RESUMEN
Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
Asunto(s)
Proteínas Nucleares , Nucleosomas , Nucleotidiltransferasas , Proteolisis , Ubiquitina-Proteína Ligasas , Animales , Humanos , Ratones , Núcleo Celular/metabolismo , Microscopía por Crioelectrón , Degrones , Infecciones por Virus ADN/inmunología , Virus ADN/inmunología , Virus ADN/metabolismo , ADN Viral/inmunología , ADN Viral/metabolismo , Inmunidad Innata , Reconocimiento de Inmunidad Innata , Interferón Tipo I/inmunología , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/ultraestructura , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Especificidad por Sustrato , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , UbiquitinaciónRESUMEN
Primary bone lymphoma (PBL) is rare and mostly represented by diffuse large B-cell lymphomas (DLBCL). Follicular lymphoma (FL), albeit commonly disseminating to the bone marrow, rarely presents primarily as bone lesions. Here, we studied 16 patients (12 men:4 women, median age 60 years) who presented with bone pain and/or skeletal radiologic abnormalities revealing bone FL. Lesions were multifocal in 11 patients (spine ± appendicular skeleton), and unifocal in 5 patients (femoral, tibial, or vertebral). An infiltrate of centrocytes and centroblasts (CD20+ CD5- CD10+ BCL2+ BCL6+) with abundant reactive T cells and an increased reticulin fibrosis massively replaced the marrow spaces between preserved bone trabeculae. The pattern was diffuse ± nodular, often with paratrabecular reinforcement and/or peripheral paratrabecular extension. Ki-67 was usually <15%. Two cases had necrosis. BCL2 rearrangement was demonstrated in 14 of 14 evaluable cases (with concomitant BCL6 rearrangement in one). High-throughput sequencing revealed BCL2, KMT2D, and TNFRSF14 to be the most frequently mutated genes. After staging, 5 qualified for PBL (3 limited stage) and 11 had stage IV systemic FL. All patients received rituximab ± polychemotherapy as firstline treatment, and 7 received local therapy (6 radiotherapy and 2 surgery). Three patients experienced transformation to DLBCL. At the last follow-up (15/16, median 48 months), 11 patients achieved complete remission, including all cases with PBL and most patients with limited extraosseous disease (3-year progression-free survival 71%). One patient died of unrelated cause (3-year overall survival 91%). FL may manifest as a localized or polyostotic bone disease. A minority represent PBL, whereas most reveal systemic disease.
Asunto(s)
Linfoma Folicular , Linfoma de Células B Grandes Difuso , Masculino , Humanos , Femenino , Persona de Mediana Edad , Linfoma Folicular/genética , Linfoma Folicular/terapia , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/patología , Rituximab , Supervivencia sin Progresión , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
NLRP3 is a pattern recognition receptor with a well-documented role in inducing inflammasome assembly in response to cellular stress. Deregulation of its activity leads to many inflammatory disorders including gouty arthritis, Alzheimer disease, and cancer. Whereas its role in the context of cancer has been mostly explored in the immune compartment, whether NLRP3 exerts functions unrelated to immunity in cancer development remains unexplored. Here, we demonstrate that NLRP3 interacts with the ATM kinase to control the activation of the DNA damage response, independently of its inflammasome activity. NLRP3 down-regulation in both broncho- and mammary human epithelial cells significantly impairs ATM pathway activation, leading to lower p53 activation, and provides cells with the ability to resist apoptosis induced by acute genotoxic stress. Interestingly, NLRP3 expression is down-regulated in non-small cell lung cancers and breast cancers, and its expression positively correlates with patient overall survival. Our findings identify a novel non-immune function for NLRP3 in maintaining genome integrity and strengthen the concept of a functional link between innate immunity and DNA damage sensing pathways to maintain cell integrity.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Humanos , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inmunidad Innata , Daño del ADN , Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismoRESUMEN
The recognition of DNA through the cGAS-STING pathway plays a critical role in antiviral immunity, but can also drive maladaptive immune responses underlying several pathological conditions. Despite its importance, understanding the organization of cGAS-STING signaling axis in the 3D space of a cell is relatively limited. In this review, we discuss recent progress in understanding the spatial coordination of DNA-induced cGAS-STING activity and its relevance in balancing innate immune responses toward (self-) DNA. We also consider the importance of context-specific co-factors and end by suggesting interesting areas for further research into spatial aspects of DNA-induced innate immunity.
