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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 NLRP1CARD 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 ASCCARD–caspase-1CARD 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.

The NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome is an immunological sensor that detects a wide range of microbial- and host-derived signals. Inflammasome activation results in the release of the potent pro-inflammatory cytokines IL-1β and IL-18 and triggers a form of inflammatory cell death known as pyroptosis. Excessive NLRP3 activity is associated with the pathogenesis of a wide range of inflammatory diseases, thus NLRP3 activation mechanisms are an area of intensive research. NLRP3 inflammasome activation is a tightly regulated process that requires both priming and activation signals. In particular, recent research has highlighted the highly complex nature of the priming step, which involves transcriptional and posttranslational mechanisms, and numerous protein binding partners. This review will describe the current understanding of NLRP3 priming and will discuss the potential opportunities for targeting this process therapeutically to treat NLRP3-associated diseases.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a family of cytosolic pattern recognition receptors that play a critical role in binding viral RNA and triggering antiviral immune responses. The RLR LGP2 (or DHX58) is a known regulator of the RIG-I signaling pathway; however, the underlying mechanism by which LGP2 regulates RIG-I signaling is poorly understood. To better understand the effects of LGP2 on RIG-I-specific signaling and myeloid cell responses, we probed RIG-I signaling using a highly specific RIG-I agonist to compare transcriptional profiles between WT and Dhx58(−/−) C57BL\6 bone marrow-derived dendritic cells. Dhx58(−/−) cells exhibited a marked increase in the magnitude and kinetics of type I interferon (IFN) induction and a broader antiviral response as early as 1 h post-treatment. We determined that LGP2 inhibited RIG-I-mediated IFN-β, IRF-3, and NF-κB promoter activities, indicating a function upstream of the RLR adaptor protein mitochondrial antiviral signaling. Mutational analysis of LGP2 revealed that RNA binding, ATP hydrolysis, and the C-terminal domain fragment were dispensable for inhibiting RIG-I signaling. Using mass spectrometry, we discovered that LGP2 interacted with the E3 ubiquitin ligase TRIM25. Finally, we determined that LGP2 inhibited the TRIM25-mediated K63-specific ubiquitination of the RIG-I N-terminus required for signaling activation.

In the adult hippocampal dentate gyrus (DG), the majority of newly generated cells are eliminated by apoptotic mechanisms. The apoptosis repressor with caspase recruitment domain (ARC), encoded by the Nol3 gene, is a potent and multifunctional death repressor that inhibits both death receptor and mitochondrial apoptotic signaling. The aim of the present study was to parse the role of ARC in the development of new granule cell neurons. Nol3 gene expression as revealed by in situ hybridization is present in the entire dentate granule cell layer. Moreover, a comparison of Nol3 expression between FACS-sorted Sox2-positive neural stem cells and Doublecortin (DCX)-positive immature neurons demonstrates upregulation of Nol3 during neurogenesis. Using ARC-deficient mice, we show that proliferation and survival of BrdU birth-dated cells are strongly reduced in the absence of ARC while neuronal-glial fate choice is not affected. Both the number of DCX-positive cells and the number of calretinin (CR)-positive immature postmitotic neurons are reduced in the hippocampus of ARC-/- mice. ARC knockout is not associated with increased numbers of microglia or with microglia activation. However, hippocampal brain-derived neurotrophic factor (BDNF) protein content is significantly increased in ARC-/- mice, possibly representing a compensatory response. Collectively, our results suggest that ARC plays a critical cell-autonomous role in preventing cell death during adult granule cell neogenesis.

Many proapoptotic and antiapoptotic proteins have been involved in the pathology of Alzheimer's disease. As the first identified antiapoptotic protein, apoptosis repressor with caspase recruitment domain (ARC) is highly expressed in terminally differentiated cells, and its functions and expressions in striated muscles and cancer cells have been widely studied. However, the expression alterations of ARC in amyloid β-induced early hippocampal neurotoxicity are less known. In this report, we not only confirm previous reports that ARC is expressed in the hippocampal neurons but also demonstrate for the first time that ARC is also expressed in the hippocampal astrocytes. Furthermore, we extend the findings to show that, contrary to the time-dependently decreased ARC levels in the hippocampal neurons, ARC in astrocytes is strikingly increased in Aβ25-35-induced early neurotoxicity. Our data suggest that ARC has distinct roles based on cell type and stimuli. Our results provide valuable information for further exploring the complicated functions and related mechanisms of ARC in amyloid-related diseases.

