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Die großzelligen B-Zell-Lymphome mit IRF4 (Interferonregulierender Faktor 4)- Translokation (LBCL+IRF4) wurden im Jahr 2016 als neuer Subtyp in die vierte Edition der WHO (Weltgesundheitsorganisation) Klassifikation maligner Lymphome aufgenommen. Diese Lymphome zeichnen sich durch eine Translokation von IRF4 neben einen Immunglobulinlokus und daraus resultierend starker Expression des IRF4/MUM1 (Multiples Myelom Onkogen 1) Proteins aus. Bis auf vereinzelte Veröffentlichungen mit geringen Fallzahlen ist über die genetischen Charakteristika der LBCL+IRF4 bisher nur wenig bekannt. Ziel dieser Arbeit war es daher die LBCL+IRF4 auf genetischer Ebene zu charakterisieren. Insbesondere sollten die Bruchpunkte der IRF4-IG-Translokation, sekundäre chromosomale Aberrationen sowie das Mutationsprofil beschrieben und im Hinblick auf ihren Beitrag zur Lymphompathogenese analysiert werden. In dieser Arbeit wurde das Tumormaterial von neun LBCL+IRF4 mit nachgewiesener IRF4- IG-Translokation aus dem MMML (Molecular mechanisms of malignant lymphoma)- Sequenzierungsprojekt des ICGC (International Cancer Genome Consortium) untersucht. Zur genauen Bruchpunktkartierung der IRF4-IG-Translokation wurden Gesamtgenomsequenzierungsdaten (WGS) (n=4) mittels SOPHIA Algorithmus ausgewertet. Die Identifikation der chromosomalen Aberrationen, bestehend aus Kopienzahlzugewinnen, Kopienzahlverlusten und Kopienzahl-neutralen Verlusten der Heterozygotie, erfolgte in Fällen, in denen keine WGS- sondern Gesamtexomsequenzierungsdaten (WES) vorlagen, mittels OncoScan-Arrays (n=5) und in Fällen mit WGS-Daten mithilfe des ACE-Seq-Algorithmus (n=4). Zur Analyse des Transkriptoms wurden RNA(Ribonukleinsäure)-Sequenzierungsdaten (n=3) verwendet. Die Detektion der Mutationen erfolgte auf Grundlage von gefilterten Sequenzierungsdaten des Tumormaterials aus WES-Daten (n=5) und WGS-Daten (n=4). Die Verifizierung der identifizierten Bruchpunkte und Mutationen erfolgte mittels PCR (Polymerasekettenreaktion) und Sangersequenzierung. Zur Untersuchung der zeitlichen Abfolge der IRF4-Mutationsentstehung wurden Klonierungsanalysen in allen LBCL+IRF4 Fällen mit mehr als einer codierenden IRF4-Mutation (n=6) durchgeführt. Mögliche funktionelle Auswirkungen der gefundenen Mutationen wurden durch bioinformatische Tools wie beispielsweise Mechismo und durch Pubmed-Recherche evaluiert. Die Bruchpunkte der IRF4-IG-Translokation wurden in drei Fällen downstream von IRF4 und in einem Fall in IRF4 lokalisiert. In drei Fällen konnte eine deutlich gesteigerte RNA- Expression von IRF4 nachgewiesen werden. Als wahrscheinlicher Entstehungsmechanismus der IRF4-IG-Translokation konnte aus den beteiligten Bruchpunktsequenzen in zwei Fällen eine V(D)J-Umlagerung und in zwei Fällen eine somatische Hypermutation identifiziert werden. Wiederkehrende sekundäre chromosomale Aberrationen waren Kopienzahlzugewinne von 7q11.23-q36.3, 11q22.3- q25, 17q12, 17q24.3-q25.3 sowie Kopienzahlverluste von 6q16.1-q27 und 17p13.3-p11.2. Das Mutationsprofil der hier untersuchten LBCL+IRF4 Fälle war gekennzeichnet durch zahlreiche und hoch rekurrente Mutationen in IRF4 (7/9), welche unter anderem die DNA- bindende Domäne von IRF4 betreffen. Zudem wurden rekurrent proteinverändernde Mutationen in CARD11 (Caspase-Rekrutierungsdomänen-haltiges Protein 11) (4/9) und in weiteren Genen des NFkB-und B-Zell-Rezeptorsignalweges detektiert. Insgesamt zeigen die Ergebnisse dieser Arbeit, dass die LBCL+IRF4 sich in ihrem genetischen Profil von follikulären Lymphomen und diffus großzelligen B-Zell-Lymphomen unterscheiden. Zusätzlich zur IRF4-IG-Translokation wurden weitere genetische Veränderungen unter anderem in potentiell pathophysiologisch relevanten Genen des NFkB-und B-Zell-Rezeptorsignalweges identifiziert. Die starke Expression von IRF4 in Kombination mit zahlreichen proteinverändernden Mutationen, unter anderem in der DNA-bindenden Domäne von IRF4, indiziert eine tragende Rolle von IRF4 in der Lymphomentstehung der LBCL+IRF4 und bietet damit möglicherweise einen therapeutischen Angriffspunkt.