Asunto(s)
Proteínas de la Membrana , Nucleotidiltransferasas , Animales , ADN , Humanos , Inmunidad Innata/genética , Proteínas de la Membrana/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de SeñalRESUMEN
Inflammasomes are molecular complexes that trigger an inflammatory response upon detection of pathogens or danger signals. Recent studies suggest that they are also involved in cancer progression. However, their roles during tumorigenesis remain poorly understood and controversial. Here, we investigated whether inflammasome activation supports mammary tumor growth. Using mouse models of invasive breast cancer, our results demonstrate that the absence of a functional inflammasome impairs tumor growth. Importantly, tumors implanted into inflammasome-deficient mice recruited significantly less neutrophils and more natural killer (NK) cells, and these latter cells displayed a more active phenotype. Interestingly, NK cell depletion abolished the anti-tumoral effect observed in inflammasome-deficient mice, although inflammasome-regulated cytokine neutralization had no effect. Thus, our work identifies a novel role for the inflammasome in supporting mammary tumor growth by attenuating NK cell recruitment and activity. These results suggest that inflammasome inhibition could be a putative target for treating invasive breast cancers.
RESUMEN
The DNA sensor cyclic GMP-AMP synthase (cGAS) initiates innate immune responses following microbial infection, cellular stress and cancer1. Upon activation by double-stranded DNA, cytosolic cGAS produces 2'3' cGMP-AMP, which triggers the induction of inflammatory cytokines and type I interferons 2-7. cGAS is also present inside the cell nucleus, which is replete with genomic DNA8, where chromatin has been implicated in restricting its enzymatic activity9. However, the structural basis for inhibition of cGAS by chromatin remains unknown. Here we present the cryo-electron microscopy structure of human cGAS bound to nucleosomes. cGAS makes extensive contacts with both the acidic patch of the histone H2A-H2B heterodimer and nucleosomal DNA. The structural and complementary biochemical analysis also find cGAS engaged to a second nucleosome in trans. Mechanistically, binding of the nucleosome locks cGAS into a monomeric state, in which steric hindrance suppresses spurious activation by genomic DNA. We find that mutations to the cGAS-acidic patch interface are sufficient to abolish the inhibitory effect of nucleosomes in vitro and to unleash the activity of cGAS on genomic DNA in living cells. Our work uncovers the structural basis of the interaction between cGAS and chromatin and details a mechanism that permits self-non-self discrimination of genomic DNA by cGAS.
Asunto(s)
Microscopía por Crioelectrón , Nucleosomas/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Células HeLa , Histonas/metabolismo , Humanos , Modelos Moleculares , Mutación , Nucleosomas/química , Nucleosomas/ultraestructura , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/ultraestructuraRESUMEN
The appearance of DNA in the cytosol is perceived as a danger signal that stimulates potent immune responses through cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS). How cells regulate the activity of cGAS toward self-DNA and guard against potentially damaging autoinflammatory responses is a fundamental biological question. Here, we identify barrier-to-autointegration factor 1 (BAF) as a natural opponent of cGAS activity on genomic self-DNA. We show that BAF dynamically outcompetes cGAS for DNA binding, hence prohibiting the formation of DNA-cGAS complexes that are essential for enzymatic activity. Upon acute loss of nuclear membrane integrity, BAF is necessary to restrict cGAS activity on exposed DNA. Our observations reveal a safeguard mechanism, distinct from physical separation, by which cells protect themselves against aberrant immune responses toward genomic DNA.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/inmunología , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Membrana Nuclear/metabolismoRESUMEN
BACKGROUND & AIMS: Low levels of toll-like receptor 3 (TLR3) in patients with hepatocellular carcinoma (HCC) are associated with poor prognosis, primarily owing to the loss of inflammatory signaling and subsequent lack of immune cell recruitment to the liver. Herein, we explore the role of TLR3-triggered apoptosis in HCC cells. METHODS: Quantitative reverse transcription PCR, western blotting, immunohistochemistry and comparative genomic hybridization were used to analyze human and mouse HCC cell lines, as well as surgically resected primary human HCCs, and to study the impact of TLR3 expression on patient outcomes. Functional analyses were performed in HCC cells, following the restoration of TLR3 by lentiviral transduction. The role of TLR3-triggered apoptosis in HCC was analyzed in vivo in a transgenic mouse model of HCC. RESULTS: Lower expression of TLR3 in tumor compared to non-tumor matched tissue was observed at both mRNA and protein levels in primary HCC, and was predictive of shorter recurrence-free survival after surgical resection in both univariate (hazard ratio [HR] 1.79; 95% CI 1.04-3.06; pâ¯=â¯0.03) and multivariate analyses (HR 1.73; CI 1.01-2.97; pâ¯=â¯0.04). Immunohistochemistry confirmed frequent downregulation of TLR3 in human and mouse primary HCC cells. None of the 6 human HCC cell lines analyzed expressed TLR3, and ectopic expression of TLR3 following lentiviral transduction not only restored the inflammatory response but also sensitized cells to TLR3-triggered apoptosis. Lastly, in the transgenic mouse model of HCC, absence of TLR3 expression was accompanied by a lower rate of preneoplastic hepatocyte apoptosis and accelerated hepatocarcinogenesis without altering the tumor immune infiltrate. CONCLUSION: Downregulation of TLR3 protects transforming hepatocytes from direct TLR3-triggered apoptosis, thereby contributing to hepatocarcinogenesis and poor patient outcome. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a heterogeneous disease associated with a poor prognosis. In patients with HCC, TLR3 downregulation is associated with reduced survival. Herein, we show that the absence of TLR3 is associated with a lower rate of apoptosis, and subsequently more rapid hepatocarcinogenesis, without any change to the immune infiltrate in the liver. Therefore, the poor prognosis associated with low TLR3 expression in HCC is likely linked to tumors ability to escape apoptosis. TLR3 may become a promising therapeutic target in TLR3-positive HCC.
