TRAM promotes Toll-like-receptor-free myddosome signal transduction

In the Toll-like receptor (TLR) signal transduction pathways, activated receptors are present for mere minutes within the initial, membrane-proximal signalling complexes called proto-myddosomes¹. Proto-myddosomes are rapidly released from TLRs to self-assemble (that is, mature) into enzyme-rich, cytosolic supramolecular organizing centres called myddosomes^2-4. Myddosomes induce hours-long inflammatory gene expression^5-10. The mechanism controlling proto-myddosome release and its effect on signal transduction are undefined. Here we identify factors that regulate the maturation process of proto-myddosomes into receptor-free cytosolic myddosomes¹¹. 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^12,13, after which TRAM dissociates MyD88 from the TLR-TIRAP complex to enable myddosome maturation and downstream signal transduction^14-19. 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^20-24 and secondary response gene expression^25-27. Chemical dissociation of cytosolic myddosomes disrupted existing inflammatory activities of TLR-stimulated cells in vitro and in vivo. Collectively, this work establishes TRAM as a regulator of myddosome maturation and signalling, providing the molecular basis for receptor-free signal transduction.