Evidence item

Here we identify factors that regulate the maturation process of proto-myddosomes into receptor-free cytosolic myddosomes. Through a genetic screen in macrophages, we identify TRAM, which was previously described to control MyD88-independent TLR signalling, as a regulator of myddosome assembly. Using biochemistry, live-cell imaging and activity reconstitution with recombinant proteins, we show that the plasma-membrane-associated adapter TIRAP seeds proto-myddosomes, after which TRAM dissociates MyD88 from the TLR–TIRAP complex to enable myddosome maturation and downstream signal transduction. In the absence of TRAM, MyD88 cannot dissociate from the TLR–TIRAP complex, resulting in abnormal and unstable MyD88 interactions with downstream signalling enzymes. These findings enabled us to identify TRAM-dependent long-lasting myddosome activities as the determinant of two TLR pathway hallmarks: hours-long NF-κB activation and secondary response gene expression. Chemical dissociation of cytosolic myddosomes disrupted existing inflammatory activities of TLR-stimulated cells in vitro and in vivo.