The inflammasome is a multiprotein complex necessary for the onset of inflammation. The adapter protein ASC assembles inflammasome components by acting as a molecular glue between danger-signal sensors and procaspase-1. The assembly is mediated by ASC self-association and protein interactions via its two Death Domains, PYD and CARD. Truncated versions of ASC have been shown to form filaments, but information on the filaments formed by full-length ASC is needed to construct a meaningful model of inflammasome assembly. To gain insights into this system, we used a combination of transmission EM, NMR, and computational analysis to investigate intact ASC structures. We show that ASC forms ∼6–7-nm-wide filaments that stack laterally to form bundles. The structural characteristics and dimensions of the bundles indicate that both PYD and CARD are integral parts of the filament. A truncated version of ASC with only the CARD domain (ASC(CARD)) forms different filaments (∼3–4-nm width), providing further evidence that both domains work in concert in filament assembly. Ring-shaped protein particles bound to pre-existing filaments match the size of ASC dimer structures generated by NMR-based protein docking, suggesting that the ASC dimer could be a basic building block for filament formation. Solution NMR binding studies identified the protein surfaces involved in the ASC(CARD)–ASC(CARD) interaction. These data provide new insights into the structural underpinnings of the inflammasome and should inform future efforts to interrogate this important biological system.

Retinoic acid inducible gene-I (RIG-I) ensures immune surveillance of viral RNAs bearing a 5’-triphosphate (5’ppp) moiety. Mutations in RIG-I (C268F and E373A) lead to impaired ATPase activity, thereby driving hyperactive signaling associated with autoimmune diseases. Here we report, using hydrogen/deuterium exchange, mechanistic models for dysregulated RIG-I proofreading that ultimately result in the improper recognition of cellular RNAs bearing 7-methylguanosine and N1-2’-O-methylation (Cap1) on the 5’ end. Cap1-RNA compromises its ability to stabilize RIG-I helicase and blunts caspase activation and recruitment domains (CARD) partial opening by threefold. RIG-I H830A mutation restores Cap1-helicase engagement as well as CARDs partial opening event to a level comparable to that of 5’ppp. However, E373A RIG-I locks the receptor in an ATP-bound state, resulting in enhanced Cap1-helicase engagement and a sequential CARDs stimulation. C268F mutation renders a more tethered ring architecture and results in constitutive CARDs signaling in an ATP-independent manner., RIG-I is a critical receptor in the induction of innate immune responses, but mutations in RIG-I can be associated with hyperactive signalling and autoimmune disease. Here Zheng et al. apply HDX-MS approaches to reveal dysregulated checkpoints that result in recognition of self-derived RNA during RIG-I mediated autoimmunity.

The NLRP3 inflammasome is a well-studied target for the treatment of multiple inflammatory diseases, but how to promote the current therapeutics remains a large challenge. CRISPR/Cas9, as a gene editing tool, allows for direct ablation of NLRP3 at the genomic level. In this study, we screen an optimized cationic lipid-assisted nanoparticle (CLAN) to deliver Cas9 mRNA (mCas9) and guide RNA (gRNA) into macrophages. By using CLAN encapsulating mCas9 and gRNA-targeting NLRP3 (gNLRP3) (CLANmCas9/gNLRP3), we disrupt NLRP3 of macrophages, inhibiting the activation of the NLRP3 inflammasome in response to diverse stimuli. After intravenous injection, CLANmCas9/gNLRP3 mitigates acute inflammation of LPS-induced septic shock and monosodium urate crystal (MSU)-induced peritonitis. In addition, CLANmCas9/gNLRP3 treatment improves insulin sensitivity and reduces adipose inflammation of high-fat-diet (HFD)-induced type 2 diabetes (T2D). Thus, our study provides a promising strategy for treating NLRP3-dependent inflammatory diseases and provides a carrier for delivering CRISPR/Cas9 into macrophages., Activation of the NLRP3 inflammasome triggers the production of inflammatory cytokines. Here, the authors inactivate NLRP3 in macrophages using CRISPR/Cas9 encapsulated in nanoparticles, and show that administration in mice is effective in preventing septic shock and peritonitis, and in improving diabetes-associated inflammation and insulin resistance.

Apoptosis repressor with caspase recruitment domain (ARC) is a highly effective and multifunctional inhibitor of apoptosis that is mainly expressed in postmitotic cells such as cardiomyocytes and skeletal muscle cells. ARC contains a C-terminal region rich in proline and glutamic acid residues and an N-terminal caspase recruitment domain (CARD). The CARD is originally described as a protein-binding motif that interacts with caspase through a CARD-CARD interaction. Initially, the inhibitory effect of ARC was only found in apoptosis, however, it was later found that ARC also played a regulatory role in other types of cell death. As a powerful cardioprotective factor, ARC can protect the heart by inhibiting the death of cardiomyocytes in various ways. ARC can reduce the cardiomyocyte apoptotic response to various stresses and injuries, including extrinsic apoptosis induced by death receptor ligands, cellular Ca2+ homeostasis and the dysregulation of endoplasmic reticulum (ER) stress, oxidative stress and hypoxia. In addition, changes in ARC transcription and translation levels in the heart can cause a series of physiological and pathological changes, and ARC can also perform corresponding functions through interactions with other molecules. Although there has been much research on ARC, the functional redundancy among proteins shows that ARC still has much research value. This review summarizes the molecular characteristics of ARC, its roles in the various death modes in cardiomyocytes and the roles of ARC in cardiac pathophysiology. This article also describes the potential therapeutic effect and research prospects of ARC.

