Mutational Analysis Identifies Residues Crucial for Homodimerization of Myeloid Differentiation Factor 88 (MyD88) and for Its Function in Immune Cells (original) (raw)
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Journal of Biological Chemistry, 2009
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-B activation upon lipopolysaccharide or PAM 2 CSK 4 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.
MyD88, an Adapter Protein Involved in Interleukin-1 Signaling
Journal of Biological Chemistry, 1998
MyD88 has a modular organization, an N-terminal death domain (DD) related to the cytoplasmic signaling domains found in many members of the tumor necrosis factor receptor (TNF-R) superfamily, and a C-terminal Toll domain similar to that found in the expanding family of Toll/interleukin-1-like receptors (IL-1R). This dual domain structure, together with the following observations, supports a role for MyD88 as an adapter in IL-1 signal transduction; MyD88 forms homodimers in vivo through DD-DD and Toll-Toll interactions. Overexpression of MyD88 induces activation of the c-Jun N-terminal kinase (JNK) and the transcription factor NF-B through its DD. A point mutation in MyD88, MyD88-lpr (F56N), which prevents dimerization of the DD, also blocks induction of these activities. MyD88-induced NF-B activation is inhibited by the dominant negative versions of TRAF6 and IRAK, which also inhibit IL-1induced NF-B activation. Overexpression of MyD88-lpr or MyD88-Toll (expressing only the Toll domain) acted to inhibit IL-1-induced NF-B and JNK activation in a 293 cell line overexpressing the IL-1RI. MyD88 coimmunoprecipitates with the IL-1R signaling complex in an IL-1-dependent manner.
Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling
Proceedings of the National Academy of Sciences, 2009
Myeloid differentiating factor 88 (MyD88) and MyD88 adaptor-like (Mal) are adaptor molecules critically involved in the Toll-like receptor (TLR) 4 signaling pathway. While Mal has been proposed to serve as a membrane-sorting adaptor, MyD88 mediates signal transduction from activated TLR4 to downstream components. The Toll/Interleukin-1 receptor (TIR) domain of MyD88 is responsible for sorting and signaling via direct or indirect TIR؊TIR interactions between Mal and TLR4. However, the molecular mechanisms involved in multiple interactions of the TIR domain remain unclear. The present study describes the solution structure of the MyD88 TIR domain. Reporter gene assays revealed that 3 discrete surface sites in the TIR domain of MyD88 are important for TLR4 signaling. Two of these sites were shown to mediate direct binding to the TIR domain of Mal. Interestingly, Mal-TIR, but not MyD88-TIR, directly binds to the cytosolic TIR domain of TLR4. These observations suggested that the heteromeric assembly of TIR domains of the receptor and adaptors constitutes the initial step of TLR4 intracellular signal transduction.
Journal of Biological Chemistry, 2005
MyD88 plays a crucial role in the signaling pathways triggered by IL-1 and Toll-like receptors in several steps of innate host defense. A crucial event in this signaling pathway is represented by dimerization of MyD88, which allows the recruitment of downstream kinases like IRAK-1 and IRAK-4. Herein, we have investigated the function of the TIR domain in MyD88 homodimerization in cell-free and in vitro experimental settings by using eptapeptides that mimic the BB-loop region of the conserved TIR domain of different proteins. By using a pull-down assay with purified GST-MyD88 TIR or coimmunoprecipitation experiments, we found that epta-peptides derived from the TIR domain of MyD88 and IL-18R are the most effective in inhibiting homodimerization with either the isolated TIR or full-length MyD88. Moreover, we demonstrated that a cell permeable analog of MyD88 eptapeptide inhibits homodimerization of MyD88 TIR domains in an in vitro cell system and significantly reduces IL-1 signaling, as assayed by activation of the downstream transcription factor NF-κB. Our results indicate that the BB-loop in TIR domain of MyD88 is a good target for specific inhibition of MyD88-mediated signaling in vivo.
