RESUMO
The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.
Assuntos
AMP Cíclico , Proteínas de Drosophila , Drosophila melanogaster , Estresse do Retículo Endoplasmático , Nucleotidiltransferases , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Animais , Camundongos , Alelos , AMP Cíclico/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Pâncreas/metabolismo , Pâncreas/fisiopatologia , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Introduction: Type I interferon (IFN) plays a vital role in the pathogenesis of systemic lupus erythematosus. Cyclic GMP AMP synthase (cGAS) is a cytosolic DNA sensor that recognizes dsDNA and creates cGAMP to activate STING-mediated type I IFN production. The activation of STING induces lupus disease in Fcgr2b deficient mice through the differentiation of dendritic cells. In contrast, Cgas-deficient mice could be generated more autoantibody production and proteinuria in pristane-induced lupus (PIL). These data suggested that the other dsDNA sensors could be involved in lupus development mechanisms. Methods: This study aimed to identify the cGAS-mediated mechanisms contributing to lupus pathogenesis in PIL. The Cgas-deficient and WT mice were induced lupus disease with pristane and subsequently analyzed autoantibody, histopathology, and immunophenotypes. The lung tissues were analyzed with the expression profiles by RT-PCR and western blot. The bone marrow-derived macrophages were stimulated with inflammasome activators and observed pyroptosis. Results: The Cgas-/- mice developed more severe pulmonary hemorrhage and autoantibody production than WT mice. The activated dendritic cells, IFN-g-, and IL-17a-producing T helper cells, and infiltrated macrophages in the lung were detected in Cgas-/- mice higher than in WT mice. We observed an increase in expression of Aim2, Casp11, and Ifi16 in the lung and serum IL-1a but IL-1b in pristane-injected Cgas-/- mice. The rise of Caspase-11 in the lung of pristane-injected Cgas-/- mice suggested noncanonical inflammasome activation. The activation of AIM2 and NLRP3 inflammasomes in bone marrow-derived macrophages (BMDMs) enhanced the number of dead cells in Cgas-/- mice compared with WT mice. Activation of the inflammasome significantly induced pyroptosis in Cgas-/- BMDMs. The dsDNA level, but not mitochondrial DNA, increased dramatically in pristane-injected Cgas-/- mice suggesting the dsDNA could be a ligand activating inflammasomes. The cGAS agonist-induced BMDM activation in the Cgas-/- mice indicated that the activation of DNA sensors other than cGAS enhanced activated macrophages. Conclusion: These findings suggested that cGAS hampers the unusual noncanonical inflammasome activation through other DNA sensors.
Assuntos
Inflamassomos , Inflamação , Lúpus Eritematoso Sistêmico , Macrófagos , Nucleotidiltransferases , Animais , Camundongos , Autoanticorpos/genética , Autoanticorpos/imunologia , Modelos Animais de Doenças , DNA/genética , DNA/imunologia , Inflamassomos/genética , Inflamassomos/imunologia , Inflamação/genética , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Macrófagos/imunologia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologiaRESUMO
Although the enhanced responses against serum cell-free DNA (cfDNA) in cases of sepsis-a life-threatening organ dysfunction due to systemic infection-are understood, the influence of the cytosolic DNA receptor cGAS (cyclic guanosine monophosphate-adenosine monophosphate (GMP-AMP) synthase) on sepsis is still unclear. Here, experiments on cGAS deficient (cGAS-/-) mice were conducted using cecal ligation and puncture (CLP) and lipopolysaccharide (LPS) injection sepsis models and macrophages. Severity of CLP in cGAS-/- mice was less severe than in wildtype (WT) mice, as indicated by mortality, serum LPS, cfDNA, leukopenia, cytokines (TNF-α, IL-6 and IL-10), organ histology (lung, liver and kidney) and spleen apoptosis. With the LPS injection model, serum cytokines in cGAS-/- mice were lower than in WT mice, despite the similar serum cfDNA level. Likewise, in LPS-activated WT macrophages, the expression of several mitochondria-associated genes (as revealed by RNA sequencing analysis) and a profound reduction in mitochondrial parameters, including maximal respiration (determined by extracellular flux analysis), DNA (mtDNA) and mitochondrial abundance (revealed by fluorescent staining), were demonstrated. These data implied the impact of cfDNA resulting from LPS-induced cell injury. In parallel, an additive effect of bacterial DNA on LPS, seen in comparison with LPS alone, was demonstrated in WT macrophages, but not in cGAS-/- cells, as indicated by supernatant cytokines (TNF-α and IL-6), M1 proinflammatory polarization (iNOS and IL-1ß), cGAS, IFN-γ and supernatant cyclic GMP-AMP (cGAMP). In conclusion, cGAS activation by cfDNA from hosts (especially mtDNA) and bacteria was found to induce an additive proinflammatory effect on LPS-activated macrophages which was perhaps responsible for the more pronounced sepsis hyperinflammation observed in WT mice compared with the cGAS-/- group.
