|Mutation Type||critical splice acceptor site (2 bp from exon)|
|Coordinate||35,198,707 bp (GRCm38)|
|Base Change||T ⇒ A (forward strand)|
|Gene Name||toll-interleukin 1 receptor (TIR) domain-containing adaptor protein|
|Synonym(s)||Mal, Mal, wyatt, MyD88-adapter-like, C130027E04Rik|
|Chromosomal Location||35,184,551-35,200,291 bp (-)|
|MGI Phenotype||Mutations in this gene lead to impaired cytokine secretion in response to TLR2 and TLR4 ligands. Homozygous null mice may also show low serum IgG3 levels, a reduced response to attenuated yellow fever vaccine, high susceptibility to bacterial infection, and altered response to myocardial infarction.|
|Amino Acid Change|
|Institutional Source||Beutler Lab|
Ensembl: ENSMUSP00000135224 (fasta)
|Gene Model||not available|
|Phenotypic Category||immune system, TLR signaling defect: TNF production by macrophages|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Recessive|
|Local Stock||Live Mice, Embryos, gDNA|
|Last Updated||03/23/2017 8:22 AM by Katherine Timer|
The torpid phenotype was identified in a G3 screen for ENU-induce mutants with altered response to Toll-like receptor (TLR) ligands (TLR Signaling Screen). Peritoneal macrophages from homozygous torpid mice fail to produce tumor necrosis factor (TNF)-α in response to peptidoglycan (TLR2/1 ligand) or Pam3CSK4 (triacyl lipopeptide, TLR2/6 ligand). However, at increasing concentrations of peptidoglycan or Pam3CSK4, torpid macrophages produce a small amount of TNF-α, indicating that TLR2 signaling is not completely abolished in torpid homozygotes (Figure 1). TNF-α production is normal in response to oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs, TLR9 ligand), poly I:C (dsRNA mimic, TLR3 ligand) and resiquimod (TLR7 ligand). Torpid macrophages produce decreased amounts of type I interferon (IFN) in response to lipopolysaccharide (LPS, TLR4 ligand).
|Nature of Mutation|
The torpid mutation mapped to Chromosome 9, and corresponds to an A to T transversion in the acceptor splice site of intron 6 (TTTCAG ->TTTCTG) in the Tirap gene (position 11943 in Genbank genomic region NC_000075). As a result, intron 6 is not removed by splicing, and instead the presence of an in-frame stop codon causes premature termination of the transcript after the anomalous addition of 11 nucleotides from the 5' end of intron 6. Tirap has 7 total exons of which only the last three are translated. This transcript yields a protein in which the six C-terminal amino acids of Tirap (encoded by exon 7) are replaced by three unrelated residues.
<--exon 6 intron 6--> <--intron 6 exon 7-->
11218 GTTATACACT GTAAGATATAG……………………TTTCAG ATCTGG 11950
233 -V--I--H-- C--K--I--* 238
The acceptor splice site of intron 6, which is destroyed by the torpid mutation, is indicated in blue lettering; the mutated nucleotide is indicated in red lettering.
Tirap (also Mal, MyD88-adapter-like) is a 249-amino acid protein that serves as an adapter in TLR2/1, TLR2/6 and TLR4 signaling (Figure 2). Like MyD88 (see records for Myd88rev1, pococurante, and lackadaisical), Tirap contains a C-terminal Toll/IL-1 receptor (TIR) domain, a conserved region of approximately 200 amino acids which mediates homo- and heterotypic protein interactions during signal transduction (1;2). TIR domains in TLRs, IL receptors and the adapter MyD88 contain 3 conserved boxes (boxes 1, 2 and 3), which are required for signaling (3). However, the conserved FW sequence in box 3 is lacking in Tirap (FY exists instead), although the (F/Y)D in box 1 and RD in box 2 are present (1). TIR domains contain six α-helices (αA, αB, αC, αC’, αD and αE) and five β-strands (βA, βB, βC, βD and βE) which are connected by seven loops. The crystal structures of the TLR1 and TLR2 TIR domains revealed that they fold into a structure with a central five-stranded parallel β-sheet surrounded by five helices (4). Modeling of the secondary structure of Tirap is reportedly consistent with this folding pattern (1). Many of the α-helices and connecting loops in the TIR domains of TLR1 and TLR2 are predicted to participate in binding partner recognition, and their mutation is expected to abrogate specific binding interactions. This is true of a proline to histidine mutation in the BB loop of TLR4, which abolishes MyD88 binding (5) and LPS-induced signaling in mice (6). This proline is conserved in Tirap, as are the three amino acids which stabilize the BB loop in TLR2 (Arg BB3, Asp BB4, Glu αA13) (1).
