Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2-and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.

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.

Summary Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryo-electron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and further confirmed by structure-based mutagenesis in filament formation in vitro, and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8, and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.

SUMMARY The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes.

Background: Pyrin domains (PYDs) mediate the assembly of inflammasome complexes, but PYD interaction modes are not well characterized. Results: Interaction sites were identified on the PYD of the inflammasome adaptor protein, ASC. Conclusion: ASC PYD has multiple binding sites allowing self-association and interaction with binding partners. Significance: Understanding molecular details of inflammasome assembly may lead to development of anti-inflammatory agents.

Inflammasomes are large protein complexes induced by a wide range of microbial, stress, and environmental stimuli that function to induce cell death and inflammatory cytokine processing. Formation of an inflammasome involves dramatic relocalization of the inflammasome adapter protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into a single speck. We have developed a flow cytometric assay for inflammasome formation, time of flight inflammasome evaluation, which detects the change in ASC distribution within the cell. The transit of ASC into the speck is detected by a decreased width or increased height of the pulse of emitted fluorescence. This assay can be used to quantify native inflammasome formation in subsets of mixed cell populations ex vivo. It can also provide a rapid and sensitive technique for investigating molecular interactions in inflammasome formation, by comparison of wild-type and mutant proteins in inflammasome reconstitution experiments.

Background: ASC mediates inflammasome assembly, recruiting procaspase-1 and procaspase-8 to initiate inflammation and cell death. Results: ASC pyrin domain (PYD) surfaces that mediate filament assembly bind procaspase-8 death effector domains (DEDs) and induce filaments. Conclusion: Procaspase-8 DED filaments are initiated from ASC PYD filaments. Significance: The data give insights into cross-talk between apoptotic and inflammatory pathways and procapase-8 activation.

The innate immune system is the first line of defense against infection and responses are initiated by pattern recognition receptors (PRRs) that detect pathogen-associated molecular patterns (PAMPs). PRRs also detect endogenous danger-associated molecular patterns (DAMPs) that are released by damaged or dying cells. The major PRRs include the Toll-like receptor (TLR) family members, the nucleotide binding and oligomerization domain, leucine-rich repeat containing (NLR) family, the PYHIN (ALR) family, the RIG-1-like receptors (RLRs), C-type lectin receptors (CLRs) and the oligoadenylate synthase (OAS)-like receptors and the related protein cyclic GMP-AMP synthase (cGAS). The different PRRs activate specific signaling pathways to collectively elicit responses including the induction of cytokine expression, processing of pro-inflammatory cytokines and cell-death responses. These responses control a pathogenic infection, initiate tissue repair and stimulate the adaptive immune system. A central theme of many innate immune signaling pathways is the clustering of activated PRRs followed by sequential recruitment and oligomerization of adaptors and downstream effector enzymes, to form higher-order arrangements that amplify the response and provide a scaffold for proximity-induced activation of the effector enzymes. Underlying the formation of these complexes are co-operative assembly mechanisms, whereby association of preceding components increases the affinity for downstream components. This ensures a rapid immune response to a low-level stimulus. Structural and biochemical studies have given key insights into the assembly of these complexes. Here we review the current understanding of assembly of immune signaling complexes, including inflammasomes initiated by NLR and PYHIN receptors, the myddosomes initiated by TLRs, and the MAVS CARD filament initiated by RIG-1. We highlight the co-operative assembly mechanisms during assembly of each of these complexes.