Assuntos
Nucleotidiltransferases/metabolismo , Sepse/metabolismo , Animais , Ceco/metabolismo , Citocinas/metabolismo , DNA/metabolismo , Interleucina-10/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidases/metabolismo , Nucleotídeos Cíclicos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Sepse/prevenção & controle , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sepsis is a life-threatening inflammatory syndrome secondary to infection. Thanks to the advances of antibiotics and life-supporting techniques, the mortality of sepsis has been decreasing in recent decades. Nevertheless, sepsis-associated encephalopathy (SAE) is still common in septic patients, which promotes the mortality of septic patients and results in cognitive dysfunction in survivors. Full understanding and effective medicine in the treatment of SAE is currently scant. Here, we revealed a novel role of cGAS signaling in the pathogenesis of SAE. Deficiency of cGas significantly restored cognitive impairment in sepsis mice model. The restoration may attribute to the recovery of neo-neuron decline that associated with the decrease of activated microglia and astrocytes in the hippocampus of cGas-deficient mice. In addition, type I interferon (IFN) signaling, a downstream of cGAS pathway, was boosted in the hippocampus of septic mice, which was dramatically attenuated by deleting cGas. Moreover, administration of recombinant IFNß markedly reversed the protection of ablation of cGas in the cognitive impairment in sepsis. Collectively, cGAS promotes the pathogenesis of SAE by up-regulating type I IFN signaling. Blocking cGAS may be a promising strategy for preventing encephalopathy in sepsis.
Assuntos
Modelos Animais de Doenças , Nucleotidiltransferases/genética , Encefalopatia Associada a Sepse/genética , Transdução de Sinais/genética , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/fisiopatologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Interferon Tipo I/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/metabolismo , Neurônios/metabolismo , Nucleotidiltransferases/deficiência , Substâncias Protetoras/metabolismo , Sepse/genética , Sepse/metabolismo , Sepse/fisiopatologia , Encefalopatia Associada a Sepse/metabolismoRESUMO
The Cytolethal Distending Toxin (CDT) is a bacterial genotoxin produced by pathogenic bacteria causing major foodborne diseases worldwide. CDT activates the DNA Damage Response and modulates the host immune response, but the precise relationship between these outcomes has not been addressed so far. Here, we show that chronic exposure to CDT in HeLa cells or mouse embryonic fibroblasts promotes a strong type I interferon (IFN) response that depends on the cytoplasmic DNA sensor cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) through the recognition of micronuclei. Indeed, despite active cell cycle checkpoints and in contrast to other DNA damaging agents, cells exposed to CDT reach mitosis where they accumulate massive DNA damage, resulting in chromosome fragmentation and micronucleus formation in daughter cells. These mitotic phenotypes are observed with CDT from various origins and in cancer or normal cell lines. Finally, we show that CDT exposure in immortalized normal colonic epithelial cells is associated to cGAS protein loss and low type I IFN response, implying that CDT immunomodulatory function may vary depending on tissue and cell type. Thus, our results establish a direct link between CDT-induced DNA damage, genetic instability and the cellular immune response that may be relevant in the context of natural infection associated to chronic inflammation or carcinogenesis.