The N-terminal region of Tirap does not show similarity to any other known protein (2). Tirap differs from MyD88 in that its N terminus is 75 amino acids shorter, and lacks a death domain (1;2), which in MyD88 recruits IL-1 receptor associated kinase (IRAK) family proteins (7).
The crystal structure of human Tirap has been recently solved [Figure 3; (8;9)]. Valkov et al. crystalized a construct comprised of residues 79-221 [(8); PDB:2Y92); diffraction at 3Å found: (i) that the TIR domain BB loop motif is conserved in Tirap, however, it is in a unique structural arrangement. (ii) There are two potential dimerization interfaces. (iii) Key residues (i.e. R196, S180, D96, and L165) involved in the binding of MyD88 are on the surface of Tirap. (iv) The TIR fold in the Tirap structure is stabilized by internal disulfide bonds, and that the formation of these bonds is unique among reported TIR domain structures. In another study, Lin et al. determined the crystal structure of Tirap at 2.4Å [(9); PDB:3UB2] and found: (i) The crystal structure was similar to the findings in Valkov et al. (ii) Tirap has a central five-stranded parallel sheet (βA-βE) surrounded by four helices (αA, αC-αE). (III) The overall topology of Tirap was similar to other TIR domains, although there were significant differences observed. (iv) Analysis of several mutant Tiraps found that mutations at several residues with the AB loop of Tirap altered TLR signaling, indicating that it is essential or proper Tirap function. In contrast to the study by Valkov et al., Lin et al. found that D96 and L165 were not necessary for MyD88 association. (v) There are two possible interfaces that mediate Tirap dimerization, supporting Valkov et al.
The torpid mutation results in a protein in which the last six C-terminal amino acids of Tirap (encoded by exon 7) are replaced by three unrelated residues. It is unclear how this mutation affects the folding, stability, or signaling properties of the protein. The closest feature to the mutated region would be box 3, although the box 3 sequence is not conserved in Tirap. The TIR domain has been shown to bind PKCδ using purified recombinant GST-fusion Tirap protein (10), suggesting that mutations of the C-terminus, as in torpid, may prevent protein-protein interactions.
Northern blot analysis reveals Tirap expression in all tissues examined, including peripheral blood leukocytes, spleen, thymus, placenta, liver, kidney, skeletal muscle and heart (1;2). Tirap transcript is also detected in mature and immature murine bone marrow-derived dendritic cells and in several monocytic/macrophage cell lines by RT-PCR analysis (1). Tirap is localized to the cytoplasm (1).
Tirap was identified during the search for TIR domain-containing adapters that could mediate MyD88-independent signaling (1;2). Such signaling, elicited by LPS, activates NF-κB, JNK and MAP kinases (11-13)), stimulates upregulation of costimulatory molecules (14;15), and activates IRF-3-mediated transcription of interferon-inducible genes (12;13;16) in MyD88-deficient macrophages or dendritic cells (Figure 4). Poly I:C treatment could also stimulate NF-κB, MAP kinase and upregulation of costimulatory molecules in a MyD88-independent manner (13;17).
