The Role of Intermediary Domain of MyD88 in Cell Activation and Therapeutic Inhibition of TLRs

Abstract

Adaptor MyD88 has a pivotal role in TLR and IL-1R signaling and is involved in mediating excessive inflammation. MyD88 is composed of a death domain and a Toll/IL-1R domain connected by an intermediary domain (INT). The alternatively spliced form of MyD88 lacking the INT prevents signaling through MyD88-dependent TLRs. We designed a peptide from the INT and showed that it inhibits TLR4 activation by LPS when linked to a cell-penetrating peptide. As a new approach for the delivery of signaling-inhibitory peptides, INT peptide acylation also provided efficient cell translocation and inhibition of activation. We determined that INT peptide targets IL-1R–associated kinase 4. Furthermore, MyD88 mutant and molecular modeling refines the MyD88– IL-1R–associated kinase 4 interaction model based on the Myddosome structure. In addition to TLR4, INT peptide also inhibited TLR5, TLR2, TLR9, and IL-1R signaling but not TLR3, which uses Toll/IL-1R domain-containing adapter inducing IFN-β signaling adaptor. Inhibition of signaling in murine and human cells was observed by decreased NF-κB activation, cytokine mRNA synthesis, and phosphorylation of downstream kinases. In the endotoxemic mouse model, INT peptide suppressed production of inflammatory cytokines and improved survival, supporting therapeutic application of INT peptides for the suppression of inflammatory conditions mediated by MyD88.

Toll/Interleukin-1 Receptor Domain Dimers as the Platform for Activation and Enhanced Inhibition of Toll-like Receptor Signaling

“…Luciferase Reporter Assay-NF-B or IFN-␤-dependent firefly luciferase and constitutive Renilla luciferase reporter were used to analyze the cell activation using a dual luciferase assay as described before (21). In the experiments with ligand stimulation, the RLU ϭ RLU (stimulated cells)-RLU (unstimulated cells), unless stated otherwise.…”

“…Immunoblotting was performed as described (21). The antibodies used were polyclonal MyD88 Ab 1:500 dilution (PRS2127, Sigma), GFP antibody 1:1000 dilution (A11122, Invitrogen), ␣␤-tubulin rabbit polyclonal Ab 1:1000 dilution (2148, NEB), polyclonal anti-Flag antibodies 1:1000 dilution (F7425, Sigma), anti-HA antibody 1:1000 dilution (H6908, Sigma).…”

Toll-like Receptors in the Vascular System: Sensing the Dangers Within

“…The delivery of TLR inhibitors as purified proteins has been suggested but the cost of this approach is currently prohibitory. Cell-penetrating moieties, including short cationic peptides and fatty acids, have been successfully added to inhibitory peptides to cross the cell membrane and target TLR signaling (Nelson et al, 2007;Avbelj et al, 2011). The use of microRNAs and inhibitors of ubiquitination to suppress excessive immune system activation and inflammation may be promising, but safety and efficacy as well as specificity have not yet been addressed.…”

“…Other inhibitory peptides including INT peptides (derived from the N terminus of the intermediary domain of MyD88) and VIPER peptides (derived from vaccinia virus protein A46) have been also used. INT peptides inhibit MyD88-dependent signaling pathways and suppress the production of inflammatory cytokines (Avbelj et al, 2011), whereas VIPER peptides target TLR4 adapters Mal/TIRAP and TRAM (Stack et al, 2005).…”

Cell Death Triggered by Yersinia enterocolitica Identifies Processing of the Proinflammatory Signal Adapter MyD88 as a General Event in the Execution of Apoptosis

“…Of interest, the MyD88 caspase-processing site D135 is highly conserved across several vertebral species, including human, dog, zebrafish, zebra finch, clawed frog, and chinese softshell turtle (Ref. 45 and data not shown). This observation supports the idea that the MyD88 caspase-processing site plays a physiological role in the course of apoptosis.…”