Abstract
BackgroundA universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)—the myddosome. Alternatively spliced variant MyD88S, lacking the intermediate domain (ID), exhibits a dominant negative effect silencing the immune response, but the mechanistic understanding is limited.MethodsLuciferase reporter assay was used to evaluate functionality of MyD88 variants and mutants. The dimerization potential of MyD88 variants and myddosome nucleation process were monitored by co-immunoprecipitation and confocal microscopy. The ID secondary structure was characterized in silico employing I-TASSER server and in vitro using nuclear magnetic resonance (NMR) and circular dichroism (CD).ResultsWe show that MyD88S is recruited to the nucleating SMOC and inhibits its maturation by interfering with incorporation of additional components. Biophysical analysis suggests that important functional role of ID is not supported by a well-defined secondary structure. Mutagenesis identifies Tyr116 as the only essential residue within ID required for myddosome nucleation and signal propagation (NF-κB activation).ConclusionsOur results argue that the largely unstructured ID of MyD88 is not only a linker separating toll-interleukin-1 receptor (TIR) homology domain and death domain (DD), but contributes intermolecular interactions pivotal in MyD88-dependent signaling. The dominant negative effect of MyD88S relies on quenching the myddosome nucleation and associated signal transduction.Dn-xKRix_iGoWWk4uZnJcGVideo abstract
Highlights
A universal adaptor protein, myeloid differentiation primary response 88 (MyD88), orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R)
Assessing the intermolecular interactions guiding the nucleation of the myddosome, we provide the mechanistic rationale for the dominant negative effect of MyD88S
Overexpression of MyD88L, containing death domain (DD), intermediate domain (ID) and toll-interleukin-1 receptor (TIR) domains, resulted in stimulation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) driven reporter expression while overexpression of MyD88S, containing only DD and TIR domains, had no effect (Fig. 1A) despite reaching protein expression levels comparable to MyD88L (Fig. 1B). These results demonstrate that the ID is essential for spontaneous MyD88 signaling in the absence of receptor stimulation
Summary
A universal adaptor protein, MyD88, orchestrates the innate immune response by propagating signals from toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R). Receptor activation seeds MyD88 dependent formation of a signal amplifying supramolecular organizing center (SMOC)—the myddosome. MyD88 is the central adaptor of innate immunity, integrating signaling via IL-1 receptor (IL-1R) and all TLRs, except TLR3. MyD88-dependent signaling relies on an assembly of a supramolecular organizing center (SMOC), a unique multiprotein complex, known as the myddosome [8, 9]. The SMOC is formed in response to pathogen mediated stimulation of TLRs/ligand mediated stimulation of IL-1 receptor [10]. Myddosome formation initiates the phosphorylation cascade which propagates the signal into the nucleus via activation of NF-κB and activator protein 1 (AP-1) leading to the production of inflammatory mediators
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