Assuntos
Toxinas Bacterianas/farmacologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Regulação para Cima/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HeLa , Humanos , Interferon Tipo I/genética , Camundongos , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genéticaRESUMO
In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.
Assuntos
Drosophila melanogaster/imunologia , Nucleotidiltransferases/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Vírus/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/virologia , Feminino , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Ligantes , Masculino , Proteínas de Membrana/metabolismo , Modelos Moleculares , NF-kappa B/metabolismo , Nucleotídeos Cíclicos/metabolismo , Nucleotidiltransferases/classificação , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , RNA de Cadeia Dupla/análise , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Receptores de Reconhecimento de Padrão/classificação , Receptores de Reconhecimento de Padrão/deficiência , Receptores de Reconhecimento de Padrão/imunologiaRESUMO
GDP-mannose-pyrophosphorylase-B (GMPPB) facilitates the generation of GDP-mannose, a sugar donor required for glycosylation. GMPPB defects cause muscle disease due to hypoglycosylation of α-dystroglycan (α-DG). Alpha-DG is part of a protein complex, which links the extracellular matrix with the cytoskeleton, thus stabilizing myofibers. Mutations of the catalytically inactive homolog GMPPA cause alacrima, achalasia, and mental retardation syndrome (AAMR syndrome), which also involves muscle weakness. Here, we showed that Gmppa-KO mice recapitulated cognitive and motor deficits. As structural correlates, we found cortical layering defects, progressive neuron loss, and myopathic alterations. Increased GDP-mannose levels in skeletal muscle and in vitro assays identified GMPPA as an allosteric feedback inhibitor of GMPPB. Thus, its disruption enhanced mannose incorporation into glycoproteins, including α-DG in mice and humans. This increased α-DG turnover and thereby lowered α-DG abundance. In mice, dietary mannose restriction beginning after weaning corrected α-DG hyperglycosylation and abundance, normalized skeletal muscle morphology, and prevented neuron degeneration and the development of motor deficits. Cortical layering and cognitive performance, however, were not improved. We thus identified GMPPA defects as the first congenital disorder of glycosylation characterized by α-DG hyperglycosylation, to our knowledge, and we have unraveled underlying disease mechanisms and identified potential dietary treatment options.
Assuntos
Distroglicanas , Guanosina Difosfato Manose , Músculo Esquelético/metabolismo , Doenças Neuromusculares , Nucleotidiltransferases/deficiência , Animais , Distroglicanas/genética , Distroglicanas/metabolismo , Glicosilação , Guanosina Difosfato Manose/genética , Guanosina Difosfato Manose/metabolismo , Humanos , Camundongos , Camundongos Knockout , Doenças Neuromusculares/dietoterapia , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Nucleotidiltransferases/metabolismoRESUMO
Radiotherapy is the most common strategy in the treatment of cancer. However, radiation-induced acute complications, in particular sepsis, render patients in a life-threatening status or lead to delay of therapy that largely influences patients' overall responses. The understanding of sepsis in radiotherapy is currently scant and effective medicine is not available by far. Here, with WT mice as control, we challenged mice deficient to cGas, Caspase-11, Gsdmd or Asc with cecal ligation and puncture (CLP, a sepsis model) after a treatment of thorax irradiation. We found that radiation robustly upgraded caspase-11 pathway in irradiated region and consequently deteriorated lung injury and mortality in the sepsis model. cGas knockout markedly attenuated radiation-upgraded caspase-11 and restored sepsis. Deficiency of non-canonical inflammasome, caspase-11 and the downstream GSDMD, rather than an AIM2 inflammasome component, ASC, dramatically protected against radiation-promoted injury and mortality in septic mice. The protection may attribute to the inhibition of caspase-11-mediated pyroptosis in endothelial cells of the lung. Thus, blocking cGAS/caspase-11 signaling would be an adjuvant treatment strategy for preventing sepsis in radiotherapy of cancer.