Host protective inflammatory caspase activity must be tightly regulated to prevent pathogens infection, however, the inflammatory caspase-engaged inflammasome activation in teleost fish remains largely unknown. In this study, we reveal a bifurcated evolutionary role of the inflammatory caspase in mediating both non-canonical and canonical inflammasome pathways in teleost fish. Through characterization of a unique inflammatory SmCaspase from the teleost Scophthalmus maximus (turbot), we found it can directly recognize cytosolic lipopolysaccharide (LPS) via its N-terminal CARD domain, resulting in caspase-5-like proteolytic enzyme activity-mediated pyroptosis in Turbot Muscle Fibroblasts. Interestingly, we also found that this inflammatory caspase can be recruited to SmNLRP3-SmASC to form the NLRP3 inflammasome complex, engaging the SmIL-1β release in Head Kidney-derived Macrophages. Consequently, the SmCaspase activation can recognize and cleave the SmGSDMEb to release its N-terminal domain, mediating both pyroptosis and bactericidal activities. Furthermore, the SmCaspase-SmGSDMEb axis-gated pyroptosis governs the bacterial clearance and epithelial desquamation in fish gill filaments in vivo. To our knowledge, this study is the first to identify an inflammatory caspase acting as a central coordinator in NLRP3 inflammasome, as well as a cytosolic LPS receptor; thus uncovering a previously unrecognized function of inflammatory caspase in turbot innate immunity.

Caspase-9 is a critical factor in the initiation of apoptosis and as a result is tightly regulated by many mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work, we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Taken together, these findings provide mechanistic insights into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate.

Inflammasomes are the multi-protein caspase-activating complexes mainly assembled by the sensor proteins (NLRs/ALRs), adaptor molecule (ASC) and effector molecule pro-caspase-1 for the production and release of proinflammatory cytokines, IL-1β and IL-18. NLRP1 is the first NLR known to assemble the multi-protein complex. Unlike NLRP3, NLRP1 has an additional effector binding domain (CARD) at the carboxyl-terminal, which is reported to interact with pro-caspase-1 (precluding the recruitment of ASC) for the transmission of danger signals. So far no direct interaction has been observed between the NLRP1 and CASP1 at the structural level. In this study, an attempt has been made to elucidate the possible mode of interaction(s) between CASP1 and NLRP1 CARDs using structural bioinformatics approaches. The results revealed that the type-Ia patch of CASP1CARD (R10, K11, and R55) is probably the favorable interface for 1:1 interaction. Moreover, the interactions mediated in the type-II and/(or) type-III interfaces of counter CARDs can also be not ruled out altogether. Overall, the findings of this study can be beneficial in understanding the underlying molecular mechanism(s) associated with NLRP1-mediated inflammasome.

The retinoic acid-inducible gene-I (RIG-I) receptor recognizes short 5′-di- and triphosphate base-paired viral RNA and is a critical mediator of the innate immune response against viruses such as influenza A, Ebola, HIV and hepatitis C. This response is reported to require an orchestrated interaction with the tripartite motif 25 (TRIM25) B30.2 protein-interaction domain. Here, we present a novel second RIG-I-binding interface on the TRIM25 B30.2 domain that interacts with CARD1 and CARD2 (caspase activation and recruitment domains) of RIG-I and is revealed by the removal of an N-terminal α-helix that mimics dimerization of the full-length protein. Further characterization of the TRIM25 coiled-coil and B30.2 regions indicated that the B30.2 domains move freely on a flexible tether, facilitating RIG-I CARD recruitment. The identification of a dual binding mode for the TRIM25 B30.2 domain is a first for the SPRY/B30.2 domain family and may be a feature of other SPRY/B30.2 family members.

Emerging evidence suggests that activation of inflammasomes plays a central mechanism in pathogenesis of periodontitis. This study aims to compare salivary levels of nod-like receptor family pyrin domain containing protein (NLRP) 3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), cysteine aspartase (caspase)-1, and interleukin (IL)-1β from individuals with aggressive (AgP) or chronic periodontitis (CP) and healthy controls (HC), as well as elucidate its association with periodontal clinical status.Saliva samples from individuals with CP (n = 75), AgP (n = 20), and HC (n = 69) were collected. Periodontal status was assessed by measurement of probing depth, clinical attachment level, and extent and severity of disease. Salivary levels of analytes were analyzed by enzyme-linked immunosorbent assay. Association between biomarkers with CP or AgP was analyzed using multivariate binary logistic regression models.Significantly higher levels of NLRP3, ASC, and IL-1β were detected in periodontitis groups in comparison to the periodontally HC group. However, no significant differences were observed for caspase-1 levels between clinical groups, and only NLRP3 salivary concentration was significantly higher in AgP compared with CP patients. Also, positive significant correlations among NLRP3, ASC, and IL-1β salivary concentrations and clinical parameters were observed. Logistic regression analyses revealed a strong/independent association of NLRP3, ASC, and IL-1β salivary levels with CP and AgP.Although the concentration of caspase-1 in saliva samples makes its determination useless for detection of periodontal disease and/or its severity, salivary levels of NLRP3, ASC, and IL-1β may act as strong/independent indicators of amount and extent of periodontal breakdown in both CP and AgP and could potentially be used for prevention and therapy of this group of diseases.