PURPOSE: The NLRP3 inflammasome, a cytoplasmic signal transduction complex that regulates inflammation, has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of visual impairment in industrialized countries. We tested the therapeutic effect of anti-inflammatory gene therapy, delivered preventively, in Liver-X-Receptor alpha knockout (LXRα(−/−)) mice, which exhibit features of dry AMD. METHODS: LXRα(−/−) mice were treated with an adeno-associated virus (AAV) vector that delivers a secretable and cell-penetrating form of the caspase activation and recruitment domain (CARD). A sGFP-FCS-TatCARD-AAV or sGFP-FCS (control) vector was delivered intravitreally to 3–5 month-old, LXRα(−/−) mice, who were then aged to 15–18 months (12–13 month treatment). Retinal function and morphology were assessed pre- and post-treatment. RESULTS: TatCARD treated LXRα(−/−) mice did not show improvement in rod and cone photoreceptor function, measured by dark adapted a- and b-wave amplitudes, and rod-saturated b-wave amplitudes. We found a sex-dependent, significant therapeutic effect in c-wave amplitudes in the TatCARD treated mice, which exhibited maintenance of amplitudes in comparison to the significant decline recorded in the control treated group, indicating a therapeutic effect mediated in part through retinal pigment epithelial (RPE) cells. Additionally, the retinas of the TatCARD treated mice exhibited a significant decline in the concentration of interleukin-1 beta (IL-1β) concomitant with modulation of several inflammatory cytokines in the retina and RPE-choroid tissues, as measured by ELISA and cytokine array, respectively. CONCLUSION: Collectively, these results support that anti-inflammatory gene constructs such as AAV-TatCARD may be considered for the treatment of inflammation in AMD and other ocular diseases of the posterior pole in which inflammation may play a role. Furthermore, our findings emphasize the need to carefully consider potential sex-different responses when assessing potential therapies in pre-clinical models.

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.

ASC is an essential adaptor of the inflammasome, a micrometer-size multiprotein complex that processes proinflammatory cytokines. Inflammasome formation depends on ASC self-association into large assemblies via homotypic interactions of its two death domains, PYD and CARD. ASCb, an alternative splicing isoform, activates the inflammasome to a lesser extent compared with ASC. Thus, it has been postulated that adaptor isoforms differentially regulate inflammasome function. At the amino acid level, ASC and ASCb differ only in the length of the linker connecting the two death domains. To understand inflammasome regulation at the molecular level, we investigated the self-association properties of ASC and ASCb using real-time NMR, dynamic light scattering (DLS), size-exclusion chromatography, and transmission electron microscopy (TEM). The NMR data indicate that ASC self-association is faster than that of ASCb; a kinetic model for this oligomerization results in differing values for both the reaction order and the rate constants. Furthermore, DLS analysis indicates that ASC self-associates into more compact macrostructures compared with ASCb. Finally, TEM data show that ASCb has a reduced tendency to form densely packed filaments relative to ASC. Overall, these differences can only be explained by an effect of the linker length, as the NMR results show structural equivalence of the PYD and CARD in both proteins. The effect of linker length was corroborated by molecular docking with the procaspase-1 CARD domain. Altogether, our results indicate that ASC's faster and less polydisperse polymerization is more efficient, plausibly explaining inflammasome activation differences by ASC isoforms at the molecular level.

