Agonist-Specific Modulation of MyD88 Interactions Governs the Temporal Dynamics of Immune Responses

Abstract Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns (PAMPs) and activate the defensive innate immune signaling networks. The adaptor protein myeloid differentiation primary response gene 88 (MyD88) functions as a critical scaffold downstream of almost all TLRs, orchestrating the assembly of a supramolecular organizing center (SMOC) called the myddosome. Here, we mapped the interactomes of endogenous MyD88 in mouse macrophages stimulated with four TLR ligands: lipopolysaccharide (LPS), Pam3CysSerLys4 (Pam3CSK4), R848 (resiquimod), and Poly(I:C) (a MyD88-independent negative control) across five time points (0, 10, 30, 60, and 120 min) using 3-plex stable isotope labeling by amino acids in cell culture (SILAC) and affinity purification-mass spectrometry (AP-MS). The results revealed a dynamic, agonist-specific MyD88 interactome, capturing canonical myddosome components (IRAK1, IRAK2, and IRAK4) and novel noncanonical signaling partners, including double-stranded RNA-dependent protein kinase (Eif2ak2/PKR), transactivation response RNA-binding protein 2 (Tarbp2/TRBP), and protein activator of PKR (Prkra/PACT). Downstream validation using targeted coimmunoprecipitation and pharmacological inhibitors confirmed that these noncanonical RNA-binding adaptors are recruited to the MyD88 niche and modulate downstream transcriptional activation, mRNA stability, and microRNA biogenesis (miR-146a, miR-21). These results highlight the modular plasticity of the myddosome, demonstrating its role as a physical scaffold and a temporal integrator that shapes context-dependent innate immune responses.