A TIR domain variant of MyD88 adapter-like (Mal)/TIRAP results in loss of MyD88 binding and reduced TLR2/TLR4 signaling

Division of Infectious Diseases and Immunology, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2009; 284(38):25742-8. DOI: 10.1074/jbc.M109.014886
Source: PubMed


The adapter protein MyD88 adapter-like (Mal), encoded by TIR-domain containing adapter protein (Tirap) (MIM 606252), is the most polymorphic of the five adapter proteins involved in Toll-like receptor signaling, harboring eight
non-synonymous single nucleotide polymorphisms in its coding region. We screened reported mutations of Mal for activity in
reporter assays to test the hypothesis that variants of Mal existed with altered signaling potential. A TIR domain variant,
Mal D96N (rs8177400), was found to be inactive. In reconstituted cell lines, Mal D96N acted as a hypomorphic mutation, with
impaired cytokine production and NF-κB activation upon lipopolysaccharide or PAM2CSK4 stimulation. Moreover, co-immunoprecipitation studies revealed that Mal D96N is unable to interact with MyD88, a prerequisite
for downstream signaling to occur. Computer modeling data suggested that residue 96 resides in the MyD88 binding site, further
supporting these findings. Genotyping of Mal D96N in three different cohorts suggested that it is a rare mutation. We, thus,
describe a rare variant in Mal that exerts its effect via its inability to bind MyD88.

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Available from: Theo S Plantinga, Apr 17, 2014
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    • "Therefore, it was proposed that PI(4,5)P2 recruits TIRAP to the plasma membrane following engagement of TLR4 ligand, thereby triggering the early phase of the MyD88-dependent pathway. In this working model, PI(4,5)P2-dependent TIRAP serves as a sorting adaptor for connecting TLR4 and MyD88, whereas MyD88 mainly functions as a signaling adaptor for TLR4 activation at the plasma membrane (52,53). A previous study demonstrated that Drosophila MyD88 harbors a PI(4,5)P2 binding motif at the C-terminus, which enables plasma membrane localization (54). "
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    • "To define the means by which TIRAP regulates TLR signaling from multiple organelles, we sought a cell type that would be easy to propagate and amenable to genetic manipulation. Immortalized BMDMs (iBMDMs) have emerged as a useful tool in this regard because they retain the signaling properties of their primary cell counterparts (Dixit et al., 2010; Halle et al., 2008), and TIRAP KO iBMDMs have been used to dissect the functions of this adaptor (Nagpal et al., 2009). Similar to our observations made in primary BMDMs, TIRAP was required for iBMDMs to respond to several substrains of HSV, including two that engage only TLR9 (Kos A and Kos CE) (Figure 2A) (Sato et al., 2006). "
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    ABSTRACT: The Toll-like receptors (TLRs) of the innate immune system are unusual in that individual family members are located on different organelles, yet most activate a common signaling pathway important for host defense. It remains unclear how this common signaling pathway can be activated from multiple subcellular locations. Here, we report that, in response to natural activators of innate immunity, the sorting adaptor TIRAP regulates TLR signaling from the plasma membrane and endosomes. TLR signaling from both locations triggers the TIRAP-dependent assembly of the myddosome, a protein complex that controls proinflammatory cytokine expression. The actions of TIRAP depend on the promiscuity of its phosphoinositide-binding domain. Different lipid targets of this domain direct TIRAP to different organelles, allowing it to survey multiple compartments for the presence of activated TLRs. These data establish how promiscuity, rather than specificity, can be a beneficial means of diversifying the subcellular sites of innate immune signal transduction.
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    • "co-immunoprecipitation studies and computer modeling data (Nagpal et al., 2009; George et al., 2010) revealed that this variation results in conformation changes in the MyD88 binding site and thus the TIRAP G286A variant is unable to interact with MyD88, a prerequisite for downstream signaling to activate the responses to Mtb. These facts lead us to suggest that the 286A allele is a risk factor for increased susceptibility to TB. "
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    ABSTRACT: Toll-interleukin 1 receptor (TIR) domain containing adaptor protein (TIRAP; also known as MAL) is an essential adaptor molecule in Toll-like receptor signaling, involved in activating the innate immune response during infection. Genetic variations in the TIRAP gene may influence human susceptibility to infectious disease. To date, in the Chinese population, a possible predisposition of TIRAP gene variants to tuberculosis has not been reported. We investigated whether TIRAP gene polymorphisms are associated with the development of tuberculosis in a Chinese population. We investigated all the single-nucleotide polymorphisms (SNPs) within the TIRAP exon 5 in a case-control study of 212 patients with tuberculosis and 215 controls in a Chinese population. Genotyping was performed to identify the polymorphisms of TIRAP gene by PCR-DNA sequencing method. Haplotypes for the TIRAP gene variants were constructed using Haplo view version 4.2. Six polymorphisms of the SNPs listed in the National Center for Biotechnology Information database were detected in these Chinese tuberculosis patients. It was found that both the frequency of the 286A allele (odds ratio (OR) = 13.37; 95% confidence interval (CI) = 0.75-238.3; P < 0.01) and the frequency of 286AG genotype (OR = 13.57; 95%CI = 0.76-242.5; P < 0.01) were significantly higher in patients than in healthy controls. However, two other SNPs, C539T and C558T, reported to be associated with tuberculosis in other populations, were found not to be associated with tuberculosis in this Chinese population. We conclude that TIRAP G286A (D96N) polymorphism is associated with susceptibility to tuberculosis and may be a new risk factor for the development of tuberculosis in China.
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