Structural basis of TIR-domain-assembly formation in MAL- and MyD88-dependent TLR4 signaling

Toll-like receptor (TLR) signaling is a key innate immunity response to pathogens. Recruitment of signaling adapters such as MAL (TIRAP) and MyD88 to the TLRs requires Toll/interleukin-1 receptor (TIR)-domain interactions, which remain structurally elusive. Here we show that MAL TIR domains spontaneously and reversibly form filaments in vitro. They also form cofilaments with TLR4 TIR domains and induce formation of MyD88 assemblies. A 7-Å-resolution cryo-EM structure reveals a stable MAL protofilament consisting of two parallel strands of TIR-domain subunits in a BB-loop-mediated head-to-tail arrangement. Interface residues that are important for the interaction are conserved among different TIR domains. Although large filaments of TLR4, MAL or MyD88 are unlikely to form during cellular signaling, structure-guided mutagenesis, combined with in vivo interaction assays, demonstrated that the MAL interactions defined within the filament represent a template for a conserved mode of TIR-domain interaction involved in both TLR and interleukin-1 receptor signaling

Prion-like Protein Polymerisation Underlies Signal Transduction in Innate Immunity: The Emergence of a universal mechanism?

The innate immune system uses a multitude of receptors and intracellular signalling pathways to sense pathogens and danger signals. Recent studies have discovered that an array of these intracellular signalling proteins assemble into oligomeric signalling platforms, consequently amplifying the transmission of signal from the stimulated receptor to the activation of relevant transcription machinery. Unexpectedly, it was also discovered that some of these proteins present in innate immune signalling can form fibrils. These fibrils can elongate in a prion-like manner to create super-sized signalling platforms. Through screening over 100 innate immune intracellular signalling proteins using single molecule fluorescence, we defined the oligomerisation and aggregation propensity of these proteins, tracking the specific signature of polymerisation. We identified multiple candidates forming super-sized protein aggregates; and were able to conclude that 19 of these proteins could self-propagate aggregation, just as prions do. 4 of these proteins had been previously published as being prion-like, leaving 15 novel prion-like polymers identified.Our observations suggest that prion-like polymerisation underlies the backbone of intracellular communication and drives the complex interplay that exists among the major signalling cascades of the innate immune system. Our disease-associated mutagenesis data highlights the crucial functions that these large signalling platforms have in the optimal execution of defence against pathogens and intracellular homeostasis and will contribute to the development of therapies aimed at aiding and regulating this critical first line of defence

Cell-free formation and interactome analysis of caveolae

Caveolae have been linked to the regulation of signaling pathways in eukaryotic cells through direct interactions with caveolins. Here, we describe a cell-free system based on () extracts for the biogenesis of caveolae and show its use for single-molecule interaction studies. Insertion of expressed caveolin-1 (CAV1) into membranes was analogous to that of caveolin in native membranes. Electron tomography showed that caveolins generate domains of precise size and curvature. Cell-free caveolae were used in quantitative assays to test the interaction of membrane-inserted caveolin with signaling proteins and to determine the stoichiometry of interactions. Binding of membrane-inserted CAV1 to several proposed binding partners, including endothelial nitric-oxide synthase, was negligible, but a small number of proteins, including TRAF2, interacted with CAV1 in a phosphorylation-(CAV1)-stimulated manner. In cells subjected to oxidative stress, phosphorylated CAV1 recruited TRAF2 to the early endosome forming a novel signaling platform. These findings lead to a novel model for cellular stress signaling by CAV1