Assuntos
Caspases Iniciadoras/metabolismo , Neoplasias/radioterapia , Nucleotidiltransferases/metabolismo , Radioterapia/efeitos adversos , Sepse/etiologia , Sepse/metabolismo , Transdução de Sinais , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/enzimologia , Neoplasias/metabolismo , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Sepse/mortalidadeRESUMO
The release of DNA into the cytoplasm upon damage to the nucleus or during viral infection triggers an interferon-mediated defense response, inflammation and cell death. In human cells cytoplasmic DNA is sensed by cyclic GMP-AMP Synthase (cGAS) and Absent In Melanoma 2 (AIM2). Here, we report the identification of a "natural knockout" model of cGAS. Comparative genomics of phylogenetically diverse mammalian species showed that cGAS and its interaction partner Stimulator of Interferon Genes (STING) have been inactivated by mutations in the Malayan pangolin whereas other mammals retained intact copies of these genes. The coding sequences of CGAS and STING1 are also disrupted by premature stop codons and frame-shift mutations in Chinese and tree pangolins, suggesting that expression of these genes was lost in a common ancestor of all pangolins that lived more than 20 million years ago. AIM2 is retained in a functional form in pangolins whereas it is inactivated by mutations in carnivorans, the phylogenetic sister group of pangolins. The deficiency of cGAS and STING points to the existence of alternative mechanisms of controlling cytoplasmic DNA-associated cell damage and viral infections in pangolins.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/genética , Fatores Reguladores de Interferon/genética , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Pangolins/genética , Animais , Sequência de Bases , Gatos , China , Códon de Terminação , Citosol/imunologia , Citosol/metabolismo , DNA/imunologia , Proteínas de Ligação a DNA/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/imunologia , Malásia , Proteínas de Membrana/deficiência , Proteínas de Membrana/imunologia , Mutação , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/imunologia , Pangolins/imunologia , Filogenia , Especificidade da EspécieRESUMO
The cyclic GMP-AMP (cGAMP) synthase (cGAS) plays a critical role in host defense by sensing cytosolic DNA derived from microbial pathogens or mis-located cellular DNA. Upon DNA binding, cGAS utilizes GTP and ATP as substrates to synthesize cGAMP, leading to MITA-mediated innate immune response. In this study, we identified the phosphatase PPP6C as a negative regulator of cGAS-mediated innate immune response. PPP6C is constitutively associated with cGAS in un-stimulated cells. DNA virus infection causes rapid disassociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 or mouse cGAS S420 in its catalytic pocket. Mutation of this serine residue of cGAS impairs its ability to synthesize cGAMP upon DNA virus infection. In vitro experiments indicate that S420-phosphorylated mcGAS has higher affinity to GTP and enzymatic activity. PPP6C-deficiency promotes innate immune response to DNA virus in various cells. Our findings suggest that PPP6C-mediated dephosphorylation of a catalytic pocket serine residue of cGAS impairs its substrate binding activity and innate immune response, which provides a mechanism for keeping the DNA sensor cGAS inactive in the absence of infection to avoid autoimmune response.