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.

Objectives This study aims to investigate whether apoptosis repressor with caspase recruitment domain (ARC) could promote survival and enhance osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Materials and methods The lentivirus transfection method was used to establish ARC-overexpressing BMSCs. The CCK-8 method was used to detect cell proliferation. The BD Pharmingen™ APC Annexin V Apoptosis Detection kit was used to detect cell apoptosis. The osteogenic capacity was investigated by OCN immunofluorescence staining, ALP analysis, ARS assays, and RT-PCR analysis. Cells were seeded into calcium phosphate cement (CPC) scaffolds and then inserted subcutaneously into nude mice and the defect area of the rat calvarium. Histological analysis was conducted to evaluate the in vivo cell apoptosis and new bone formation of the ARC-overexpressing BMSCs. RNA-seq was used to detect the possible mechanism of the effect of ARC on BMSCs. Results ARC promoted BMSC proliferation and inhibited cell apoptosis. ARC enhanced BMSC osteogenic differentiation in vitro. An in vivo study revealed that ARC can inhibit BMSC apoptosis and increase new bone formation. ARC regulates BMSCs mainly by activating the Fgf-2/PI3K/Akt pathway. Conclusions The present study suggests that ARC is a powerful agent for promoting bone regeneration of BMSCs and provides a promising method for bone tissue engineering.

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.

Objectives Nod-like receptor pyrin domain containing 3 (NLRP3) is one of the well characterized inflammasome that controls the maturation of pro-inflammatory cytokines and thereby the inflammation in pancreas which could be a promising target for anti-inflammatory drugs. The present study is aimed to explore whether luteolin can target the NLRP3 inflammasome and modulate its activity through the signaling protein, HSP70 in the ethanol-cerulein model of experimental pancreatitis. Methods Male albino Wistar rats were divided into four groups. Groups 1 and 2 rats received normal diet. Groups 3 and 4 rats received isocalorically adjusted diet containing ethanol for 5 weeks and cerulein (20 μg/kg body weight i.p., thrice weekly for the last 3 weeks of the experimental period). Additionally, group 2 and 4 rats received 2 mg/kg body weight of luteolin orally from third week. Results Luteolin co-administration decreased the serum levels of HSP70, oxidative stress markers, myeloperoxidase, GSH/GSSG and GST with concomitant downregulation in the mRNA expression of HSP70, caspase-1, ASC-NLRP3 and NF-κB. Spearman’s rank correlation test showed that serum HSP70 has positive correlation with the expression of ASC-NLRP3, caspase-1, NF-κB and 4-hydroxynonenal and negative correlation with GSH:GSSG ratio. Conclusions The modulating effect of luteolin on the expression of HSP70, NF-κB and thereby on ASC-NLRP3 complex may be claimed for its pancreato-protective activity.

Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the beta coronavirus subfamily and causes severe morbidity and mortality in humans especially when infected patients have underlying diseases such chronic obstructive pulmonary disease (COPD). Previously, we demonstrated that MERS-CoV-encoded ORF8b strongly inhibits MDA5- and RIG-I-mediated induction of the interferon beta (IFN-β) promoter activities. Here, we report that ORF8b seem to regulate MDA5 or RIG-I differentially as protein levels of MDA5 were significantly down-regulated while those of RIG-I were largely unperturbed. In addition, ORF8b seemed to efficiently suppress phosphorylation of IRF3 at the residues of 386 and 396 in cells transfected with RIG-I while total endogenous levels of IRF3 remained largely unchanged. Furthermore, ORF8b was able to inhibit all forms of RIG-I; full-length, RIG-I-1-734, and RIG-I-1-228, last of which contains only the CARD domains. Taken together, it is tempting to postulate that ORF8b may interfere with the CARD-CARD interactions between RIG-I and MAVS. Further detailed analysis is required to delineate the mechanisms of how ORF8b inhibits the MDA5/RIG-I receptor signaling pathway.

A critical role of influenza A virus nonstructural protein 1 (NS1) is to antagonize the host cellular antiviral response. NS1 accomplishes this role through numerous interactions with host proteins, including the cytoplasmic pathogen recognition receptor, retinoic acid–inducible gene I (RIG-I). Although the consequences of this interaction have been studied, the complete mechanism by which NS1 antagonizes RIG-I signaling remains unclear. We demonstrated previously that the NS1 RNA-binding domain (NS1RBD) interacts directly with the second caspase activation and recruitment domain (CARD) of RIG-I. We also identified that a single strain-specific polymorphism in the NS1RBD (R21Q) completely abrogates this interaction. Here we investigate the functional consequences of an R21Q mutation on NS1's ability to antagonize RIG-I signaling. We observed that an influenza virus harboring the R21Q mutation in NS1 results in significant up-regulation of RIG-I signaling. In support of this, we determined that an R21Q mutation in NS1 results in a marked deficit in NS1's ability to antagonize TRIM25-mediated ubiquitination of the RIG-I CARDs, a critical step in RIG-I activation. We also observed that WT NS1 is capable of binding directly to the tandem RIG-I CARDs, whereas the R21Q mutation in NS1 significantly inhibits this interaction. Furthermore, we determined that the R21Q mutation does not impede the interaction between NS1 and TRIM25 or NS1RBD's ability to bind RNA. The data presented here offer significant insights into NS1 antagonism of RIG-I and illustrate the importance of understanding the role of strain-specific polymorphisms in the context of this specific NS1 function.

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.

Background: Aerobic capacity, as measured by peak oxygen uptake (V˙O2), is one of the most powerful predictors of prognosis in heart failure (HF). Inflammation is a key factor contributing to alterations in aerobic capacity, and interleukin (IL)-1 cytokines are implicated in this process. The adaptor protein ASC is necessary for inflammasome activation of IL-1β and IL-18. ASC expression is controlled through epigenetic modification; lower ASC methylation is associated with worse outcomes in HF. The purpose of this study is to examine the relationships between ASC methylation, IL-1β, and IL-18 with V˙O2peak in persons with HF. Methods: This study examined the relationship between ASC methylation, IL-1β, and IL-18 with V˙O2peak in 54 stable outpatients with HF. All participants were NYHA class II or III, not engaged in an exercise program, and physically able to complete an exercise treadmill test. Results: Mean V˙O2peak was 16.68 ± 4.7 mL·kg−1·min−1. V˙O2peak was positively associated with mean percent ASC methylation (r = 0.47, P = 0.001) and negatively associated with IL-1β (r = −0.38, P = 0.007). Multiple linear regression models demonstrated that V˙O2peak increased by 2.30 mL·kg−1·min−1 for every 1% increase in ASC methylation and decreased by 1.91 mL·kg−1·min−1 for every 1 pg·mL−1 increase in plasma IL-1β. Conclusions: Mean percent ASC methylation and plasma IL-1β levels are associated with clinically meaningful differences in V˙O2peak in persons with HF. Inflammasome activation may play a mechanistic role in determining aerobic capacity. ASC methylation is a potentially modifiable mechanism for reducing the inflammatory response, thereby improving aerobic capacity in HF.