Mitochondrial antiviral signaling protein (MAVS) is an adaptor protein of the innate immune system of higher vertebrate. In this paper, the transcription profile of black carp MAVS (bcMAVS) in host cells in response to spring viremia of carp virus (SVCV) and grass carp reovirus (GCRV) infection was identified. EPC cells expressing bcMAVS possessed obviously enhanced antiviral activity against both SVCV and GCRV. Immunofluorescence (IF) staining data demonstrated that bcMAVS molecules were redistributed and formed aggregates on the mitochondria of EPC cells after virus infection. Co-immunoprecipitation (co-IP) assay in HEK293T cells demonstrated that bcMAVS proteins bound to each other, which suggested that this fish protein owned self-association in vivo. IF assay identified that the transmembrane (TM) domain of bcMAVS was crucial for its mitochondrial localization. Co-IP assays among bcMAVS mutants demonstrated that both N-terminal caspase recruitment domain (CARD) and TM domain were indispensible for dimerization of bcMAVS. It was interesting that Truncated-bcMAVS possessed much enhanced interferon-inducing activity and antiviral ability than wild type bcMAVS, which only contains CARD and TM. All the data generated in this study support the idea that oligomerization of bcMAVS on mitochondrion is crucial for the antiviral ability of bcMAVS, which is depend on both CARD and TM domain of this fish MAVS orthologue.

The caspase recruitment domain (CARD), a well‑known protein interaction module, belongs to the death domain (DD) superfamily, which includes DDs, death effector domains, and pyrin domains. The DD superfamily mediates the protein interactions necessary for apoptosis and immune cell signaling pathways. Among these domains, the CARD has been studied extensively as it mediates important cellular signaling events that are associated with various human diseases including cancer, neuro‑degenerative diseases and immune disorders. Homo‑type and hetero‑type CARD‑CARD interactions mediate the formation of large signaling complexes, including caspase‑activating complexes and downstream signaling complexes. The present review summarizes and discusses the results of structural studies of various CARDs and their complexes. These studies shed light on the mechanisms that control the assembly and disassembly of signaling complexes and provide an improved understanding of cellular signaling processes.

Lipopolysaccharides (LPS) are a major component of the outer membrane of Gram-negative bacteria and are pathogen-associated molecular patterns recognized by the TLR4/MD2 complex that induces an inflammatory response. Recently, the cytosolic receptors caspase-4/-5/-11 that bind LPS inside the cell and trigger inflammasome activation or pyroptosis, have been identified. Despite the important roles of caspase-4 in human immune responses, few studies have investigated its biochemical characteristics and interactions with LPS. Since caspase-4 (C258A) purified from an Escherichia coli host forms aggregates, monomeric proteins including full-length caspase-4, caspase-4 (C258A), and the CARD domain of caspase-4 have been purified from the insect cell system. Here, we report the overexpression and purification of monomeric caspase-4 (C258A) and CARD domain from E. coli and demonstrate that purified caspase-4 (C258A) and CARD domain bind large LPS micelles and disaggregate them to small complexes. As the molar ratio of caspase-4 to LPS increases, the size of the caspase-4/LPS complex decreases. Our results present a new function of caspase-4 and set the stage for structural and biochemical studies, and drug discovery targeting LPS/caspase-4 interactions by establishing a facile purification method to obtain large quantities of purified caspase-4 (C258A) and the CARD domain.

The innate immune system offers the first line of defense against invading microbial pathogens through the recognition of conserved pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). The host innate immune system through PRRs, the sensors for PAMPs, induces the production of cytokines. Among different families of PRRs, the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), and its mitochondrial adaptor ie, the mitochondrial antiviral-signaling (MAVS) protein, are crucial for RLR-triggered interferon (IFN) antiviral immunity. Recent studies have shown that the N-terminal caspase recruitment domain (CARD) and transmembrane domain play a pivotal role in oligomerization of black carp MAVS (BcMAVS), crucial for the host innate immune response against viral invasion. In this study, we have used molecular modeling, docking, and molecular dynamics (MD) simulation approaches to shed molecular insights into the oligomerization mechanism of BcMAVSCARD . MD simulation and interaction analysis portrayed that the type-I surface patches of BcMAVS CARD make the major contribution to the interaction. Moreover, the evidence from surface patches and critical residues involved in the said interaction is found to be similar to that of the human counterpart and requires further investigation for legitimacy. Altogether, our study provided crucial information on oligomerization of BcMAVS CARDs and might be helpful for clarifying the innate immune response against pathogens and downstream signaling in fishes.