Assuntos
DNA Viral/imunologia , Imunidade Inata/imunologia , Nucleotidiltransferases/imunologia , Fosfoproteínas Fosfatases/imunologia , Animais , Sítios de Ligação , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Fosfoproteínas Fosfatases/deficiência , Fosforilação , Especificidade por Substrato , Células THP-1RESUMO
Schistosomiasis is a human parasitic disease responsible for serious consequences for public health, as well as severe socioeconomic impacts in developing countries. Here, we provide evidence that the adaptor molecule STING plays an important role in Schistosoma mansoni infection. S. mansoni DNA is sensed by cGAS leading to STING activation in murine embryonic fibroblasts (MEFs). Sting-/- and C57BL/6 (WT) mice were infected with schistosome cercariae in order to assess parasite burden and liver pathology. Sting-/- mice showed worm burden reduction but no change in the number of eggs or granuloma numbers and area when compared to WT animals. Immunologically, a significant increase in IFN-γ production by the spleen cells was observed in Sting-/- animals. Surprisingly, Sting-/- mice presented an elevated percentage of neutrophils in lungs, bronchoalveolar lavage, and spleens. Moreover, Sting-/- neutrophils exhibited increased survival rate, but similar ability to kill schistosomula in vitro when stimulated with IFN-γ when compared to WT cells. Finally, microbiota composition was altered in Sting-/- mice, revealing a more inflammatory profile when compared to WT animals. In conclusion, this study demonstrates that STING signaling pathway is important for S. mansoni DNA sensing and the lack of this adaptor molecule leads to enhanced resistance to infection.
Assuntos
Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/metabolismo , Esquistossomose mansoni/parasitologia , Animais , DNA de Protozoário/imunologia , Modelos Animais de Doenças , Microbioma Gastrointestinal , Interações Hospedeiro-Patógeno/imunologia , Imunidade Celular , Imunidade Humoral , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , Especificidade de Órgãos/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Clearance of apoptotic cells by macrophages prevents excessive inflammation and supports immune tolerance. Here, we examined the effect of blocking apoptotic cell clearance on anti-tumor immune response. We generated an antibody that selectively inhibited efferocytosis by phagocytic receptor MerTK. Blockade of MerTK resulted in accumulation of apoptotic cells within tumors and triggered a type I interferon response. Treatment of tumor-bearing mice with anti-MerTK antibody stimulated T cell activation and synergized with anti-PD-1 or anti-PD-L1 therapy. The anti-tumor effect induced by anti-MerTK treatment was lost in Stinggt/gt mice, but not in Cgas-/- mice. Abolishing cGAMP production in Cgas-/- tumor cells, depletion of extracellular ATP, or inactivation of the ATP-gated P2X7R channel also compromised the effects of MerTK blockade. Mechanistically, extracellular ATP acted via P2X7R to enhance the transport of extracellular cGAMP into macrophages and subsequent STING activation. Thus, MerTK blockade increases tumor immunogenicity and potentiates anti-tumor immunity, which has implications for cancer immunotherapy.
Assuntos
Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Nucleotídeos Cíclicos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , c-Mer Tirosina Quinase/imunologia , Trifosfato de Adenosina/metabolismo , Animais , Apoptose , Antígeno B7-H1/imunologia , Células Cultivadas , Feminino , Imunidade Inata , Imunoterapia , Interferon Tipo I/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/metabolismo , Fagocitose , Receptor de Morte Celular Programada 1/imunologia , Receptores Purinérgicos P2X7/deficiência , Transdução de Sinais/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , c-Mer Tirosina Quinase/genéticaRESUMO
Toxoplasma gondii is an important neurotropic pathogen that establishes latent infections in humans that can cause toxoplasmosis in immunocompromised individuals. It replicates inside host cells and has developed several strategies to manipulate host immune responses. However, the cytoplasmic pathogen-sensing pathway that detects T. gondii is not well-characterized. Here, we found that cyclic GMP-AMP synthase (cGAS), a sensor of foreign dsDNA, is required for activation of anti-T. gondii immune signaling in a mouse model. We also found that mice deficient in STING (Stinggt/gt mice) are much more susceptible to T. gondii infection than WT mice. Of note, the induction of inflammatory cytokines, type I IFNs, and interferon-stimulated genes in the spleen from Stinggt/gt mice was significantly impaired. Stinggt/gt mice exhibited more severe symptoms than cGAS-deficient mice after T. gondii infection. Interestingly, we found that the dense granule protein GRA15 from T. gondii is secreted into the host cell cytoplasm and then localizes to the endoplasmic reticulum, mediated by the second transmembrane motif in GRA15, which is essential for activating STING and innate immune responses. Mechanistically, GRA15 promoted STING polyubiquitination at Lys-337 and STING oligomerization in a TRAF protein-dependent manner. Accordingly, GRA15-deficient T. gondii failed to elicit robust innate immune responses compared with WT T. gondii. Consequently, GRA15-/-T. gondii was more virulent and caused higher mortality of WT mice but not Stinggt/gt mice upon infection. Together, T. gondii infection triggers cGAS/STING signaling, which is enhanced by GRA15 in a STING- and TRAF-dependent manner.