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.

Apoptosis plays important roles in maintaining normal development and homeostasis and in the pathophysiological processes of various diseases.Previous studies have shown that cardiomyocyte apoptosis is involved in the development of various cardiovascular diseases.The apoptosis repressor with caspase recruitment domain(ARC)is abundantly expressed in heart,which makes ARC a unique and central cardioprotective factor via anti-apoptotic pathway.This article reviews the structure and characteristics of ARC as well as the roles and mechanisms of ARC in cardiovascular diseases.

Infection by enteroviruses can cause severe neurological complications in humans. The interactions between the enteroviral and host proteins may facilitate the virus replication and be involved in the pathogenicity of infected individuals. It has been shown that human enteroviruses possess various mechanisms to suppress host innate immune responses in infected cells. Previous studies showed that infection by enterovirus 71 (EV71) causes the degradation of MDA5, which is a critical cytoplasmic pathogen sensor in the recognition of picornaviruses for initiating transcription of type I interferons. In the present study, we demonstrated that the RNA-dependent RNA polymerase (RdRP; also denoted 3D(pol)) encoded by EV71 interacts with the caspase activation and recruitment domains (CARDs) of MDA5 and plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation and mRNA expression. In addition, we found that the 3D(pol) protein encoded by coxsackievirus B3 also interacted with MDA5 and downregulated the antiviral signaling initiated by MDA5. These findings indicate that enteroviral RdRP may function as an antagonist against the host antiviral innate immune response. IMPORTANCE Infection by enteroviruses causes severe neurological complications in humans. Human enteroviruses possess various mechanisms to suppress the host type I interferon (IFN) response in infected cells to establish viral replication. In the present study, we found that the enteroviral 3D(pol) protein (or RdRP), which is a viral RNA-dependent RNA polymerase for replicating viral RNA, plays a role in the inhibition of MDA5-mediated beta interferon (IFN-β) promoter activation. We further demonstrated that enteroviral 3D(pol) protein interacts with the caspase activation and recruitment domains (CARDs) of MDA5. These findings indicate that enteroviral RdRP functions as an antagonist against the host antiviral response.

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.

BACKGROUND: CARD11 encodes a scaffold protein in lymphocytes that links antigen receptor engagement with downstream signaling to NF-κB, JNK, and mTORC1. Germline CARD11 mutations cause several distinct primary immune disorders in humans, including SCID (biallelic null mutations), B cell Expansion with NF-κB and T cell Anergy (BENTA; heterozygous, gain-of-function mutations), and severe atopic disease (loss-of-function, heterozygous, dominant interfering mutations), which has focused attention on CARD11 mutations discovered by whole exome sequencing. OBJECTIVES: To determine the molecular actions of an extended allelic series of CARD11, and to characterize the expanding range of clinical phenotypes associated with heterozygous CARD11 loss-of-function alleles. METHODS: Cell transfections and primary T cell assays were utilized to evaluate signaling and function of CARD11 variants. RESULTS: Here we report on an expanded cohort of patients harboring novel heterozygous CARD11 mutations that extend beyond atopy to include other immunologic phenotypes not previously associated with CARD11 mutations. In addition to (and sometimes excluding) severe atopy, heterozygous missense and indel mutations in CARD11 presented with immunologic phenotypes similar to those observed in STAT3-LOF, DOCK8 deficiency, common variable immune deficiency (CVID), neutropenia, and immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX)-like syndrome. Pathogenic variants exhibited dominant negative activity, and were largely confined to the CARD or coiled-coil domains of the CARD11 protein. CONCLUSION: These results illuminate a broader phenotypic spectrum associated with CARD11 mutations in humans, and underscore the need for functional studies to demonstrate that rare gene variants encountered in expected and unexpected phenotypes must nonetheless be validated for pathogenic activity.

ファイル公開：2020-04-01

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.