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1.
Signal Transduct Target Ther ; 6(1): 382, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732709

RESUMO

The global coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense RNA virus. How the host immune system senses and responds to SARS-CoV-2 infection remain largely unresolved. Here, we report that SARS-CoV-2 infection activates the innate immune response through the cytosolic DNA sensing cGAS-STING pathway. SARS-CoV-2 infection induces the cellular level of 2'3'-cGAMP associated with STING activation. cGAS recognizes chromatin DNA shuttled from the nucleus as a result of cell-to-cell fusion upon SARS-CoV-2 infection. We further demonstrate that the expression of spike protein from SARS-CoV-2 and ACE2 from host cells is sufficient to trigger cytoplasmic chromatin upon cell fusion. Furthermore, cytoplasmic chromatin-cGAS-STING pathway, but not MAVS-mediated viral RNA sensing pathway, contributes to interferon and pro-inflammatory gene expression upon cell fusion. Finally, we show that cGAS is required for host antiviral responses against SARS-CoV-2, and a STING-activating compound potently inhibits viral replication. Together, our study reported a previously unappreciated mechanism by which the host innate immune system responds to SARS-CoV-2 infection, mediated by cytoplasmic chromatin from the infected cells. Targeting the cytoplasmic chromatin-cGAS-STING pathway may offer novel therapeutic opportunities in treating COVID-19. In addition, these findings extend our knowledge in host defense against viral infection by showing that host cells' self-nucleic acids can be employed as a "danger signal" to alarm the immune system.


Assuntos
COVID-19/imunologia , Cromatina/imunologia , Citoplasma/imunologia , Imunidade Inata , Nucleotidiltransferases/imunologia , SARS-CoV-2/imunologia , Animais , COVID-19/genética , Cromatina/genética , Citoplasma/genética , Modelos Animais de Doenças , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Transgênicos , Nucleotidiltransferases/genética , SARS-CoV-2/genética
2.
Rejuvenation Res ; 24(5): 397-402, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34694148

RESUMO

Numerous studies demonstrate a global decrease in nicotinamide adenine dinucleotide (NAD+) with aging. This decline is associated with the development of several of the hallmarks of aging such as reduced mitophagy and neuroinflammation, processes thought to play a significant role in the progression of Alzheimer's disease (AD). Augmentation of NAD+ by oral administration of a precursor, nicotinamide riboside (NR), reduces senescence of affected cells, attenuates DNA damage and neuroinflammation in the transgenic APP/PS1 murine model of AD. Inflammation mediated by microglial cells plays an important role in progression of AD and other neurodegenerative diseases. The cytoplasmic DNA sensor, cyclic GMP-AMP synthase (cGAS) and downstream stimulator of interferon genes (STING), generates an interferon signature characteristic of senescence and inflammaging in the brain of AD mice. Elevated cGAS-STING observed in the AD mouse brains and human AD fibroblasts was normalized by NR. This intervention also increased mitophagy with improved cognition and behavior in the APP/PS1 mice. These studies suggest that modulation of the cGAS-STING pathway may benefit AD patients and possibly other disorders characterized by compromised mitophagy and excessive neuroinflammation.


Assuntos
Doença de Alzheimer , Animais , Humanos , Proteínas de Membrana , Camundongos , Niacinamida/análogos & derivados , Nucleotidiltransferases , Compostos de Piridínio
3.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3201-3210, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622628

RESUMO

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.


