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NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1 CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASC CARD –caspase-1 CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling. Pathogen triggered N-terminal degradation of NLRP1 and CARD8 by the proteasome releases their C-terminal UPA-CARD fragments (CT) to form the inflammasome, which in turn activates caspase-1. Here, the authors present the cryo-EM structures of the NLRP1-CT and CARD8-CT helical filaments as well as the ASC − caspase-1 octamer structure, which together with in vitro and cell based assays provide further insights into the architecture and specificity of the active NLRP1 and CARD8 inflammasomes.

Multiple innate immune sensors undergo rapid assembly into large complexes known as signalosomes. This is an essential step during cellular responses to microbes and danger signals. How this process is regulated to avoid accumulation of potentially toxic protein aggregates remains poorly understood. Abdel-Nour et al. identified a pathway, dependent on heme-regulated inhibitor, eukaryotic initiation factor 2α, activating transcription factor 4, and heat shock protein B8, which controls the folding and scaffolding of innate immune sensors, allowing optimal proinflammatory signaling (see the Perspective by Pierre). The pathway appears to mirror the endoplasmic reticulum unfolded protein response (UPR), and so was named the cytosolic UPR (cUPR). The cUPR may represent a general mechanism to control protein misfolding in cells. Science , this issue p. eaaw4144 ; see also p. 28 A cytosolic unfolded protein response controls the scaffolding and signaling of innate immune complexes Multiple cytosolic innate sensors form large signalosomes after activation, but this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We found that the eIF2α kinase heme-regulated inhibitor (HRI) controls NOD1 signalosome folding and activation through a process requiring eukaryotic initiation factor 2α (eIF2α), the transcription factor ATF4, and the heat shock protein HSPB8. The HRI/eIF2α signaling axis was also essential for signaling downstream of the innate immune mediators NOD2, MAVS, and TRIF but dispensable for pathways dependent on MyD88 or STING. Moreover, filament-forming α-synuclein activated HRI-dependent responses, which suggests that the HRI pathway may restrict toxic oligomer formation. We propose that HRI, eIF2α, and HSPB8 define a novel cytosolic unfolded protein response (cUPR) essential for optimal innate immune signaling by large molecular platforms, functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR.

Inflammasomes are multiprotein complexes that orchestrate proinflammatory cytokine secretion and cell death. Proteases such as anthrax lethal factor can activate an inflammasome known as NLRP1B, but the mechanism for this activation has been unclear. Chui et al. used genome-wide knockout screens to show that proteolysis of NLRP1B by lethal factor induces proteasomal degradation of the amino-terminal domains of NLRP1B and eventual cell death. Sandstrom et al. found that degradation of the amino-terminal domains of NLRP1B resulted in the release of a carboxyl-terminal fragment that activates caspase-1. This process, called “functional degradation,” allows the immune system to detect pathogen-associated activities, much as it recognizes pathogen-associated antigens. Science , this issue p. 82 , p. eaau1330 Pathogen-associated proteasomal degradation is detected by innate immune sensor NLRP1B. Inflammasomes are multiprotein platforms that initiate innate immunity by recruitment and activation of caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. In this study, we find that cleavage results in proteasome-mediated degradation of the amino-terminal domains of NLRP1B, liberating a carboxyl-terminal fragment that is a potent caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

Innate immune receptors such as RIG-I, cGAS, and Toll-like receptors bind microbial fragments and alert the immune system to an infection. Each receptor type signals through a different adapter protein. These signals activate the protein kinase TBK1 and the transcription factor IRF3, which tells cells to secrete interferon proteins (IFNs) important for host defense. Liu et al. now report a common signaling mechanism used by all three types of innate immune receptor-adaptor protein pairs to activate IRF3 and generate IFNs. This is important because cells must regulate their IFN production carefully to avoid inflammation and autoimmunity. Science , this issue 10.1126/science.aaa2630 Diverse innate immune receptors use a common signaling mechanism to activate type I interferons. During virus infection, the adaptor proteins MAVS and STING transduce signals from the cytosolic nucleic acid sensors RIG-I and cGAS, respectively, to induce type I interferons (IFNs) and other antiviral molecules. Here we show that MAVS and STING harbor two conserved serine and threonine clusters that are phosphorylated by the kinases IKK and/or TBK1 in response to stimulation. Phosphorylated MAVS and STING then bind to a positively charged surface of interferon regulatory factor 3 (IRF3) and thereby recruit IRF3 for its phosphorylation and activation by TBK1. We further show that TRIF, an adaptor protein in Toll-like receptor signaling, activates IRF3 through a similar phosphorylation-dependent mechanism. These results reveal that phosphorylation of innate adaptor proteins is an essential and conserved mechanism that selectively recruits IRF3 to activate the type I IFN pathway.