Activation of the innate immune pattern recognition receptor NOD2 by the bacterial muramyl-dipeptide peptidoglycan fragment triggers recruitment of the downstream adaptor kinase RIP2, eventually leading to NF-κB activation and proinflammatory cytokine production. Here we show that full-length RIP2 can form long filaments mediated by its caspase recruitment domain (CARD), in common with other innate immune adaptor proteins. We further show that the NOD2 tandem CARDs bind to one end of the RIP2 CARD filament, suggesting a mechanism for polar filament nucleation by activated NOD2. We combine X-ray crystallography, solid-state NMR and high-resolution cryo-electron microscopy to determine the atomic structure of the helical RIP2 CARD filament, which reveals the intermolecular interactions that stabilize the assembly. Using structure-guided mutagenesis, we demonstrate the importance of RIP2 polymerization for the activation of NF-κB signalling by NOD2. Our results could be of use to develop new pharmacological strategies to treat inflammatory diseases characterised by aberrant NOD2 signalling., Binding of bacterial peptidoglycan muramyl dipeptides induces NOD2 activation and signalling via the downstream adaptor kinase RIP2. Here the authors show that RIP2 forms filaments via its CARD domain, analyse the structure of the CARD filaments and demonstrate the requirement of RIP2 polymerisation for the activation of NF-κB by NOD2.

Nod like receptors (NLRs) are a large group of cytoplasmic PRRs believed to play an important role in bacterial recognition in higher vertebrates. In this study, a novel Nod like receptor C3 (AsNLRC3) has been identified, cloned and characterised from Asian seabass, Lates calcarifer. The full-length AsNLRC3 transcript composed of a 4142 bp nucleic acid sequence encode for a protein of 1134 deduced amino acids. Three signature domains identified are conserved NACHT-domain, C-terminal LLR domain and N-terminal CARD effector domain. From the domain architecture and phylogenetic analysis, it was quite evident that AsNLRC3 is different from the NLR subfamily C of other teleosts. AsNLRC3 expressed in all the 11 tissues tested but highly expressed in tissues facing external environment such as gill, hindgut and midgut. The ontogenic expression profile of this receptor showed constitutive expression throughout the embryonic and larval developmental stages, which could be an innate immune strategy against different marine pathogens for larval survival. Infection with Vibrio alginolyticus and poly I:C induction showed an alteration of expression pattern in different tissues but did not show significant alteration in expression with Staphylococcus aureus infection. In vitro study in Asian seabass kidney cell line (SISK) stimulated with different ligands such as LPS, PGN and poly I:C showed considerable up-regulation at some of the time-points tested. These results suggest that AsNLRC3 can be a pivotal cytosolic innate immune receptor for recognizing wide array of pathogens in a euryhaline teleost model like Asian seabass in diverse environmental conditions.

Summary The poly(ADP-ribose) polymerase (PARP) Tankyrase (TNKS and TNKS2) is paramount to Wnt-β-catenin signaling and a promising therapeutic target in Wnt-dependent cancers. The pool of active β-catenin is normally limited by destruction complexes, whose assembly depends on the polymeric master scaffolding protein AXIN. Tankyrase, which poly(ADP-ribosyl)ates and thereby destabilizes AXIN, also can polymerize, but the relevance of these polymers has remained unclear. We report crystal structures of the polymerizing TNKS and TNKS2 sterile alpha motif (SAM) domains, revealing versatile head-to-tail interactions. Biochemical studies informed by these structures demonstrate that polymerization is required for Tankyrase to drive β-catenin-dependent transcription. We show that the polymeric state supports PARP activity and allows Tankyrase to effectively access destruction complexes through enabling avidity-dependent AXIN binding. This study provides an example for regulated signal transduction in non-membrane-enclosed compartments (signalosomes), and it points to novel potential strategies to inhibit Tankyrase function in oncogenic Wnt signaling., Graphical Abstract, Highlights • SAM domain crystal structures reveal mechanism of Tankyrase polymerization • Catalysis-independent Tankyrase scaffolding drives Wnt-β-catenin signaling • Tankyrase polymerization supports PARP activity and AXIN binding, Catalysis-independent scaffolding by Tankyrase supports Wnt-β-catenin signaling. Scaffolding is mediated by AXIN-binding ankyrin repeat clusters and a polymerizing SAM domain. In a structure-function approach, Mariotti et al. show that polymerization supports Tankyrase PARP activity and enables avidity-dependent AXIN binding.

