Activation of lymphoma-associated MyD88 mutations via allostery-induced TIR-domain oligomerization.

Myeloid differentiation 88 (MyD88) is the key signaling adapter of Toll-like and interleukin-1 receptors. Recurrent lymphoma-associated mutations, particularly Leu265Pro (L265P), within the MyD88 Toll/interleukin-1 receptor (TIR) domain sustain lymphoma cell survival due to constitutive nuclear factor κB signaling. We found that mutated TIR domains displayed an intrinsic propensity for augmented oligomerization and spontaneous formation of cytosolic Myddosome aggregates in lymphoma cell lines, mimicking the effect of dimerized TIR domains. Blocking of MyD88 oligomerization induced apoptosis. The L265P TIR domain can recruit the endogenous wild-type MyD88 for oligomer formation and hyperactivity. Molecular dynamics simulations and analysis of additional mutations suggest that constitutive activity is caused by allosteric oligomerization.

Toll/interleukin-1 receptor domain dimers as the platform for activation and enhanced inhibition of Toll-like receptor signaling.

TIR (Toll/IL-1 receptor) domains mediate interactions between TLR (Toll-like) or IL-1 family receptors and signaling adapters. While homotypic TIR domain interactions mediate receptor activation they are also usurped by microbial TIR domain containing proteins for immunosuppression. Here we show the role of a dimerized TIR domain platform for the suppression as well as for the activation of MyD88 signaling pathway. Coiled-coil dimerization domain, present in many bacterial TCPs, potently augments suppression of TLR/IL-1R signaling. The addition of a strong coiled-coil dimerization domain conferred the superior inhibition against the wide spectrum of TLRs and prevented the constitutive activation by a dimeric TIR platform. We propose a molecular model of MyD88-mediated signaling based on the dimerization of TIR domains as the limiting step.

Suppression of TLR signaling by targeting TIR domain-containing proteins.

Toll-like receptors (TLRs) recognize molecules specific to pathogens and endogenous danger signals. Binding of agonists to the ectodomain of the receptor initiates TLR activation and is followed by the association of receptor cytosolic Toll/Interleukin-1 receptor (TIR) domains with TIR domains of adapter proteins leading to the assembly of signaling cascade of protein kinases that ultimately trigger the activation of transcription factors and expression of genes involved in the immune response. Excessive activation of TIR-domain mediated signaling has been implicated in inflammatory diseases (e.g. rheumatoid arthritis, systemic lupus erythematosus, colitis) as well as in the development of cancer. Targeting receptor-adapter interactions represents a potential strategy for the therapeutic TLR/IL-1R-specific inhibition due to the unique interacting domains involved. Peptide and protein-domain binding TLR inhibitors originating from the interacting surfaces of TIR-domain containing proteins can bind to the site on their target interacting protein thereby preventing the assembly of the functional signaling complex. Here we review protein-domain, peptide and peptidomimetic inhibitors targeting TIR-domain mediated interactions and their application demonstrated on in vitro and in vivo models. Recent structural data and elucidation of the molecular mechanisms of TIR-domain mediated signaling enabled the development of peptide inhibitors from TIR domains of TLRs and adapters, MyD88 intermediary domain as well as improved protein inhibitors based on TIR domain dimerization, mimicking bacterial TIR-domain containing immunosuppressors (TCPs) which we discuss with challenges concerning the delivery and specificity of inhibitors targeting TLR adapters.

Inhibition and protection of cholinesterases by methanol and ethanol.

The cholinesterases have been investigated in terms of the effects of methanol and ethanol on substrate and carbamate turnover, and on their phosphorylation. It was found: 1) that at low substrate concentrations the two alcohols inhibit all three tested cholinesterases and that the optimum activities are shifted towards higher substrate concentrations, but with a weak effect on horse butyrylcholinesterase; 2) that methanol slows down carbamoylation by eserine and does not influence decarbamoylation of vertebrate and insect acetylcholinesterase and 3) that ethanol decreases the rate of phosphorylation of vertebrate acetylcholinesterase by DFP. Our results are in line with the so-called 'approach-and-exit' hypothesis. By hindering the approach of substrate and the exit of products, methanol and ethanol decrease cholinesterase activity at low substrate concentrations and allow for the substrate inhibition only at higher substrate concentrations. Both effects appears to be a consequence of the lower ability of substrate to substitute alcohol rather than water. It also seems that during substrate turnover in the presence of alcohol the transacetylation is negligible.