Natural Loss-of-function Mutation of Myeloid Differentiation Protein 88 Disrupts Its Ability to Form Myddosomes

Abstract

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.Toll-like receptors (TLRs) 4 are a part of the innate immune system and play a critical role in host defense against a variety of pathogens. Ten functional TLRs in humans and 12 in mice have been identified to date (1). Each TLR recognizes different categories of evolutionarily conserved patterns called pathogen-associated molecular patterns in microbes. For example, TLR4 recognizes lipopolysaccharide (LPS) from the cell walls of bacteria, whereas CpG-rich DNA from bacteria and viruses acts as the ligand for TLR9.Much of the diversity of TLR pathways can be attributed to differential use of the TIR domain-containing adapter proteins. There are four adapter proteins: MyD88, MyD88 adapter-like (Mal), also called TIR domain-containing adapter protein (TIRAP), TIR domain-containing adapter-inducing interferon-␤ (TRIF), and TRIF-related adaptor molecule (TRAM).MyD88 is a key adapter protein involved in signaling from all of the TLRs except TLR3 and the TRIF-mediated arm of TLR4 signaling. Thus, depending on its use, TLR pathways can be divided into MyD88-dependent and MyD88-independent pathways. The engagement of a particular TLR by its ligand triggers a signaling cascade, which culminates in the secretion of various proinflammatory cytokines and interferons.MyD88 is a 296-amino acid protein with a modular domain structure. In addition to the N-terminal TIR domain, it has a death domain at its C terminus. The crystal structure of the MyD88 death domain has recently been resolved, and it reveals that MyD88 exists in oligomeric form in solution (2). These oligomers of MyD88 can recruit the death domains of interleukin-1 receptor-associated kinase (IRAK)4 and IRAK2 to form a ternary complex called the Myddosome.The importance of single nucleotide polymorphisms (SNPs) in TLR-related proteins has recently been brought to the forefront, with many studies implicating SNPs in disease susceptibility and outcome (3-7). Mal and MyD88 are the most polymorphic of the four adapter proteins, and Mal has received the most scrutiny in this regard (8 -10). However, other than a study by Von Bernuth et al., where children with MyD88 ...

MyD88 Interacts with Interferon Regulatory Factor (IRF) 3 and IRF7 in Atlantic Salmon (Salmo salar)

“…In mammals, it is well known that transgenic SsMyD88 accumulates in granules and larger, condensed structures distributed both throughout the nucleus and the cytoplasm (50). Earlier studies have failed to identify whether the aggregates are associated with any particular organelles, and it has more recently been proposed that these might represent myddosomes (51). In the current study, the up-regulation of the endogenous SsMyD88 protein varied considerably between individuals (Fig.…”

Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of Listeria monocytogenes Infection

“…To then establish the role of NF-κB activation in the observed antibacterial activity of MYD88, we tested the function of a naturally occurring single nucleotide polymorphism of MYD88 (rs1319438) that confers a S34Y substitution. While this mutation does not affect the interaction of MYD88 with IRAK1, IRAK4, and Mal, it disrupts MYD88 signaling and NF-κB activation by preventing oligomeric Myddosome complex formation required for downstream signaling (Fig 3B) [31, 32]. The cellular expression level of MYD88 S34Y was comparable to wild type MYD88 (Fig 3C), however the mutated protein failed to activate NF-κB (Fig 3D).…”

Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96

“…To test whether Semapimod affects this event, we examined changes in intracellular localization of MyD88 following stimulation with LPS, with or without pre-treatment with Semapimod. In untreated cells, MyD88 localized to “myddosomes”, granule-like structures dispersed throughout the cell (37). LPS caused pronounced localization of MyD88 to the cell surface in the absence, but not in the presence of Semapimod (Fig.…”