Assuntos
Circovirus , Interferon Tipo I , Macrófagos Alveolares , Transdução de Sinais , Animais , Células Cultivadas , Interferon Tipo I/genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Suínos
4.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(10): 977-980, 2021 Oct 10.
Artigo em Chinês | MEDLINE | ID: mdl-34625936

RESUMO

OBJECTIVE: To detect variant of TRNT1 gene in a child featuring sideroblastic anemia with B-cell immunodeficiency, periodic fever and developmental delay (SIFD). METHODS: The proband and his parents were analyzed through trio-whole exome sequencing. Sanger sequencing and bioinformatic analysis were carried out to verify the candidate variant sites associated with the clinical phenotype. RESULTS: Genetic testing showed that the proband has carried compound heterozygous variants of the TRNT1 gene, namely c.88A>G(p.Met30Val) and c.363G>T(p.Glu121Asp). Sanger sequencing confirmed that the variants were respectively inherited from his father and mother. The variants were unreported previously. By bioinformatic analysis, both variants were predicted to affect the stability of binding of the TRNT1 protein with tRNA. Based on the American College of Medical Genetics and Genomics standards and guidelines, c.88A>G and c.363G>T variants of TRNT1 gene were predicted to be uncertain significance (PM2+PP3+PP4) and likely pathogenic (PM1+PM2+PP3+PP4), respectively. CONCLUSION: The c.88A>G (p.Met30Val) and c.363G>T(p.Glu121Asp) compound heterozygous variants of the TRNT1 gene probably underlay the disease in this patient. Above finding has enriched the spectrum of TRNT1 gene variants.


Assuntos
Testes Genéticos , Nucleotidiltransferases , Humanos
5.
Biol Direct ; 16(1): 20, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674770

RESUMO

SARS-CoV-2 infection could cause severe acute respiratory syndrome, largely attributed to dysregulated immune activation and extensive lung tissue damage. However, the underlying mechanisms are not fully understood. Here, we reported that viral infection could induce syncytia formation within cells expressing ACE2 and the SARS-CoV-2 spike protein, leading to the production of micronuclei with an average rate of about 4 per syncytium (> 93%). Remarkably, these micronuclei were manifested with a high level of activation of both DNA damage response and cGAS-STING signaling, as indicated by micronucleus translocation of γH2Ax and cGAS, and upregulation of their respective downstream target genes. Since activation of these signaling pathways were known to be associated with cellular catastrophe and aberrant immune activation, these findings help explain the pathological effects of SARS-CoV-2 infection at cellular and molecular levels, and provide novel potential targets for COVID-19 therapy.


Assuntos
COVID-19/genética , COVID-19/metabolismo , Dano ao DNA , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , SARS-CoV-2/patogenicidade , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/virologia , Células Gigantes/metabolismo , Células Gigantes/virologia , Células HeLa , Humanos , Testes para Micronúcleos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transdução de Sinais , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
PLoS Pathog ; 17(10): e1009841, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34648591

RESUMO

In general, in mammalian cells, cytosolic DNA viruses are sensed by cyclic GMP-AMP synthase (cGAS), and RNA viruses are recognized by retinoic acid-inducible gene I (RIG-I)-like receptors, triggering a series of downstream innate antiviral signaling steps in the host. We previously reported that measles virus (MeV), which possesses an RNA genome, induces rapid antiviral responses, followed by comprehensive downregulation of host gene expression in epithelial cells. Interestingly, gene ontology analysis indicated that genes encoding mitochondrial proteins are enriched among the list of downregulated genes. To evaluate mitochondrial stress after MeV infection, we first observed the mitochondrial morphology of infected cells and found that significantly elongated mitochondrial networks with a hyperfused phenotype were formed. In addition, an increased amount of mitochondrial DNA (mtDNA) in the cytosol was detected during progression of infection. Based on these results, we show that cytosolic mtDNA released from hyperfused mitochondria during MeV infection is captured by cGAS and causes consequent priming of the DNA sensing pathway in addition to canonical RNA sensing. We also ascertained the contribution of cGAS to the in vivo pathogenicity of MeV. In addition, we found that other viruses that induce downregulation of mitochondrial biogenesis as seen for MeV cause similar mitochondrial hyperfusion and cytosolic mtDNA-priming antiviral responses. These findings indicate that the mtDNA-activated cGAS pathway is critical for full innate control of certain viruses, including RNA viruses that cause mitochondrial stress.


