Key Points• The hyperactive phenotype of lymphoma-associated mutations is caused by increased oligomerization propensity of the MyD88 TIR domain.• The TIR domain of mutants interacts with wild-type MyD88, explaining why heterozygous mutation could be sufficient as a driver mutation.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 kB 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. (Blood. 2014;124(26):3896-3904) IntroductionMyeloid differentiation 88 (MyD88) is a pivotal signaling protein in the innate immune system, participating in Toll-like receptor (TLR) signaling pathways during a host's response to infection. 1 Patients with germline MYD88 loss-of-function mutations suffer from severe susceptibility to pyogenic bacterial infection during childhood and only survive into adulthood on a strict therapy of antibiotics.2 Conversely, somatic MyD88 gain-of-function mutations were recently discovered in diffuse large B-cell lymphoma (DBLCL), Waldenström's macroglobulinemia (WM), and chronic lymphocytic leukemia (supplemental Tables 1 and 2 available on the Blood Web site). These data suggest that such mutations, particularly the most prominent Leu265Pro (L265P) mutation, are oncogenic driver mutations that sustain B-cell survival and thus oncogenesis. Indeed, MyD88-mutated cells (cell lines and primary cells) could be selectively killed by ablation of MyD88 downstream signaling using short hairpin RNA or pharmacologic inhibition. 3,4 Although hyperactivation of nuclear factor kB (NF-kB) has been described as a key feature of MyD88-mutated B cells, the molecular mechanism of how the amino acid alterations in lymphomaassociated MyD88 mutations exert this phenotype has to be resolved.MyD88 is composed of an N-terminal death domain (DD) 5 and a highly conserved C-terminal Toll/interleukin-1 receptor (TIR) domain. 6 Whereas the DD and an intermediate linker domain 7 are responsible for downstream signal propagation via kinases of the interleukin-1 receptor-associated kinases (IRAKs), 8 the MyD88 TIR domain integrates signals from upstream TLR and interleukin-1 receptor. Association of TLRs mediated by their agonists triggers the formation of TIR-domain dimers, which results in downstream signaling and expression of genes involved in the host defense system. 1,9 TIR domains comprise 135-...

Background: TIR domains mediate TLR signaling and are hijacked for immunosuppression by bacteria. Results: Tethering of TIR domains or their dimerization by fusion with coiled-coil segment strongly improved inhibition of TLR signaling. Conclusion:The presence of a coiled-coil segment in bacterial TCPs potentiates inhibition while preventing the constitutive activity of TIR domain dimer. Significance: Dimeric TIR domain represents the platform for the formation of Myddosome.

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.

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.

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