Background/Aims: Viral infections represent a global health problem with the need for new viral therapies and better understanding of the immune response during infection. The most immediate and potent anti-viral defense mechanism is the production of type I interferon (IFN-I) which are activated rapidly following recognition of viral infection by host pathogen recognition receptors (PRR). The mechanisms of innate cellular signaling downstream of PRR activation remain to be fully understood. In the present study, we demonstrate that CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD/RAIDD) is a critical component in type I IFN production. Methods: The role of RAIDD during IFN-I production was investigated using western blot, shRNA mediated lentiviral knockdown, immunoprecipitation and IFN-I driven dual luciferase assay. Results: Immunoprecipitation analysis revealed the molecular interaction of RAIDD with interferon regulatory factor 7 (IRF7) and its phosphorylating kinase IKKε. Using an IFN-4α driven dual luciferase analysis in RAIDD deficient cells, type I IFN activation by IKKε and IRF7 was dramatically reduced. Furthermore, deletion of either the caspase recruitment domain (CARD) or death domain (DD) of RAIDD inhibited IKKε and IRF7 mediated interferon-4α activation. Conclusion: We have identified that the adaptor molecule RAIDD coordinates IKKε and IRF7 interaction to ensure efficient expression of type I interferon.

A major challenge in regenerative medicine is the repair of injured neurons. Regeneration of laser-cut C. elegans neurons requires early action of core apoptosis activator CED-4/Apaf1 and CED-3/caspase. While testing models for CED-4 as a candidate calcium-sensitive activator of repair, we unexpectedly discovered that amino acid substitutions affecting alpha-helix-6 within the CED-4 caspase recruitment domain (CARD) confer a CED-4 gain-of-function (gf) activity that increases axonal regrowth without disrupting CED-4 apoptosis activity. The in vivo caspase reporter CA-GFP reveals a rapid localized increase in caspase activity upon axotomy, which is absent in ced-4 and ced-3 loss-of-function mutants but present in the ced-4(gf) mutant. The ced-3 loss-of-function mutation can significantly suppress the axonal regrowth of the ced-4(gf) mutant, indicating that CED-4(gf) regeneration depends on CED-3 caspase. Thus, we identified a subdomain within the CED-4 CARD that regulates the dynamic and controlled caspase activity required for efficient regeneration.

Inflammation contributes to heart failure (HF) progression and the interleukin (IL)-1 cytokine IL-1β is implicated in this process. The adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is necessary for inflammasome activation of IL-1β. Lower ASC methylation is associated with worse outcomes in HF. The purpose of this study was to examine the effects of exercise on changes in ASC methylation and activation of the IL-1 family cytokine IL-1β in persons with HF.Participants (N = 54) were randomized to receive exercise intervention (n = 38) or attention control (n = 16) for 3 months. Percent methylation of the ASC gene, plasma IL-1β, and ASC mRNA and were obtained at baseline, 3 months, and 6 months.ASC methylation was higher in the exercise group as compared to control at 3 months (6.10% ± 0.5% vs 5.80% ± 0.4%; P = 0.04) and 6 months (6.07 ± 0.4 vs 5.82 ± 0.4; P = 0.04). Plasma IL-1β was lower in the exercise group at 3 months (1.43 ± 0.5 pg·mL vs 2.09 ± 1.3 pg·mL; P = 0.02) and 6 months (1.49 ± 0.5 pg·mL vs 2.13 ± 1.4 pg·mL; P = 0.004). ASC mRNA expression was negatively associated with ASC methylation at baseline (r = -0.97, P = 0.001), 3 months (r = -0.90, P = 0.001), and 6 months (r = -0.81, P = 0.001). ASC mRNA was lower than baseline at 3 months (P = 0.004) and 6 months (P = 0.002) among those in the exercise group. ASC methylation was positively associated with 6-min walk test at baseline (r = 0.517, P0.001), 3 months (r = 0.464, P = 0.004), and 6 months (r = 497, P = 0.05).Exercise was related to increased mean percent ASC methylation and decreased IL-1β and ASC mRNA gene expression in HF. Epigenetic regulation of ASC can be a biological mechanism by which exercise can promote better outcomes in HF.

Summary Receptor interacting protein 2 (RIP2) plays a role in sensing intracellular pathogens, but its function in T cells is unclear. We show that RIP2 deficiency in CD4+ T cells resulted in chronic and severe interleukin-17A-mediated inflammation during Chlamydia pneumoniae lung infection, increased T helper 17 (Th17) cell formation in lungs of infected mice, accelerated atherosclerosis, and more severe experimental autoimmune encephalomyelitis. While RIP2 deficiency resulted in reduced conventional Th17 cell differentiation, it led to significantly enhanced differentiation of pathogenic (p)Th17 cells, which was dependent on RORα transcription factor and interleukin-1 but independent of nucleotide oligomerization domain (NOD) 1 and 2. Overexpression of RIP2 resulted in suppression of pTh17 cell differentiation, an effect mediated by its CARD domain, and phenocopied by a cell-permeable RIP2 CARD peptide. Our data suggest that RIP2 has a T cell-intrinsic role in determining the balance between homeostatic and pathogenic Th17 cell responses.