Sequence homology searches based on the TIR domain identified a protein with a C terminal TIR domain and homology to MyD88, which was designated Tirap or Mal (1;2). Overexpression of Tirap in HEK 293 cells leads to activation of NF-κB, JNK and MAP kinases, but dominant negative Tirap constructs (for example, the TIR domain of Tirap alone or Tirap P125H, which harbors the same substitution in the corresponding residue of TLR4 that renders mice LPS-unresponsive) inhibit TLR4-dependent NF-κB activation (1;2). Tagged versions of TLR4 and Tirap can be coimmunoprecipitated when co-expressed in HEK 293 cells (1;2). Together, these data suggest that Tirap is an adaptor in the TLR4 signaling pathway.
Dominant negative Tirap constructs fail to inhibit IL-1R- or TLR9-dependent NF-κB activation (1;2), which are known to rely solely on MyD88 (18;19). In addition, dominant negative Tirap (a 14-amino acid Tirap peptide encompassing the P125H mutation) blocked LPS-induced upregulation of costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in wild type and MyD88-deficient dendritic cells (2). Thus, Tirap was advocated as the missing adaptor mediating the MyD88-independent pathway (1;2).
Subsequent study of Tirap-deficient mice (20;21) demonstrated Tirap does not mediate MyD88-independent TLR4 signaling, but that functions with or in parallel to MyD88. Tirap-/- macrophages display the same intact but delayed LPS-dependent activation of NF-κB, JNK and MAP kinases observed in MyD88-deficient macrophages (11;20;21). In addition, expression of interferon-inducible genes encoding IP-10, GARG-16 and IRG-1 is still observed in Tirap-null and MyD88-/-Tirap-/- double knockout mice (21). Tirap-/- and MyD88-/-Tirap-/- DCs also upregulate CD86 and CD40 normally in response to LPS, indicating that DC maturation requires neither Tirap nor MyD88 (20;21). However, torpid macrophages exhibit decreased production of type I IFN in response to LPS, suggesting that some crosstalk may occur between the MyD88-independent pathway and Tirap; such a connection has not been tested.
Tirap serves as an adaptor for TLR4 and TLR2 signaling (20;21). Whereas DC or macrophage IL-6, IL-12 and TNF-α production is normal in response to CpG ODN and R-848 (TLR7 ligand), it is significantly reduced in response to LPS, Pam3CSK4, MALP-2 (diacyl lipopeptide, TLR2/6 ligand) or peptidoglycan (20;21). Tirap signaling in TLR4 and TLR2 pathways involves IRAK-1 (interleukin-1 receptor-associated kinase 1), one of several required kinase components that interact with a MyD88 complex at the receptor. IRAK-1 fails to become activated in response to LPS or MALP-2 in Tirap-/- macrophages, demonstrating that Tirap acts upstream or at the same level as IRAK-1 during TLR4 and -2 signaling (21). Tirap has been shown to form heterodimers with MyD88 by yeast two-hybrid analysis and coimmunoprecipitation, further supporting the idea that Tirap functions in a MyD88 complex (1).
Tirap-mediated signaling events are still under study. Recently, Tirap has been shown to interact via its TIR domain with caspase-1, which processes the precursors of IL-1β and IL-18 (22). Caspase-1 cleaves Tirap at aspartic acid 198, a cleavage required for optimal TLR2- and TLR4-mediated NF-κB and MAP kinase activation (22). LPS- and MALP-2-induced, but not R-848-induced, NF-κB and MAP kinase activation are diminished in caspase-1-deficient macrophages (22).
Tirap function may also require phosphorylation by Bruton’s tyrosine kinase (Btk) (23). A Tirap construct in which two putative tyrosine phosphorylation sites are mutated to phenylalanine acts as a dominant negative inhibitor of LPS-induced NF-κB activation, and treatment of the monocytic cell line THP-1 with a Btk inhibitor blocks LPS- or MALP-2-induced phosphorylation on Tirap (23).