Assuntos
Imunidade Inata/imunologia , Sarampo/metabolismo , Mitocôndrias/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Regulação para Baixo , Humanos , Vírus do Sarampo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/virologia , Biogênese de Organelas , Infecções por Vírus de RNA/metabolismo , Vírus de RNA
7.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576144

RESUMO

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3'-UTR processing, we observed a high association of Star-PAP at the 3'-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3'-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.


Assuntos
Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Regulação para Baixo/genética , Genoma Humano , Células HEK293 , Meia-Vida , Humanos , Modelos Biológicos , Ligação Proteica , Processamento Pós-Transcricional do RNA/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , Transdução de Sinais , Transcriptoma/genética , Regulação para Cima/genética
8.
J Immunol ; 207(8): 2155-2166, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34526378

RESUMO

Cytosolic DNA from pathogens activates the DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) that produces the second messenger, cGAMP. cGAMP triggers a signal cascade leading to type I IFN expression. Host DNA is normally restricted in the cellular compartments of the nucleus and mitochondria. Recent studies have shown that DNA virus infection triggers mitochondrial stress, leading to the release of mitochondrial DNA to the cytosol and activation of cGAS; however, the regulatory mechanism of mitochondrial DNA-mediated cGAS activation is not well elucidated. In this study, we analyzed cGAS protein interactome in mouse RAW264.7 macrophages and found that cGAS interacted with C1QBP. C1QBP predominantly localized in the mitochondria and leaked into the cytosol during DNA virus infection. The leaked C1QBP bound the NTase domain of cGAS and inhibited cGAS enzymatic activity in cells and in vitro. Overexpression of the cytosolic form of C1QBP inhibited cytosolic DNA-elicited innate immune responses and promoted HSV-1 infection. By contrast, deficiency of C1QBP led to the elevated innate immune responses and impaired HSV-1 infection. Taken together, our study suggests that C1QBP is a novel cGAS inhibitor hidden in the mitochondria.


Assuntos
DNA , Nucleotidiltransferases , Animais , Citosol/metabolismo , DNA/metabolismo , Imunidade Inata , Camundongos , Mitocôndrias , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo
9.
Biochem Biophys Res Commun ; 577: 38-44, 2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34507063

RESUMO

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/metabolismo
10.
PLoS Pathog ; 17(9): e1009940, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34543359

RESUMO

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.


Assuntos
Infecções por Circoviridae/metabolismo , Circovirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Infecções por Circoviridae/imunologia , Circovirus/imunologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/metabolismo , Células HEK293 , Humanos , Interferon Tipo I/imunologia , Nucleotidiltransferases/imunologia , Suínos , Doenças dos Suínos/virologia
11.
J Hematol Oncol ; 14(1): 152, 2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34556152

RESUMO

Chimeric antigen receptor T-cell (CAR-T) therapy has shown tremendous success in eradicating hematologic malignancies. However, this success has not yet been extrapolated to solid tumors due to the limited infiltration and persistence of CAR-T cells in the tumor microenvironment (TME). In this study, we screened a novel anti-CD70 scFv and generated CD70 CAR-T cells that showed effective antitumor functions against CD70+ renal carcinoma cells (RCCs) both in vitro and in vivo. We further evaluated the effect and explored the molecular mechanism of a PARP inhibitor (PARPi) in CAR-T cell immunotherapy by administering the PARPi to mouse xenografts model derived from human RCC cells. Treatment with the PARPi promoted CAR-T cell infiltration by stimulating a chemokine milieu that promoted CAR-T cell recruitment and the modulation of immunosuppression in the TME. Moreover, our data demonstrate that PARPi modulates the TME by activating the cGAS-STING pathway, thereby altering the balance of immunostimulatory signaling and enabling low-dose CAR-T cell treatment to induce effective tumor regression. These data demonstrate the application of CD70 CAR-T cell therapeutic strategies for RCC and the cross-talk between targeting DNA damage responses and antitumor CAR-T cell therapy. These findings provide insight into the mechanisms of PARPis in CAR-T cell therapy for RCC and suggest a promising adjuvant therapeutic strategy for CAR-T cell therapy in solid tumors.


