TcpB

BtpA/Btp1/TcpB is a virulence factor produced by Brucella species that possesses a Toll interleukin-1 receptor (TIR) domain. Once delivered into the host cell, BtpA interacts with MyD88 to interfere with TLR signalling and modulates microtubule dynamics. Here the crystal structure of the BtpA TIR domain at 3.15 Å is presented. The structure shows a dimeric arrangement of a canonical TIR domain, similar to the Paracoccus denitrificans Tir protein but secured by a unique long N-terminal α-tail that packs against the TIR:TIR dimer. Structure-based mutations and multi-angle light scattering experiments characterized the BtpA dimer conformation in solution. The structure of BtpA will help with studies to understand the mechanisms involved in its interactions with MyD88 and with microtubules.     

A novel splice variant of interleukin-1 receptor (IL-1R)-associated kinase 1 plays a negative regulatory role in Toll/IL-1R-induced inflammatory signaling

The interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK1) is a member of the IRAK kinase family that plays a pivotal role in the Toll/IL-1 receptor (TIR) family signaling cascade. We have identified a novel splice variant, IRAK1c, which lacks a region encoded by exon 11 of the IRAK1 gene. IRAK1c expression was confirmed by both RNA and protein detection. Although both IRAK1 and IRAK1c are expressed in most tissues tested, IRAK1c is the predominant form of IRAK1 expressed in the brain. Unlike IRAK1, IRAK1c lacks kinase activity and cannot be phosphorylated by IRAK4. However, IRAK1c retains the ability to strongly interact with IRAK2, MyD88, Tollip, and TRAF6. Overexpression of IRAK1c suppressed NF-kappaB activation and blocked IL-1beta-induced IL-6 as well as lipopolysaccharide- and CpG-induced tumor necrosis factor alpha production in multiple cellular systems. Mechanistically, we provide evidence that IRAK1c functions as a dominant negative by failing to be phosphorylated by IRAK4, thus remaining associated with Tollip and blocking NF-kappaB activation. The presence of a regulated, alternative splice variant of IRAK1 that functions as a kinase-dead, dominant-negative protein adds further complexity to the variety of mechanisms that regulate TIR signaling and the subsequent inflammatory response.     

Crystal structure of the Toll/interleukin-1 receptor domain of human IL-1RAPL

The Toll/interleukin-1 receptor (TIR) domain is conserved in the intracellular regions of Toll-like receptors (TLRs) and interleukin-1 receptors (IL-1Rs) as well as in several cytoplasmic adapter molecules. This domain has crucial roles in signal transduction by these receptors for host immune response. Here we report the crystal structure at 2.3-A resolution of the TIR domain of human IL-1RAPL, the first structure of a TIR domain of the IL-1R superfamily. There are large structural differences between this TIR domain and that of TLR1 and TLR2. Helix alphaD in IL-1RAPL is almost perpendicular to its equivalent in TLR1 or TLR2. The BB loop contains a hydrogen bond unique to IL-1RAPL between Thr residues at the 8th and 10th positions. The structural and sequence diversity among these domains may be important for specificity in the signal transduction by these receptors. A dimer of the TIR domain of IL-1RAPL is observed in the crystal, although this domain is monomeric in solution. Residues in the dimer interface are mostly unique to IL-1RAPL, which is consistent with the distinct functional roles of this receptor. Our functional studies show IL-1RAPL can activate JNK but not the ERK or the p38 MAP kinases, whereas its close homolog, TIGIRR, cannot activate JNK. Deletion mutagenesis studies show that the activation of JNK by IL-1RAPL does not depend on the integrity of its TIR domain, suggesting a distinct mechanism of signaling through this receptor.     

Evolution of the TIR domain-containing adaptors in humans: swinging between constraint and adaptation

Natural selection is expected to act strongly on immune system genes as hosts adapt to novel, diverse, and coevolving pathogens. Population genetic studies of host defense genes with parallel functions in model organisms have revealed distinct evolutionary histories among the different components-receptors, adaptors, and effectors-of the innate immune system. In humans, however, detailed evolutionary studies have been mainly confined to the receptors and in particular to Toll-like receptors (TLRs). By virtue of a toll/interleukin-1 receptor (TIR) domain, TLRs activate distinct signaling pathways, which are mediated by the five TIR-containing adaptors: myeloid differentiation factor-88 (MyD88), myeloid differentiation factor-88 adaptor-like protein (MAL), toll/interleukin-1 receptor domain-containing adaptor protein inducing interferon (IFN)β (TRIF), toll/interleukin-1 receptor domain-containing adaptor protein inducing IFNβ-related adaptor molecule (TRAM), and sterile α- and armadillo motif-containing protein (SARM). Here, we have examined the extent to which natural selection has affected immune adaptors in humans, using as a paradigm the TIR-containing adaptors. To do so, we characterized their levels of naturally occurring genetic variation in various human populations. We found that MyD88 and TRIF have mainly evolved under purifying selection, suggesting that their role in the early stages of signal transduction is essential and nonredundant for host survival. In addition, the adaptors have been targeted by multiple episodes of positive selection, differing in timing and spatial location. MyD88 and SARM display signatures of a selective sweep that has occurred in all humans, whereas for the other three adaptors, we detected signatures of adaptive evolution that are restricted to specific populations. Our study provides evidence that the contemporary diversity of the five TIR-containing adaptors results from the intermingling of different selective events, swinging between constraint and adaptation.