Finally, Tirap may interact with TRAF6 (24), an E3 ubiquitin ligase that also coordinates the activation of several kinases including TAK-1, and in turn MAP kinases and the IKK complex leading to NF-κB activation. Tirap contains a P-X-E-X-X-acidic/aromatic putative TRAF6 binding motif (24). Mutant Tirap in which E190 in the putative TRAF6 binding motif is mutated to alanine inhibits TLR2- or TLR4-dependent activation of an NF-κB reporter in HEK 293 cells (24). It remains unknown whether Tirap interactions with caspase-1, Btk or TRAF6 are important for TLR4 or -2 signaling during microbial infections in vivo.
The single nucleotide polymorphism (SNP) resulting in substitution of leucine for Ser180 of TIRAP, in heterozygous form, is reported to be associated with a protective effect against invasive pneumococcal disease, bacteremia, malaria and tuberculosis (25). In a case-control study of 6,106 individuals from the UK, Vietnam, and several African countries, TIRAP S180L heterozygotes were significantly more common in control than in disease groups (25). Using reconstituted Tirap-/- mouse embryo fibroblasts, the authors demonstrate that MALP-2-induced IκBα degradation, NF-κB reporter activation and IL-6 production failed to occur when cells expressed Tirap S180L, in contrast to wild type Tirap (25). Notably, co-expression of Tirap S180L with wild type Tirap blocked NF-κB reporter activation. Tirap S180L failed to bind TLR2 in in vitro binding assays, although it retained the ability to bind both itself and MyD88. The authors suggest that the TIRAP S180L heterozygous genotype provides the optimal amount of TLR signaling, leading to a balanced inflammatory response (25). However, it remains unknown how this variant affects the signaling pathway between TLR2 or -4 and NF-κB.
|Primers||Primers cannot be located by automatic search.|
The torpid mutation destroys a Bgl II restriction enzyme site in the Tirap genomic DNA sequence. Torpid genotyping is performed by amplifying the region containing the mutation using PCR, followed by Bgl II restriction enzyme digestion.
Torp(F): 5’-GTGAAAGGTAACAGAAACCAGTCACCTCC -3’
1) 94°C 2:00
2) 94°C 0:30
3) 55°C 0:30
4) 72°C 1:00
5) repeat steps (2-4) 40X
6) 72°C 10:00
7) 4°C ∞
The following sequence of 553 nucleotides (from Genbank genomic region NC_000075 for linear DNA sequence of Tirap) is amplified:
11784 gtgaaag gtaacagaaa ccagtcacct cctgggagtg
11821 tgttaaagga gggacattgt agcaggtggg gcagaaaaca ggatggaact ggaagagggg
11881 agaaacacat gtgctgggtt tgggcagagt ggcaagcctt gtgattgtct gtggtctctt
11941 tcagatctgg agacactaag ctgacacttg ggctttcata agaaaagctg ggaatagctc
12001 acagcagtca ttaatccagt atatacagca gagccagagc cagctgcacc ctgtttatga
12061 cacaggatca gatcgtcacc agcttccatt atcgtgggcc cacaagatgg ccaggctgaa
12121 gatgggaacc acccccgcaa ggagaccagg aagaagatgg ggactcccca agaagggcag
12181 gaggaagcca gggactcccc caggaagaca acgaggaagt cagaaattcg agttgtcaag
12241 ggactgtgct atcgggaagg tcagcagtgt cccctctgcg ctgaactgga taggaagctg
12301 atacatggct gtcttcttcc tctggcatca ggcacg
The primer binding sites are underlined; the Bgl II site destroyed by the torpid mutation is highlighted in gray; the mutated A is shown in red text.
Digest PCR reactions with Bgl II. Run on 2% agarose gel with heterozygous and C57BL/6J controls.
Products: Torpid allele- 553 bp. Wild type allele- 160 bp, 393 bp.
1. Fitzgerald, K. A., Palsson-McDermott, E. M., Bowie, A. G., Jefferies, C. A., Mansell, A. S., Brady, G., Brint, E., Dunne, A., Gray, P., Harte, M. T., McMurray, D., Smith, D. E., Sims, J. E., Bird, T. A., and O'Neill, L. A. (2001) Mal (MyD88-Adapter-Like) is Required for Toll-Like Receptor-4 Signal Transduction. Nature. 413, 78-83.
