Two Human MYD88 Variants, S34Y and R98C, Interfere with MyD88-IRAK4-Myddosome Assembly

Abstract

Innate immune receptors detect microbial pathogens and subsequently activate adaptive immune responses to combat pathogen invasion. MyD88 is a key adaptor molecule in both Toll-like receptor (TLR) and IL-1 receptor superfamily signaling pathways. This is illustrated by the fact that human individuals carrying rare, naturally occurring MYD88 point mutations suffer from reoccurring life-threatening infections. Here we analyzed the functional properties of six reported non-synonymous single nucleotide polymorphisms of MYD88 in an in vitro cellular system. Two variants found in the MyD88 death domain, S34Y and R98C, showed severely reduced NF-κB activation due to reduced homo-oligomerization and IRAK4 interaction. Structural modeling highlights Ser-34 and Arg-98 as residues important for the assembly of the Myddosome, a death domain (DD) post-receptor complex involving the DD of MyD88, IRAK4, and IRAK2 or IRAK1. Using S34Y and R98C as functional probes, our data show that MyD88 homo-oligomerization and IRAK4 interaction is modulated by the MyD88 TIR and IRAK4 kinase domain, demonstrating the functional importance of non-DD regions not observed in a recent Myddosome crystal structure. The differential interference of S34Y and R98C with some (IL-1 receptor, TLR2, TLR4, TLR5, and TLR7) but not all (TLR9) MyD88-dependent signaling pathways also suggests that receptor specificities exist at the level of the Myddosome. Given their detrimental effect on signaling, it is not surprising that our epidemiological analysis in several case-control studies confirms that S34Y and R98C are rare variants that may drastically contribute to susceptibility to infection in only few individuals.

A Coding IRAK2 Protein Variant Compromises Toll-like receptor (TLR) Signaling and Is Associated with Colorectal Cancer Survival

“…Luminescence-based Mammalian Interactome Mapping (LUMIER)-As described in a recent Myddosome study (9), the avidity of a protein-protein interaction is measured by co-expressing two interacting proteins with Protein A (bait) and Renilla luciferase (prey) tags, respectively (see the legend to . 48 h post-transfection, cells were lysed, raw Renilla activity was measured, and Protein A-tagged proteins and bound Renilla-tagged interactors were purified using magnetic IgG beads.…”

“…HEK293T Signaling Assays and ELISA-HEK293T were transfected with an NF-B firefly luciferase reporter (100 ng; Stratagene), pRL-Tk Renilla luciferase control reporter (10 ng; Promega), pC1-EGFP (100 ng; Clontech), and IRAK2 plasmids (as indicated) and assayed as described (9). Luciferase measurements were performed in triplicate Ϯ S.D.…”

“…Gateway-compatible entry clones for IRAK2 or IRAK4 full-length or DD-only or TRAF6 (gift from S. Wiemann, German Cancer Research Center) were transferred via Gateway LR-reaction (Invitrogen) to a pT-Rex-Dest30-based plasmid containing N-terminal Renilla or Protein A tags (9). Alternatively, the IRAK2 full-length and DD constructs were transferred to a pcDNA5/FRT/TO-based plasmid to add an N-or C-terminal Strep-HA tag (T. Bürckstümmer (CeMM, Vienna) and M. Gstaiger (ETH Zurich)).…”

“…Co-immunoprecipitation and Expression Analysis-Immunoprecipitation experiments were done as described (9). TRAF6 ubiquitination analysis was performed as described (15).…”

“…MyD88-IRAK interactions take place in the context of the so-called "Myddosome" postreceptor complex, a hierarchical DD assembly of MyD88, IRAK4, and IRAK2 (7). Myddosome formation is initiated by MyD88 DD oligomerization into a ringlike structure that enables the recruitment of a second DD ring consisting of IRAK4 DD (8), a process blocked by naturally occurring MYD88 mutations (9). The four IRAK4 DDs in turn provide a charge-complementary docking site for a homo-oligomeric ring of four IRAK2 DDs (8).…”