Biochemical and Biophysical Research Communications, 2009
Homotypic interactions of death domains (DD) mediate complex formation between MyD88 and IL-1 receptor-associated kinases (IRAKs). A truncated splice variant of MyD88, MyD88s, cannot recruit IRAK-4 and fails to elicit inflammatory responses. We have generated recombinant DD of MyD88 and IRAK-4, both alone and extended by the linkers to TIR or kinase domains. We show that both MyD88 DD variants bind to the linker-extended IRAK-4 DD and pull-down full-length IRAK-4 from monocyte extracts. By contrast, residues up to Glu 116 from the DD-kinase connector of IRAK-4 are needed for strong interactions with the adaptor. Our findings indicate that residues 110-120, which form a C-terminal extra helix in MyD88, but not the irregular linker between DD and TIR domains, are required for IRAK-4 recruitment, and provide a straightforward explanation for the negative regulation of innate immune responses mediated by MyD88s.
Journal of Biological Chemistry, 2011
Myeloid differentiation protein 88 (MyD88) is a key signaling adapter in Toll-like receptor (TLR) signaling. MyD88 is also one of the most polymorphic adapter proteins. We screened the reported nonsynonymous coding mutations in MyD88 to identify variants with altered function. In reporter assays, a death domain variant, S34Y, was found to be inactive. Importantly, in reconstituted macrophage-like cell lines derived from knockout mice, MyD88 S34Y was severely compromised in its ability to respond to all MyD88-dependent TLR ligands. Unlike wildtype MyD88, S34Y is unable to form distinct foci in the cells but is present diffused in the cytoplasm. We observed that IRAK4 co-localizes with MyD88 in these aggregates, and thus these foci appear to be "Myddosomes." The MyD88 S34Y lossof-function mutant demonstrates how proper cellular localization of MyD88 to the Myddosome is a feature required for MyD88 function.
FEBS Letters, 2007
MyD88 is a cytoplasmic adaptor protein that is critical for Toll-like receptor (TLR) signaling. The subcellular localization of MyD88 is characterized as large condensed forms in the cytoplasm. The mechanism and significance of this localization with respect to the signaling function, however, are currently unknown. Here, we demonstrate that MyD88 localization depends on the entire non-TIR region and that the correct cellular targeting of MyD88 is indispensable for its signaling function. The Toll-interleukin I receptor-resistance (TIR) domain does not determine the subcellular localization, but it mediates interaction with specific TLRs. These findings reveal distinct roles for the TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88.
Journal of Biological Chemistry, 2005
Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1, a key inflammatory cytokine. IL1 receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1 signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1induced and NFB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix ␣A, 195-197 in Box 2, and 275 in E-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro 200 by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a threedimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed. The importance of the Toll-like receptor (TLR) 1 family in innate immune response to microbial surfaces and nucleic acids is well established. Surprisingly, two members of this family recognize key inflammatory cytokines interleukin (IL) 1 and IL18. These cytokines induce genes that encode other mediators of inflammation such as pleiotropic inflammatory cytokine IL6 and interferon ␥, respectively (1-5).
Modeling MyD88 Deficiency In Vitro Provides New Insights in Its Function
Frontiers in Immunology
Inherited defects in MyD88 and IRAK4, two regulators in Toll-like receptor (TLR) signaling, are clinically highly relevant, but still incompletely understood. MyD88- and IRAK4-deficient patients are exceedingly susceptible to a narrow spectrum of pathogens, with ∼50% lethality in the first years of life. To better understand the underlying molecular and cellular characteristics that determine disease progression, we aimed at modeling the cellular response to pathogens in vitro. To this end, we determined the immunophenotype of monocytes and macrophages derived from MyD88- and IRAK4-deficient patients. We recognized that macrophages derived from both patients were particularly poorly activated by streptococci, indicating that both signaling intermediates are essential for the immune response to facultative pathogens. To characterize this defect in more detail, we generated induced pluripotent stem cells (iPSCs) of fibroblasts derived from an MyD88-deficient patient. The underlying ge...