Assuntos
Imunidade Inata , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Interferon gama/metabolismo , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Multimerização Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Baço/metabolismo , Taxa de Sobrevida , Toxoplasma/patogenicidade , Toxoplasmose/mortalidade , Toxoplasmose/parasitologia , Toxoplasmose/patologia , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , UbiquitinaçãoRESUMO
Riboflavin (vitamin B2), a water-soluble vitamin, is an essential nutrient in higher organisms as it is not endogenously synthesised, with requirements being met principally by dietary intake. Tissue-specific transporter proteins direct riboflavin to the intracellular machinery responsible for the biosynthesis of the flavocoenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). These flavocoenzymes play a vital role in ensuring the functionality of a multitude of flavoproteins involved in bioenergetics, redox homeostasis, DNA repair, chromatin remodelling, protein folding, apoptosis, and other physiologically relevant processes. Hence, it is not surprising that the impairment of flavin homeostasis in humans may lead to multisystem dysfunction including neuromuscular disorders, anaemia, abnormal fetal development, and cardiovascular disease. In this review, we provide an overview of riboflavin absorption, transport, and metabolism. We then focus on the clinical and biochemical features associated with biallelic FLAD1 mutations leading to FAD synthase deficiency, the only known primary defect in flavocoenzyme synthesis, in addition to providing an overview of clinical disorders associated with nutritional deficiency of riboflavin and primary defects of riboflavin transport. Finally, we give a brief overview of disorders of the cellular flavoproteome. Because riboflavin therapy may be beneficial in a number of primary or secondary disorders of the cellular flavoproteome, early recognition and prompt management of these disorders is imperative.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Redes e Vias Metabólicas/genética , Nucleotidiltransferases/deficiência , Riboflavina/metabolismo , Animais , Transporte Biológico/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Homeostase , Humanos , Proteínas de Membrana Transportadoras/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismoRESUMO
Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.
Assuntos
Núcleo Celular/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotidiltransferases/metabolismo , Reparo de DNA por Recombinação , Transporte Ativo do Núcleo Celular , Adulto , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/enzimologia , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Nucleotidiltransferases/deficiência , Fosforilação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Ligação Proteica/efeitos dos fármacos , Reparo de DNA por Recombinação/genética , Quinases da Família src/metabolismoRESUMO
Enterotoxigenic Escherichia coli (ETEC) is one of the most common causes of diarrheal illness in third world countries and it especially affects children and travelers visiting these regions. ETEC causes disease by adhering tightly to the epithelial cells in a concerted effort by adhesins, flagella, and other virulence-factors. When attached ETEC secretes toxins targeting the small intestine host-cells, which ultimately leads to osmotic diarrhea. HldE is a bifunctional protein that catalyzes the nucleotide-activated heptose precursors used in the biosynthesis of lipopolysaccharide (LPS) and in post-translational protein glycosylation. Both mechanisms have been linked to ETEC virulence: Lipopolysaccharide (LPS) is a major component of the bacterial outer membrane and is needed for transport of heat-labile toxins to the host cells, and ETEC glycoproteins have been shown to play an important role for bacterial adhesion to host epithelia. Here, we report that HldE plays an important role for ETEC virulence. Deletion of hldE resulted in markedly reduced binding to the human intestinal cells due to reduced expression of colonization factor CFA/I on the bacterial surface. Deletion of hldE also affected ETEC motility in a flagella-dependent fashion. Expression of both colonization factors and flagella was inhibited at the level of transcription. In addition, the hldE mutant displayed altered growth, increased biofilm formation and clumping in minimal growth medium. Investigation of an orthogonal LPS-deficient mutant combined with mass spectrometric analysis of protein glycosylation indicated that HldE exerts its role on ETEC virulence both through protein glycosylation and correct LPS configuration. These results place HldE as an attractive target for the development of future antimicrobial therapeutics.