Key Points Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a newly described family of intracellular sensors of microbial infections and danger signals. NLRs detect microbial motifs such as bacterial peptidoglycan (sensed by NOD1 and NOD2) or bacterial flagellin (sensed by NLR family, CARD-domain-containing 4 (NLRC4; also known as IPAF)) and NLR family, apoptosis inhibitory protein 5 (NAIP5). NLRs also sense danger signals, including uric acid, K + efflux, extracellular ATP, silica, asbestos and β-amyloid peptide through NLR family, pyrin domain-containing 3 (NLRP3). NLRs trigger innate immune responses by inducing signalling pathways, such nuclear factor-κB, mitogen-activated protein kinases, and the caspase 1 inflammasome. This results in the activation of inflammatory cytokines and/or chemokines. NLRs also work in synergy with Toll-like receptors to potentiate signal transduction pathways. Mutations in several NLR genes are associated with autoinflammatory disorders, including NOD2 (associated with Crohn's disease and Blau syndrome), NLRP3 (associated with Muckle–Wells syndrome, chronic infantile neurologic cutaneous and articular syndrome, and familial cold urticaria), NOD1 (associated with asthma, allergy and atopic eczema) and NLRP1 (associated with vitiligo). NLRs have a crucial role in the detection of molecules that were initially known as adjuvants, such as muramyl peptides and complete Freund's adjuvant (sensed by NOD1 and NOD2) and aluminium hydroxide (sensed by NLRP3). On detection of these molecules, NLRs shape the immune response to antigens, highlighting the link between NLRs and adaptive immunity. Because of their importance in innate immunity and adjuvanticity, NLRs and NLR-triggered pathways are promising target candidates for therapeutic strategies against autoinflammatory disorders. The recent development of interleukin 1-specific strategies against gout and Muckle–Wells syndrome illustrates the translation of NLR basic research into clinical practice. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. This Review discusses the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections. Nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are a family of intracellular sensors that have key roles in innate immunity and inflammation. Whereas some NLRs — including NOD1, NOD2, NAIP (NLR family, apoptosis inhibitory protein) and NLRC4 — detect conserved bacterial molecular signatures within the host cytosol, other members of this family sense 'danger signals', that is, xenocompounds or molecules that when recognized alert the immune system of hazardous environments, perhaps independently of a microbial trigger. In the past few years, remarkable progress has been made towards deciphering the role and the biology of NLRs, which has shown that these innate immune sensors have pivotal roles in providing immunity to infection, adjuvanticity and inflammation. Furthermore, several inflammatory disorders have been associated with mutations in human NLRgenes. Here, we discuss the effect that research on NLRs will have on vaccination, treatment of chronic inflammatory disorders and acute bacterial infections.

Cell-surface receptors frequently use scaffold proteins to recruit cytoplasmic targets, but the rationale for this is uncertain. Activated receptor tyrosine kinases, for example, engage scaffolds such as Shc1 that contain phosphotyrosine (pTyr)-binding (PTB) domains. Using quantitative mass spectrometry, here we show that mammalian Shc1 responds to epidermal growth factor (EGF) stimulation through multiple waves of distinct phosphorylation events and protein interactions. After stimulation, Shc1 rapidly binds a group of proteins that activate pro-mitogenic or survival pathways dependent on recruitment of the Grb2 adaptor to Shc1 pTyr sites. Akt-mediated feedback phosphorylation of Shc1 Ser 29 then recruits the Ptpn12 tyrosine phosphatase. This is followed by a sub-network of proteins involved in cytoskeletal reorganization, trafficking and signal termination that binds Shc1 with delayed kinetics, largely through the SgK269 pseudokinase/adaptor protein. Ptpn12 acts as a switch to convert Shc1 from pTyr/Grb2-based signalling to SgK269-mediated pathways that regulate cell invasion and morphogenesis. The Shc1 scaffold therefore directs the temporal flow of signalling information after EGF stimulation. The Shc1 scaffold mediates a switch in the signaling output of the epidermal growth factor receptor tyrosine kinase over time through recruitment of successive waves of proteins with distinct biological functions. More than a support role for scaffold proteins Receptor-associated scaffolds are generally thought of as relatively static components of signalling pathways that link an activated receptor to downstream targets and expand the receptor's range and potency. An example is the scaffold protein Shc1, which binds to the activated EGF receptor tyrosine kinase. Here, Tony Pawson and colleagues use a quantitative proteomics approach to demonstrate that Shc1 is more than just a simple adaptor; it recruits successive waves of proteins with distinct functions and thereby switches the signalling output of the EGF receptor over time.

