RESUMEN
Caspase-1 location in cells has been studied with fluorochrome-labeled inhibitors of caspase-1 (FLICA reagents). We report that FLICA reagents have limited cell-membrane permeability. This impacts experimental design as cells with intact membranes, including caspase-1 knockout cells, are not appropriate controls for cells with inflammasome-induced gasdermin D membrane pores.
Asunto(s)
Caspasa 1 , Inhibidores de Caspasas , Permeabilidad de la Membrana Celular , Colorantes Fluorescentes , Inflamasomas , Macrófagos , Caspasa 1/metabolismo , Animales , Macrófagos/inmunología , Macrófagos/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ratones , Inflamasomas/metabolismo , Inhibidores de Caspasas/farmacología , Ratones Noqueados , Proteínas de Unión a Fosfato/metabolismo , HumanosRESUMEN
Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.