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

Julie George,Precious G Motshwene,Hui Wang,Andriy V Kubarenko,Anna Rautanen,Tara C Mills,Adrian V.S Hill,Nicholas J Gay,Alexander N.R Weber

doi:10.1074/jbc.m110.159996

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.

Highlights

myeloid differentiation primary response gene 88 (MyD88)-dependent signaling involves three functional domains; that is, a C-terminal, evolutionarily conserved Toll/IL-1 receptor (TIR) domain present in all Toll-like receptor (TLR) adaptors that allows for upstream homotypic interactions with stimulated TLR and IL-1 receptor complexes, a central so-called intermediate domain (ID; residues 110 –154) of unknown protein architecture that is absent in an inhibitory, shorter splice variant, MyD88s [6], and an N-terminal domain belonging to the death domain (DD; residues 19 –109) superfamily required for recruitment and activation of downstream DD-containing kinases of the IL-1 receptor-associated kinase (IRAK)-family
Two Naturally Occurring Mutations in the MyD88 Death Domain Are Dysfunctional in Downstream Signaling—In the human MYD88 gene more than 40 single nucleotide polymorphisms (SNPs) have been reported, six of which result in amino acid substitutions in different MyD88 domains: S34Y, L35V, L74M, R98C (DD), K115N (ID), and M178I (TIR)
To functionally characterize the genetic variants reported for MyD88, Myc-tagged expression constructs corresponding to the nsSNP-associated amino acid changes (“MyD88 mutants”) were generated

Summary

Introduction

The expression levels were similar (Fig. 1B), and the MyD88 mutants were tested for their ability to activate the NF-␬B signaling pathway using a dual luciferase assay in HEK293 cells. We conducted LUMIER experiments, and this showed that the interaction of MyD88 WT FL with IRAK4 FL was comparable with MyD88 FL or DD interactions with IRAK4 DDs. By contrast, no interaction of IRAK4 FL could be detected with the S34Y and R98C mutants (Fig. 5B, Panel 3), implying that the presence of the kinase domain influences the assembly of the Myddosome in vitro.

Results

Conclusion