Redundancy of cancer cells towards ROS-mediated apoptosis despite expressing proline-rich p66shc abundantly needs to be investigated properly. P66shc, an adapter protein, is indispensable both for initiating ROS-mediated apoptosis and subsequent ROS generation through Rac-1 activation. P66shc gets phosphorylated at Ser-36 that triggers its translocation to the mitochondria and subsequent release of Cytochrome c in response to oxidative stress. It also aids in Rac-1 dependent NADPH oxidase activation, leading to the generation of cytosolic ROS that can perform diverse functions depending on its concentration. This study has identified the multi-faceted anti-apoptotic protein BAG3 as an interacting partner of p66shc. BAG3 utilizes its WW domain to bind to the proline-rich motifs of p66shc. BAG3, through its WW domain, antagonizes p66shc mediated apoptosis, by inhibiting both the expression and phosphorylation of p66shc under normal and oxidative stress conditions. This results in significant protection against ROS-mediated apoptosis. BAG3-mediated reduction in p66shc expression increases cell proliferation and metastasis. The increase in cell proliferation is attributed to the impact of BAG3 on Rac-1 activation and ROS production under normal conditions. This study has unraveled an interactor of p66shc that enhances pro-survival role while simultaneously suppressing its apoptotic role. Graphical Abstract Highlights BAG3 & p66shc interact with each other. This interaction is mediated by WW domain of BAG3 & certain proline residues located at position 47–50 on CH2 domain of p66shc. BAG3 curtails p66shc mediated apoptosis by inhibiting its expression and its subsequent Ser-36 phosphorylation. Besides this, BAG3 is possibly also involved in elevating oncogenic activities of p66shc as can be held enhanced Rac-1 activation. P66shc also acts reciprocally on expression of BAG3 thereby further confirming their contradictory roles. BAG3 mediated curtailment in p66shc apoptotic activity results in enhanced proliferation and metastasis and diminished apoptosis under in-vitro conditions.

Interferons (IFNs) are a group of cytokines with a well-established antiviral function. They can be induced by viral infection, are secreted and bind to specific receptors on the same or neighbouring cells to activate the expression of hundreds of IFN stimulated genes (ISGs) with antiviral function. Type I IFN has been known for more than half a century. However, more recently, type III IFN (IFNλ, IL-28/29) was shown to play a similar role and to be particularly important at epithelial surfaces. Here we show that airway epithelia, the primary target of influenza A virus, produce both IFN I and III upon infection, and that induction of both depends on the RIG-I/MAVS pathway. While IRF3 is generally regarded as the transcription factor required for initiation of IFN transcription and the so-called \"priming loop\", we find that IRF3 deficiency has little impact on IFN expression. In contrast, lack of IRF7 reduced IFN production significantly, and only IRF3(-/-)IRF7(-/-) double deficiency completely abolished it. The transcriptional response to influenza infection was largely dependent on IFNs, as it was reduced to a few upregulated genes in epithelia lacking receptors for both type I and III IFN (IFNAR1(-/-)IL-28Rα(-/-)). Wild-type epithelia and epithelia deficient in either the type I IFN receptor or the type III IFN receptor exhibit similar transcriptional profiles in response to virus, indicating that none of the induced genes depends selectively on only one IFN system. In chimeric mice, the lack of both IFN I and III signalling in the stromal compartment alone significantly increased the susceptibility to influenza infection. In conclusion, virus infection of airway epithelia induces, via a RIG-I/MAVS/IRF7 dependent pathway, both type I and III IFNs which drive two completely overlapping and redundant amplification loops to upregulate ISGs and protect from influenza infection.

The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer’s disease, deposition of amyloid-β is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-β and increase the formation of amyloid-β oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-β pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-β pathology in transgenic double-mutant APP Swe PSEN1 dE9 mice. By contrast, homogenates from brains of APP Swe PSEN1 dE9 mice failed to induce seeding and spreading of amyloid-β pathology in ASC-deficient APP Swe PSEN1 dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-β pathology in APP Swe PSEN1 dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-β pathology in patients with Alzheimer’s disease. Deposition and spreading of amyloid-β pathology in mice requires binding to microglia-released ASC specks. ASC specks bind to amyloid-β Innate immune activation in Alzheimer's disease involves the inflammasome-dependent formation of specks of adapter protein ASC (an apoptosis-associated speck-like protein containing a caspase recruitment domain) in microglial cells. Here it is shown that ASC specks released by microglia bind to amyloid-β and increase amyloid-β oligomer and aggregate formation, acting as an inflammation-driven cross-seed for amyloid-β pathology.