Assuntos
Ligante CD27/antagonistas & inibidores , Carcinoma de Células Renais/terapia , Imunoterapia Adotiva/métodos , Neoplasias Renais/terapia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Animais , Ligante CD27/imunologia , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/imunologia , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/imunologia , Proteínas de Membrana/imunologia , Camundongos , Nucleotidiltransferases/imunologia , Transdução de Sinais
12.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34546869

RESUMO

Advances in proteomics have enabled a comprehensive understanding of host-pathogen interactions. Here we have characterized Japanese encephalitis virus (JEV) infection-driven changes in the mouse embryonic fibroblast (MEF) proteome. Through tandem mass tagging (TMT)-based mass spectrometry, we describe changes in 7.85 % of the identified proteome due to JEV infection. Pathway enrichment analysis showed that proteins involved in innate immune sensing, interferon responses and inflammation were the major upregulated group, along with the immunoproteasome and poly ADP-ribosylation proteins. Functional validation of several upregulated anti-viral innate immune proteins, including an active cGAS-STING axis, was performed. Through siRNA depletion, we describe a crucial role of the DNA sensor cGAS in restricting JEV replication. Further, many interferon-stimulated genes (ISGs) were observed to be induced in infected cells. We also observed activation of TLR2 and inhibition of TLR2 signalling using TLR1/2 inhibitor CU-CPT22-blocked production of inflammatory cytokines IL6 and TNF-α from virus-infected N9 microglial cells. The major proteins that were downregulated by infection were involved in cell adhesion (collagens), transport (solute carrier and ATP-binding cassette transporters), sterol and lipid biosynthesis. Several collagens were found to be transcriptionally downregulated in infected MEFs and mouse brain. Collectively, our data provide a bird's-eye view into how fibroblast protein composition is rewired following JEV infection.


Assuntos
Vírus da Encefalite Japonesa (Espécie)/fisiologia , Encefalite Japonesa/metabolismo , Encefalite Japonesa/virologia , Fibroblastos/metabolismo , Fibroblastos/virologia , Proteoma , Animais , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Colágeno/genética , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Encefalite Japonesa/genética , Encefalite Japonesa/imunologia , Fibroblastos/imunologia , Interações Hospedeiro-Patógeno , Imunidade Inata/genética , Inflamação , Interferons/imunologia , Metabolismo dos Lipídeos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Proteínas/metabolismo , Proteômica , Transdução de Sinais , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Regulação para Cima
13.
Am J Physiol Lung Cell Mol Physiol ; 321(5): L859-L871, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34524912

RESUMO

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5-7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.


Assuntos
Células Epiteliais Alveolares/patologia , Senescência Celular/fisiologia , Dano ao DNA/genética , Fibrose Pulmonar Idiopática/patologia , Nucleotidiltransferases/metabolismo , Células A549 , Benzofuranos/farmacologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocinas/biossíntese , DNA Mitocondrial/metabolismo , Desoxirribonuclease I/farmacologia , Molécula de Adesão da Célula Epitelial/metabolismo , Etoposídeo/farmacologia , Humanos , Mitocôndrias/genética , Mitocôndrias/patologia , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia
14.
Yi Chuan ; 43(8): 802-812, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34413019

RESUMO

The genetically modified (GM) maize 'Shuangkang'12-5 has good insect resistance and herbicide tolerance, which is one of the first series of GM maizes obtained a safety certificate in China, and it has broad application prospect in the future. This study established an on-site rapid detection method for GM 'Shuangkang'12-5 based on recombinase polymerase amplification (RPA) technology, which primes and probe were designed according to the specific flank sequence. Then the best combination of primers and probe was obtained through a screeing process. The amplification results of fluorescence RPA can be directly visualized under blue light. The results showed that the visual detection system of GM 'Shuangkang'12-5 with high specificity, and the detection sensitivity of the method could reached 10 copies. Further research found that the RPA amplification system had a wide range of temperature (34℃-46℃). According to this property, the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system. Finally, we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM 'Shuangkang'12-5, and compared the results with the detection results of qPCR. The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR. Moreover, the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish. The rapid on-site visual detection method for GM 'Shuangkang' 12-5 established in this study has high specificity, high sensitivity and convenience. It not only meets the needs of on-site rapid detection of GM 'Shuangkang'12-5, but also provides highlight for the development of other on-site rapid detection methods.