Asunto(s)
Carcinogénesis/metabolismo , Carcinoma Hepatocelular , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas , Pronóstico , Receptor Toll-Like 3/genética , Animales , Apoptosis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/terapia , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Femenino , Hepatectomía/métodos , Hepatectomía/mortalidad , Humanos , Estimación de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/terapia , Masculino , Ratones , Persona de Mediana Edad , Transducción de SeñalRESUMEN
Cellular senescence is triggered by various distinct stresses and characterized by a permanent cell cycle arrest. Senescent cells secrete a variety of inflammatory factors, collectively referred to as the senescence-associated secretory phenotype (SASP). The mechanism(s) underlying the regulation of the SASP remains incompletely understood. Here we define a role for innate DNA sensing in the regulation of senescence and the SASP. We find that cyclic GMP-AMP synthase (cGAS) recognizes cytosolic chromatin fragments in senescent cells. The activation of cGAS, in turn, triggers the production of SASP factors via stimulator of interferon genes (STING), thereby promoting paracrine senescence. We demonstrate that diverse stimuli of cellular senescence engage the cGAS-STING pathway in vitro and we show cGAS-dependent regulation of senescence following irradiation and oncogene activation in vivo. Our findings provide insights into the mechanisms underlying cellular senescence by establishing the cGAS-STING pathway as a crucial regulator of senescence and the SASP.
Asunto(s)
Senescencia Celular , Cromatina/enzimología , Citosol/enzimología , Inmunidad Innata , Nucleotidiltransferasas/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Senescencia Celular/efectos de la radiación , Cromatina/inmunología , Cromatina/efectos de la radiación , Citosol/inmunología , Citosol/efectos de la radiación , Activación Enzimática , Femenino , Genotipo , Inmunidad Innata/efectos de la radiación , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/inmunología , Estrés Oxidativo , Comunicación Paracrina , Fenotipo , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
The caspase-1 enzymatic activity plays a major role in the innate immune response as it regulates the maturation of two major proinflammatory cytokines, the interleukin-1beta (IL-1ß) and IL-18. In this chapter, we describe the technique of Western blot to assess caspase-1 activation. This method provides multiple information within one experiment. It allows the detection of both unprocessed and processed caspase-1 and substrates.
Asunto(s)
Caspasa 1/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Animales , Western Blotting , Células Cultivadas , Inmunidad Innata , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , RatonesRESUMEN
During cell life, proteins often misfold, depending on particular mutations or environmental changes, which may lead to protein aggregates that are toxic for the cell. Such protein aggregates are the root cause of numerous diseases called "protein conformational diseases," such as myofibrillar myopathy and familial amyotrophic lateral sclerosis. To fight against aggregates, cells are equipped with protein quality control mechanisms. Here we report that NFκB transcription factor is activated by misincorporation of amino acid analogues into proteins, inhibition of proteasomal activity, expression of the R120G mutated form of HspB5 (associated with myofibrillar myopathy), or expression of the G985R and G93A mutated forms of superoxide dismutase 1 (linked to familial amyotrophic lateral sclerosis). This noncanonical stimulation of NFκB triggers the up-regulation of BAG3 and HspB8 expression, two activators of selective autophagy, which relocalize to protein aggregates. Then NFκB-dependent autophagy allows the clearance of protein aggregates. Thus NFκB appears as a central and major regulator of protein aggregate clearance by modulating autophagic activity. In this context, the pharmacological stimulation of this quality control pathway might represent a valuable strategy for therapies against protein conformational diseases.
Asunto(s)
Autofagia/fisiología , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células HeLa , Humanos , Neuronas Motoras/metabolismo , Conformación Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Activación Transcripcional , Regulación hacia Arriba , Cadena B de alfa-Cristalina/genética , Cadena B de alfa-Cristalina/metabolismoRESUMEN
Inflammasomes are caspase-1-activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1ß and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1ß cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11(-/-) cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.