Abstract
IL-36 cytokines signal through the IL-36 receptor (IL-36R) and a shared subunit, IL-1RAcP (IL-1 receptor accessory protein). The activation mechanism for the IL-36 pathway is proposed to be similar to that of IL-1 in that an IL-36R agonist (IL-36α, IL-36β, or IL-36γ) forms a binary complex with IL-36R, which then recruits IL-1RAcP. Recent studies have shown that IL-36R interacts with IL-1RAcP even in the absence of an agonist. To elucidate the IL-36 activation mechanism, we considered all possible binding events for IL-36 ligands/receptors and examined these events in direct binding assays. Our results indicated that the agonists bind the IL-36R extracellular domain with micromolar affinity but do not detectably bind IL-1RAcP. Using surface plasmon resonance (SPR), we found that IL-1RAcP also does not bind IL-36R when no agonist is present. In the presence of IL-36α, however, IL-1RAcP bound IL-36R strongly. These results suggested that the main pathway to the IL-36R·IL-36α·IL-1RAcP ternary complex is through the IL-36R·IL-36α binary complex, which recruits IL-1RAcP. We could not measure the binding affinity of IL-36R to IL-1RAcP directly, so we engineered a fragment crystallizable-linked construct to induce IL-36R·IL-1RAcP heterodimerization and predicted the binding affinity during a complete thermodynamic cycle to be 74 μm The SPR analysis also indicated that the IL-36R antagonist IL-36Ra binds IL-36R with higher affinity and a much slower off rate than the IL-36R agonists, shedding light on IL-36 pathway inhibition. Our results reveal the landscape of IL-36 ligand and receptor interactions, improving our understanding of IL-36 pathway activation and inhibition.
Highlights
Two significant figures are reported for the averages
To complete the thermodynamic cycle, we needed to measure the affinity of IL-36␣ binding to a preassociated IL-36R1⁄7IL-1RAcP heterodimer (Fig. 1)
To test for the ability of IL-36 cytokines to bind to IL-36R monomer and IL-36R1⁄7IL-1RAcP heterodimer in solution; we developed a TR–FRET assay using the biotinylated AVI-tagged version of IL-36␣ and IL-36Ra
Summary
It has been shown that IL-36 agonists do not activate NF-B unless both IL-36R and IL-1RAcP are present [1]. Recent studies have shown that loss of function mutations in IL-36Ra leads to a severe and rare form of skin inflammation: generalized pustular psoriasis [5]. An orthogonal time-resolved (TR)–FRET assay was applied to evaluate direct binding. Through these measurements, we were able to establish the temporal order of binding events associated with IL-36 ligands and receptors. We measured direct binding of IL-36Ra1⁄7IL-36R, which was much stronger than the agonist1⁄7IL-36R These results offer a biochemical basis for elucidating IL-36 activation and inhibition
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