Assuntos
Recombinases , Zea mays , Primers do DNA , Técnicas de Amplificação de Ácido Nucleico , Nucleotidiltransferases , Zea mays/genética
16.
Arch Biochem Biophys ; 710: 109001, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352244

RESUMO

The enzyme cGAS functions as a sensor that recognizes the cytosolic DNA from foreign pathogen. The activation of the protein triggers the transcription of inflammatory genes, leading into the establishment of an antipathogen state. An interesting new discovery is that the detection of DNA by cGAS induced the formation of liquid-like droplets. However how cells regulate the formation of these droplets is still not fully understood. In order to unravel the molecular mechanism beneath the DNA-mediated phase separation of cGAS, we developed a polymer-based coarse-grained model which takes into accounts the basic structural organization in DNA and cGAS, as well as the binding properties between these biomolecules. This model was further integrated into a hybrid simulation algorithm. With this computational method, a multi-step kinetic process of aggregation between cGAS and DNA was observed. Moreover, we systematically tested the model under different concentrations and binding parameters. Our simulation results show that phase separation requires both cGAS dimerization and protein-DNA interactions, whereas polymers can be kinetically trapped in small aggregates under strong binding affinities. Additionally, we demonstrated that supramolecular assembly can be facilitated by increasing the number of functional modules in protein or DNA polymers, suggesting that multivalency and intrinsic disordered regions play positive roles in regulating phase separation. This is consistent to previous experimental evidences. Taken together, this is, to the best of our knowledge, the first computational model to study condensation of cGAS-DNA complexes. While the method can reach the timescale beyond the capability of atomic-level MD simulations, it still includes information about spatial arrangement of functional modules in biopolymers that is missing in the mean-field theory. Our work thereby adds a useful dimension to a suite of existing experimental and computational techniques to study the dynamics of phase separation in biological systems.


Assuntos
DNA/química , DNA/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Algoritmos , Simulação por Computador , Humanos , Cinética , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Agregados Proteicos , Transdução de Sinais
17.
Nat Commun ; 12(1): 5004, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408154

RESUMO

The endoplasmic reticulum (ER) Hsp70 chaperone BiP is regulated by AMPylation, a reversible inactivating post-translational modification. Both BiP AMPylation and deAMPylation are catalysed by a single ER-localised enzyme, FICD. Here we present crystallographic and solution structures of a deAMPylation Michaelis complex formed between mammalian AMPylated BiP and FICD. The latter, via its tetratricopeptide repeat domain, binds a surface that is specific to ATP-state Hsp70 chaperones, explaining the exquisite selectivity of FICD for BiP's ATP-bound conformation both when AMPylating and deAMPylating Thr518. The eukaryotic deAMPylation mechanism thus revealed, rationalises the role of the conserved Fic domain Glu234 as a gatekeeper residue that both inhibits AMPylation and facilitates hydrolytic deAMPylation catalysed by dimeric FICD. These findings point to a monomerisation-induced increase in Glu234 flexibility as the basis of an oligomeric state-dependent switch between FICD's antagonistic activities, despite a similar mode of engagement of its two substrates - unmodified and AMPylated BiP.