Augmented apoptotic signaling can result in degradation of skeletal muscle proteins and loss of myonuclei, ultimately contributing to muscle atrophy and contractile dysfunction. Apoptosis repressor with caspase recruitment domain (ARC) is an anti-apoptotic protein highly expressed in skeletal muscle. Here we examined the role of ARC on age-related skeletal muscle apoptosis and wasting by utilizing an ARC-deficient mouse model. Aged mice displayed a number of morphological, phenotypic, and contractile alterations in both soleus and plantaris muscle with aging. Although no differences were found in proteolytic enzyme activity, ARC protein decreased while several anti-apoptotic proteins (e.g., BCL2, BCLXL, HSP70, and XIAP) and the release of mitochondrial housed protein (i.e., SMAC, AIF) increased in aged muscle. Importantly, ARC KO mice had low muscle weights and fewer fibers in soleus, with 2-year-old ARC KO mice displaying lower mitochondrial BCL2 protein along with augmented release of CYTC and SMAC in red/oxidative muscle. Overall, these results indicate that aged skeletal muscle undergoes atrophy as well as contractile and fiber type composition alterations despite an increase in anti-apoptotic protein expression. Although some mitochondrial-specific apoptotic alterations occurred in skeletal muscle due to ARC ablation over the lifespan, our data suggest that ARC may not have a large influence during skeletal muscle aging.

Sensing and responding to pathogens and tissue damage is a core mechanism of innate immune host defense, and inflammasomes represent a central cytosolic pattern recognition receptor pathway leading to the generation of the pro-inflammatory cytokines interleukin-1β and interleukin-18 and pyroptotic cell death that causes the subsequent release of danger signals to propagate and perpetuate inflammatory responses. While inflammasome activation is essential for host defense, deregulated inflammasome responses and excessive release of inflammatory cytokines and danger signals are linked to an increasing spectrum of inflammatory diseases. In this review, we will discuss recent developments in elucidating the role of PYRIN domain-only proteins (POPs) and the related CARD-only proteins (COPs) in regulating inflammasome responses and their impact on inflammatory disease.

The apoptosis repressor with caspase recruiting domain (ARC) is aberrantly overexpressed in various cancers. ARC contains a CARD domain that is the main mediator of protein-protein interactions. Mutation of Leu31 within the CARD of ARC to Phe (ARC_L31F) is widely used as a functionally defective mutant of ARC despite a lack of clear experimental evidence regarding how its functionality is lost. In this study, we show that L31 in helix 2 (H2) is critical for stabilization of the helix bundle fold in the CARD domain. In addition, the L31F mutation disrupts homodimer formation that is critical to ARC functions. Our current study reveals the molecular basis for the L31F mutation disrupting the ARC CARD functions. This article is protected by copyright. All rights reserved.

Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.

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.

Nucleotide binding oligomerization domain 2 (NOD2), a protein involved in the first line defence mechanism has a pivotal role in innate immunity. Impaired function of this protein is implicated in disorders such as Blau syndrome and Crohn's disease. Since an altered function is linked to protein's structure, we framed a systematic strategy to interpret the structure-function relationship of the protein. Initiated with mutation-based pattern prediction and identified a distant ortholog (DO) of NOD2 from which the intra-residue interaction network was elucidated. The network was used to identify hotspots that serve as critical points to maintain the stable architecture of the protein. Structural comparison of NOD2 domains with a DO revealed the minimal number of intra-protein interactions required by the protein to maintain the structural fold. In addition, the conventional molecular dynamics simulation emphasized the conformational transitions at hot spot residues between native NOD2 domains and its respective mutants (G116R, R42W and R54A) structures. The analysis of intra-protein interactions globally and the displacement of residues locally around the mutational site revealed loss of several critical bonds and residues vital for the protein's function. Conclusively we report, about 10 residues in leucine-rich repeat, 13 residues in NOD and 6 residues in CARD domain are required by the NOD2 to maintain its function. This protocol will help the researchers to achieve for more prospective studies to attest druggable site utility in discovering novel drug candidates.

Caspases are key enzymes responsible for mediating apoptotic cell death. Across species, caspase-2 is the most conserved caspase and stands out due to unique features. Apart from cell death, caspase-2 also regulates autophagy, genomic stability and ageing. Caspase-2 requires dimerization for its activation which is primarily accomplished by recruitment to high molecular weight protein complexes in cells. Here, we demonstrate that apoptosis inhibitor 5 (API5/AAC11) is an endogenous and direct inhibitor of caspase-2. API5 protein directly binds to the caspase recruitment domain (CARD) of caspase-2 and impedes dimerization and activation of caspase-2. Interestingly, recombinant API5 directly inhibits full length but not processed caspase-2. Depletion of endogenous API5 leads to an increase in caspase-2 dimerization and activation. Consistently, loss of API5 sensitizes cells to caspase-2-dependent apoptotic cell death. These results establish API5/AAC-11 as a direct inhibitor of caspase-2 and shed further light onto mechanisms driving the activation of this poorly understood caspase.