2. Horng, T., Barton, G. M., and Medzhitov, R. (2001) TIRAP: An Adapter Molecule in the Toll Signaling Pathway. Nat. Immunol.. 2, 835-841.
3. Slack, J. L., Schooley, K., Bonnert, T. P., Mitcham, J. L., Qwarnstrom, E. E., Sims, J. E., and Dower, S. K. (2000) Identification of Two Major Sites in the Type I Interleukin-1 Receptor Cytoplasmic Region Responsible for Coupling to Pro-Inflammatory Signaling Pathways. J. Biol. Chem.. 275, 4670-4678.
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5. Rhee, S. H., and Hwang, D. (2000) Murine TOLL-Like Receptor 4 Confers Lipopolysaccharide Responsiveness as Determined by Activation of NF Kappa B and Expression of the Inducible Cyclooxygenase. J. Biol. Chem.. 275, 34035-34040.
6. Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., Galanos, C., Freudenberg, M., Ricciardi-Castagnoli, P., Layton, B., and Beutler, B. (1998) Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4 Gene. Science. 282, 2085-2088.
7. Wesche, H., Henzel, W. J., Shillinglaw, W., Li, S., and Cao, Z. (1997) MyD88: An Adapter that Recruits IRAK to the IL-1 Receptor Complex. Immunity. 7, 837-847.
8. Valkov, E., Stamp, A., Dimaio, F., Baker, D., Verstak, B., Roversi, P., Kellie, S., Sweet, M. J., Mansell, A., Gay, N. J., Martin, J. L., and Kobe, B. (2011) Crystal Structure of Toll-Like Receptor Adaptor MAL/TIRAP Reveals the Molecular Basis for Signal Transduction and Disease Protection. Proc. Natl. Acad. Sci. U. S. A.. 108, 14879-14884.
9. Lin, Z., Lu, J., Zhou, W., and Shen, Y. (2012) Structural Insights into TIR Domain Specificity of the Bridging Adaptor Mal in TLR4 Signaling. PLoS One. 7, e34202.
10. Kubo-Murai, M., Hazeki, K., Sukenobu, N., Yoshikawa, K., Nigorikawa, K., Inoue, K., Yamamoto, T., Matsumoto, M., Seya, T., Inoue, N., and Hazeki, O. (2007) Protein Kinase Cdelta Binds TIRAP/Mal to Participate in TLR Signaling. Mol. Immunol.. 44, 2257-2264.
11. Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999) Unresponsiveness of MyD88-Deficient Mice to Endotoxin. Immunity. 11, 115-122.
12. Hoshino, K., Kaisho, T., Iwabe, T., Takeuchi, O., and Akira, S. (2002) Differential Involvement of IFN-Beta in Toll-Like Receptor-Stimulated Dendritic Cell Activation. Int. Immunol.. 14, 1225-1231.
13. Hoebe, K., Du, X., Georgel, P., Janssen, E., Tabeta, K., Kim, S. O., Goode, J., Lin, P., Mann, N., Mudd, S., Crozat, K., Sovath, S., Han, J., and Beutler, B. (2003) Identification of Lps2 as a Key Transducer of MyD88-Independent TIR Signalling. Nature. 424, 743-748.
14. Kaisho, T., Takeuchi, O., Kawai, T., Hoshino, K., and Akira, S. (2001) Endotoxin-Induced Maturation of MyD88-Deficient Dendritic Cells. J. Immunol.. 166, 5688-5694.
15. Hoebe, K., Janssen, E. M., Kim, S. O., Alexopoulou, L., Flavell, R. A., Han, J., and Beutler, B. (2003) Upregulation of Costimulatory Molecules Induced by Lipopolysaccharide and Double-Stranded RNA Occurs by Trif-Dependent and Trif-Independent Pathways. Nat. Immunol.. 4, 1223-1229.