Assuntos
Monofosfato de Adenosina/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Biocatálise , Dimerização , Proteínas de Choque Térmico/genética , Humanos , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Processamento de Proteína Pós-Traducional
19.
Nucleic Acids Res ; 49(16): 9389-9403, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34387695

RESUMO

Cyclic GMP-AMP synthase (cGAS) is a key DNA sensor that detects aberrant cytosolic DNA arising from pathogen invasions or genotoxic stresses. Upon binding to DNA, cGAS is activated and catalyzes the synthesis of cyclic GMP-AMP (cGAMP), which induces potent antimicrobial and antitumor responses. Kaposi sarcoma-associated herpesvirus (KSHV) is a human DNA tumor virus that causes Kaposi sarcoma and several other malignancies. We previously reported that KSHV inhibitor of cGAS (KicGAS) encoded by ORF52, inhibits cGAS enzymatic activity, but the underlying mechanisms remained unclear. To define the inhibitory mechanisms, here we performed in-depth biochemical and functional characterizations of KicGAS, and mapped its functional domains. We found KicGAS self-oligomerizes and binds to double stranded DNA cooperatively. This self-oligomerization is essential for its DNA binding and cGAS inhibition. Interestingly, KicGAS forms liquid droplets upon binding to DNA, which requires collective multivalent interactions with DNA mediated by both structured and disordered domains coordinated through the self-oligomerization of KicGAS. We also observed that KicGAS inhibits the DNA-induced phase separation and activation of cGAS. Our findings reveal a novel mechanism by which DNA viruses target the host protein phase separation for suppression of the host sensing of viral nucleic acids.


Assuntos
Herpesvirus Humano 8/genética , Interações Hospedeiro-Patógeno/genética , Nucleotidiltransferases/genética , Sarcoma de Kaposi/genética , Citosol/enzimologia , Citosol/microbiologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , DNA Viral/genética , Proteínas de Ligação a DNA/genética , Herpesvirus Humano 8/patogenicidade , Humanos , Evasão da Resposta Imune/efeitos dos fármacos , Imunidade Inata/genética , Nucleotídeos Cíclicos/genética , Nucleotidiltransferases/antagonistas & inibidores , Sarcoma de Kaposi/tratamento farmacológico , Sarcoma de Kaposi/virologia , Proteínas Virais/genética
20.
J Immunol ; 207(3): 784-798, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34290106

RESUMO

In mammals, cyclic GMP-AMP synthase (cGAS) recognizes cytosolic dsDNA to induce the type I IFN response. However, the functional role of cGAS in the IFN response of fish remains unclear or controversial. In this study, we report that cGAS orthologs from crucian carp Carassius auratus (CacGAS) and grass carp Ctenopharyngodon idellus (CicGAS) target the dsRNA sensor retinoic acid-inducible gene I (RIG-I) for negative regulation of the IFN response. First, poly(deoxyadenylic-deoxythymidylic) acid-, polyinosinic-polycytidylic acid-, and spring viremia of carp virus-induced IFN responses were impaired by overexpression of CacGAS and CicGAS. Then, CacGAS and CicGAS interacted with CiRIG-I and CiMAVS and inhibited CiRIG-I- and CiMAVS-mediated IFN induction. Moreover, the K63-linked ubiquitination of CiRIG-I and the interaction between CiRIG-I and CiMAVS were attenuated by CacGAS and CicGAS. Finally, CacGAS and CicGAS decreased CiRIG-I-mediated the cellular antiviral response and facilitated viral replication. Taken together, data in this study identify CacGAS and CicGAS as negative regulators in RIG-I-like receptor signaling, which extends the current knowledge regarding the role of fish cGAS in the innate antiviral response.


Assuntos
Proteínas de Peixes/genética , Interferon Tipo I/metabolismo , Nucleotidiltransferases/genética , Infecções por Rhabdoviridae/imunologia , Rhabdoviridae/fisiologia , Animais , Carpas , Cyprinidae , Proteínas de Peixes/imunologia , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Carpa Dourada , Células HEK293 , Humanos , Imunidade Inata/genética , Nucleotidiltransferases/imunologia , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Ubiquitinação , Proteínas de Peixe-Zebra/genética
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