RIP2, one of the RIP kinases, interacts with p75 neurotrophin receptor, regulating the neuron survival, and with NOD1 and NOD2 proteins, causing the innate immune response against gram-negative and gram-positive bacteria via its caspase recruitment domain (CARD). This makes RIP2 a prospective target for novel therapies, aimed to modulate the inflammatory diseases and neurogenesis/neurodegeneration. Several studies report the problems with the stability of human RIP2 CARD and its production in bacterial hosts, which is a prerequisite for the structural investigation with solution NMR spectroscopy. In the present work, we report the high yield production and refolding protocols and resolve the structure of rat RIP2 CARD. The structure reveals the important differences to the previously published conformation of the homologous human protein. Using solution NMR, we characterized the intramolecular mobility and pH-dependent behavior of RIP2 CARD, and found the propensity of the protein to form high-order oligomers at physiological pH while being monomeric under acidic conditions. The oligomerization of protein may be explained, based on the electrostatic properties of its surface. Analysis of the structure and sequences of homologous proteins reveals the residues which are significant for the unusual fold of RIP2 CARD domains from different species. The high-throughput protein production/refolding protocols and proposed explanation for the protein oligomerization, provide an opportunity to design the stabilized variants of RIP2 CARD, which could be used to study the structural details of RIP2/NOD1/NOD2 interaction and perform the rational drug design.

17 páginas, 10 figuras.-- Sylwia Tyrkalska ... et al.-- This work is licensed under a Creative Commons Attribution 4.0 International License, Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections, This work was supported by the Spanish Ministry of Economy and Competiveness (grants BIO2011-23400 and BIO2014-52655-R to V.M., AGL2012-39674 to M.P.S., and BIO2013-45336-R to A.S.-F. and PhD fellowships to S.C., D.A. and V.G.-A., all co-funded with Fondos Europeos de Desarrollo Regional/European Regional Development Funds), the European 7th Framework Initial Training Network FishForPharma (PhD fellowship to S.D.T., PITG-GA-2011-289209), the Sara Borrell postdoc grant from the Instituto Salud Carlos III to F.M.-S. (CD12/00523) and the European Research Council (ERC-2013-CoG 614578 to P.P.)

Tuberculosis is a global health problem and at least one-third of the world’s population is infected with Mycobacterium tuberculosis (MTB). MTB is a successful pathogen that enhances its own intracellular survival by inhibiting inflammation and arresting phago-lysosomal fusion. We previously demonstrated that Toxoplasma gondii (T. gondii) dense granule antigen (GRA) 7 interacts with TNF receptor-associated factor 6 via Myeloid differentiation primary response gene 88, enabling innate immune responses in macrophages. To extend these studies, we found that GRA7 interacts with host proteins involved in antimicrobial host defense mechanisms as a therapeutic strategy for tuberculosis. Here, we show that protein kinase C (PKC)α-mediated phosphorylation of T. gondii GRA7-I (Ser52) regulates the interaction of GRA7 with PYD domain of apoptosis-associated speck-like protein containing a carboxy-terminal CARD, which is capable of oligomerization and inflammasome activation can lead to antimicrobial defense against MTB. Furthermore, GRA7-III interacted with the PX domain of phospholipase D1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7-III (Ser135) phosphorylation-dependent manner via PKCα. Taken together, these results underscore a previously unrecognized role of GRA7 in modulating antimicrobial host defense mechanism during mycobacterial infection., Author Summary We previously demonstrated that Toxoplasma gondii (T. gondii) dense granule antigen (GRA) 7 interacts with TRAF6 via MyD88, enabling innate immune responses in macrophages and effective protection against T. gondii infection in vivo. However, its exact role and how it regulates host innate immune responses have not been fully explained. Herein, we show that PKCα-mediated phosphorylation of GRA7 is essential for the interaction between GRA7 and ASC or PLD1, which can promote antimicrobial defense against Mycobacterium tuberculosis (MTB). Notably, PKCα specifically phosphorylated Ser52 and Ser135 of GRA7 in vitro and in vivo, indicating that GRA7 is a substrate of PKCα. The N-terminal of GRA7 (GRA7-I) was sufficient for interaction with the PYD domain of ASC, which is capable of ASC oligomerization and inflammasome activation. Furthermore, GRA7-III interacted with the PX domain of PLD1, facilitating its enzyme activity, phago-lysosomal maturation, and subsequent antimicrobial activity in a GRA7 phosphorylation-dependent manner. Interestingly, phosphomimetic mutation in GRA7 overcame the need for PKCα. Collectively, these results provide novel insight into how GRA7 can promote ASC and PLD1 activation in a PKCα-dependent manner as an antimicrobial host defense mechanism.