16. Kawai, T., Takeuchi, O., Fujita, T., Inoue, J., Muhlradt, P. F., Sato, S., Hoshino, K., and Akira, S. (2001) Lipopolysaccharide Stimulates the MyD88-Independent Pathway and Results in Activation of IFN-Regulatory Factor 3 and the Expression of a Subset of Lipopolysaccharide-Inducible Genes. J. Immunol.. 167, 5887-5894.
17. Alexopoulou, L., Holt, A. C., Medzhitov, R., and Flavell, R. A. (2001) Recognition of Double-Stranded RNA and Activation of NF-kappaB by Toll-Like Receptor 3. Nature. 413, 732-738.
18. Schnare, M., Holt, A. C., Takeda, K., Akira, S., and Medzhitov, R. (2000) Recognition of CpG DNA is Mediated by Signaling Pathways Dependent on the Adaptor Protein MyD88. Curr. Biol.. 10, 1139-1142.
19. Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., and Akira, S. (1998) Targeted Disruption of the MyD88 Gene Results in Loss of IL-1- and IL-18-Mediated Function. Immunity. 9, 143-150.
20. Horng, T., Barton, G. M., Flavell, R. A., and Medzhitov, R. (2002) The Adaptor Molecule TIRAP Provides Signalling Specificity for Toll-Like Receptors. Nature. 420, 329-333.
21. Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., Takeuchi, O., Kobayashi, M., Fujita, T., Takeda, K., and Akira, S. (2002) Essential Role for TIRAP in Activation of the Signalling Cascade Shared by TLR2 and TLR4. Nature. 420, 324-329.
22. Miggin, S. M., Palsson-McDermott, E., Dunne, A., Jefferies, C., Pinteaux, E., Banahan, K., Murphy, C., Moynagh, P., Yamamoto, M., Akira, S., Rothwell, N., Golenbock, D., Fitzgerald, K. A., and O'Neill, L. A. (2007) NF-kappaB Activation by the Toll-IL-1 Receptor Domain Protein MyD88 Adapter-Like is Regulated by Caspase-1. Proc. Natl. Acad. Sci. U. S. A.. 104, 3372-3377.
23. Gray, P., Dunne, A., Brikos, C., Jefferies, C. A., Doyle, S. L., and O'Neill, L. A. (2006) MyD88 Adapter-Like (Mal) is Phosphorylated by Bruton's Tyrosine Kinase during TLR2 and TLR4 Signal Transduction. J. Biol. Chem.. 281, 10489-10495.
24. Mansell, A., Brint, E., Gould, J. A., O'Neill, L. A., and Hertzog, P. J. (2004) Mal Interacts with Tumor Necrosis Factor Receptor-Associated Factor (TRAF)-6 to Mediate NF-kappaB Activation by Toll-Like Receptor (TLR)-2 and TLR4. J. Biol. Chem.. 279, 37227-37230.
25. Khor, C. C., Chapman, S. J., Vannberg, F. O., Dunne, A., Murphy, C., Ling, E. Y., Frodsham, A. J., Walley, A. J., Kyrieleis, O., Khan, A., Aucan, C., Segal, S., Moore, C. E., Knox, K., Campbell, S. J., Lienhardt, C., Scott, A., Aaby, P., Sow, O. Y., Grignani, R. T., Sillah, J., Sirugo, G., Peshu, N., Williams, T. N., Maitland, K., Davies, R. J., Kwiatkowski, D. P., Day, N. P., Yala, D., Crook, D. W., Marsh, K., Berkley, J. A., O'Neill, L. A., and Hill, A. V. (2007) A Mal Functional Variant is Associated with Protection Against Invasive Pneumococcal Disease, Bacteremia, Malaria and Tuberculosis. Nat. Genet.. 39, 523-528.
|Science Writers||Eva Marie Y. Moresco, Anne Murray|
|Illustrators||Diantha La Vine, Katherine Timer|
|Authors||Michael Berger, Bruce Beutler|