Assuntos
Escherichia coli Enterotoxigênica/patogenicidade , Complexos Multienzimáticos/metabolismo , Nucleotidiltransferases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fatores de Virulência/metabolismo , Aderência Bacteriana , Células CACO-2 , Escherichia coli Enterotoxigênica/fisiologia , Células Epiteliais/microbiologia , Proteínas de Fímbrias/metabolismo , Deleção de Genes , Humanos , Locomoção , Complexos Multienzimáticos/deficiência , Nucleotidiltransferases/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/deficiênciaRESUMO
Mycobacterium tuberculosis (Mtb) infection remains a major public health concern. The STING (stimulator of interferon genes) pathway contributes to the cytosolic surveillance of host cells. Most studies on the role of STING activation in Mtb infection have focused on macrophages. Moreover, a detailed investigation of the role of STING during Mtb infection in vivo is required. Here, we deciphered the involvement of STING in the activation of dendritic cells (DCs) and the host response to Mtb infection in vivo. In DCs, this adaptor molecule was important for Ifn-ß expression and IL-12 production as well as for the surface expression of the activation markers CD40 and CD86. We also documented that Mtb DNA induces STING activation in murine fibroblasts. In vivo Mtb aerogenic infection induced the upregulation of the STING and cGAS (cyclic GMP-AMP synthase) genes, and Ifn-ß pulmonary expression was dependent on both sensors. However, mice deficient for STING or cGAS presented a similar outcome to wild-type controls, with no major alterations in body weight gain, bacterial burden, or survival. Lung inflammation, proinflammatory cytokine production, and inflammatory cell recruitment were similar in STING- and cGAS-deficient mice compared to wild-type controls. In summary, although the STING pathway seems to be crucial for DC activation during Mtb infection, it is dispensable for host protection in vivo.
Assuntos
Células Dendríticas/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/fisiologia , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Tuberculose/microbiologia , Animais , Células Cultivadas , Citocinas/metabolismo , Citosol/metabolismo , Células Dendríticas/microbiologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/microbiologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Masculino , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium tuberculosis/genética , Nucleotidiltransferases/deficiência , Tuberculose/metabolismo , Tuberculose/patologia , Regulação para Cima/genéticaRESUMO
Nucleic acids, which constitute the genetic material of all organisms, are continuously exposed to endogenous and exogenous damaging agents, representing a significant challenge to genome stability and genome integrity over the life of a cell or organism. Unrepaired DNA lesions, such as single- and double-stranded DNA breaks (SSBs and DSBs), and single-stranded gaps can block progression of the DNA replication fork, causing replicative stress and/or cell cycle arrest. However, translesion synthesis (TLS) DNA polymerases, such as Rev1, have the ability to bypass some DNA lesions, which can circumvent the process leading to replication fork arrest and minimize replicative stress. Here, we show that Rev1-deficiency in mouse embryo fibroblasts or mouse liver tissue is associated with replicative stress and mitochondrial dysfunction. In addition, Rev1-deficiency is associated with high poly(ADP) ribose polymerase 1 (PARP1) activity, low endogenous NAD+, low expression of SIRT1 and PGC1α and low adenosine monophosphate (AMP)-activated kinase (AMPK) activity. We conclude that replication stress via Rev1-deficiency contributes to metabolic stress caused by compromized mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.
Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias Hepáticas/genética , Nucleotidiltransferases/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Poli(ADP-Ribose) Polimerases/genética , Sirtuína 1/genética , Animais , DNA Polimerase Dirigida por DNA , Embrião de Mamíferos , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação da Expressão Gênica , Fígado/enzimologia , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Knockout , Mitocôndrias Hepáticas/enzimologia , NAD/metabolismo , Nucleotidiltransferases/deficiência , Fosforilação Oxidativa , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Cultura Primária de Células , Transdução de Sinais , Sirtuína 1/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Alcohol abuse impairs immune defense. To study the effect of chronic-plus-binge alcohol exposure on the granulopoietic response, acute alcohol intoxication (intraperitoneal injection of 5 g alcohol/kg body weight) was introduced to mice chronically fed on the Lieber-DeCarli low-fat liquid alcohol diet for 5 weeks. Bacteremia was induced by intravenous injection of Escherichia coli Bacteremia caused a remarkable increase in marrow lin- c-kit+ Sca-1+ cells. Activation of cell proliferation supported the increase in marrow lin- c-kit+ Sca-1+ cells. Alcohol administration inhibited this activation of lin- c-kit+ Sca-1+ cells. The bone marrow of pair-fed control mice receiving intraperitoneal saline stored a large number of mature granulocytes expressing a high level of Gr1 (Gr1hi cells). The proportion of Gr1hi cells and the total number of Gr1+ cells were markedly reduced in the bone marrow, along with an increase in the ratio of Gr1+ granulocytes in peripheral white blood cells following bacteremia. E. coli infection stimulated proliferation of granulopoietic precursor cells, resulting in a marked increase in the ratio of immature Gr1lo cells in the bone marrow. Alcohol administration itself triggered marrow release of Gr1+ cells, resulting in reduction of the marrow granulocyte reserve with an elevation of granulocytes in the circulation. Alcohol also impaired activation of granulopoietic precursor proliferation following bacteremia. Alcohol disrupted lipopolysaccharide (LPS)-TLR4-ERK1/2-cyclin D1 signaling and inhibited upregulation of Sca-1 and C/EBPß expression by lineage-negative marrow cells in response to bacteremia. These results indicate that chronic-plus-binge alcohol exposure inhibits the granulopoietic response by disrupting key cell signaling for hematopoietic precursor cell activation and commitment to granulocyte lineage development.
Assuntos
Bacteriemia/imunologia , Consumo Excessivo de Bebidas Alcoólicas/imunologia , Infecções por Escherichia coli/imunologia , Etanol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Bacteriemia/genética , Bacteriemia/patologia , Consumo Excessivo de Bebidas Alcoólicas/genética , Consumo Excessivo de Bebidas Alcoólicas/patologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/imunologia , Ciclina D1/genética , Ciclina D1/imunologia , Modelos Animais de Doenças , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/imunologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/patologia , Regulação da Expressão Gênica/imunologia , Granulócitos/efeitos dos fármacos , Granulócitos/imunologia , Granulócitos/patologia , Hematopoese/genética , Hematopoese/imunologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Nucleotidiltransferases/deficiência , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
A fundamental principle in biology is that the program for early development is established during oogenesis in the form of the maternal transcriptome. How the maternal transcriptome acquires the appropriate content and dosage of transcripts is not fully understood. Here we show that 3' terminal uridylation of mRNA mediated by TUT4 and TUT7 sculpts the mouse maternal transcriptome by eliminating transcripts during oocyte growth. Uridylation mediated by TUT4 and TUT7 is essential for both oocyte maturation and fertility. In comparison to somatic cells, the oocyte transcriptome has a shorter poly(A) tail and a higher relative proportion of terminal oligo-uridylation. Deletion of TUT4 and TUT7 leads to the accumulation of a cohort of transcripts with a high frequency of very short poly(A) tails, and a loss of 3' oligo-uridylation. By contrast, deficiency of TUT4 and TUT7 does not alter gene expression in a variety of somatic cells. In summary, we show that poly(A) tail length and 3' terminal uridylation have essential and specific functions in shaping a functional maternal transcriptome.