Mutant p53R211* specifically suppresses Th17 cell population and ameliorates the arthritic condition in AIA rat via direct binding of TBK1 and inhibition of TBK1-IRF3-STING signaling cascade
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
PROVIDER: GSE221158 | GEO | 2023/11/22
REPOSITORIES: GEO
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Project description:Mutant p53R211* specifically suppresses Th17 cell population and ameliorates the arthritic condition in AIA rat via direct binding of TBK1 and inhibition of TBK1-IRF3-STING signaling cascade
| PRJNA913129 | ENA
Project description:Cytosolic double-stranded DNA (dsDNA) stimulates the production of type I interferon (IFN) through the endoplasmic reticulum (ER)-resident adaptor protein STING (stimulator of IFN genes), which activates the transcription factor interferon regulatory factor 3 (IRF3); however, how STING activates IRF3 is unclear. Here, we showed that STING stimulates phosphorylation of IRF3 by the kinase TBK1 (TANK-binding kinase 1) in an in vitro reconstitution system. With this system, we identified a carboxyl-terminal region of STING that was both necessary and sufficient to activate TBK1 and stimulate the phosphorylation of IRF3. We also found that STING interacted with both TBK1 and IRF3 and that mutations in STING that selectively disrupted its binding to IRF3 abrogated phosphorylation of IRF3 without impairing the activation of TBK1. These results suggest that STING functions as a scaffold protein to specify and promote the phosphorylation of IRF3 by TBK1. This scaffolding function of STING (and possibly of other adaptor proteins) may explain why IRF3 is activated in only a subset of signaling pathways that activate TBK1.
| S-EPMC3549669 | biostudies-literature
Project description:Cyclic GMP-AMP synthase (cGAS) is an important cytosolic DNA sensor that plays a crucial role in triggering STING-dependent signal and inducing type I interferons (IFNs). cGAS is important for intracellular bacterial recognition and innate immune responses. However, the regulating effect of the cGAS pathway for bone marrow-derived dendritic cells (BMDCs) during Mycobacterium bovis (M. bovis) infection is still unknown. We hypothesized that the maturation and activation of BMDCs were modulated by the cGAS/STING/TBK1/IRF3 signaling pathway. In this study, we found that M. bovis promoted phenotypic maturation and functional activation of BMDCs via the cGAS signaling pathway, with the type I IFN and its receptor (IFNAR) contributing. Additionally, we showed that the type I IFN pathway promoted CD4⁺ T cells' proliferation with BMDC during M. bovis infection. Meanwhile, the related cytokines increased the expression involved in this signaling pathway. These data highlight the mechanism of the cGAS and type I IFN pathway in regulating the maturation and activation of BMDCs, emphasizing the important role of this signaling pathway and BMDCs against M. bovis. This study provides new insight into the interaction between cGAS and dendritic cells (DCs), which could be considered in the development of new drugs and vaccines against tuberculosis.
| S-EPMC6412216 | biostudies-literature
Project description:BackgroundAn important signaling pathway connecting illness and natural immunity is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but aberrant activation of this pathway is associated with the development of autoimmune and inflammatory diseases. Hence, targeted inhibition of the activation of the cGAS-STING pathway is potentially valuable in the treatment of disease. The primary active component of Salvia miltiorrhiza is total tanshinone (TTN). Research has indicated that TTN possesses noteworthy anti-inflammatory properties. However, the protective mechanism of TTN against acute liver injury (ALI) and autoimmune diseases is unknown.MethodsA model of aberrant activation of the cGAS-STING pathway was established in various cells and treated with TTN, and the expression of cGAS-STING pathway-related proteins, type I interferon, interferon stimulated genes and inflammatory factors was assessed by western blotting, real-time qPCR. Immunofluorescence analysis of the effect of TTN on the entry of associated proteins into the nucleus following aberrant activation of the cGAS-STING pathway. The effect of TTN on STING oligomerisation was investigated using 2'-3'-cyclic GMP-AMP (2',3'-cGAMP) to induce STING oligomerisation. Western blotting was used to examine the impact of TTN on the interactions of STING, tank-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) after HA or Flag-labelled plasmids were transfected into HEK-293 T cells. A dimethylxanthenone-4-acetic acid (DMXAA) -induced activation model of the cGAS-STING pathway in mice was established to study the effect of TTN on aberrant activation of the cGAS-STING pathway in vivo. On the other hand, an animal model of lipopolysaccharide/D-galactosamine (LPS/D-GaIN)-induced ALI and an autoimmune disease model induced by trex1 knockout were established to study the effects of TTN on inflammatory and autoimmune diseases mediated by the cGAS-STING pathway in vivo.ResultsIn several models of aberrant activation of the cGAS-STING pathway, TTN significantly inhibited the phosphorylation of STING and IRF3, thereby suppressing the expression of type I interferon, interferon-stimulated genes and inflammatory factors. Additionally, TTN prevented P65 and IRF3 from entering the nucleus after the cGAS-STING signalling pathway was abnormally activated. Subsequent research indicated that TTN was not involved in the oligomerization of STING or the integration of STING-TBK1 and TBK1-IRF3. However, TTN was found to have a substantial effect on the binding process between STING and IRF3. On the other hand, DMXAA-induced STING activation and activation of downstream signalling in vivo are inhibited by TTN. Furthermore, TTN exhibits positive treatment effects on autoimmune diseases caused by deficiency of trex1 and LPS/D-GaIN-induced ALI.ConclusionOur research indicates that TTN effectively treats ALI and autoimmune illnesses mediated by the cGAS-STING pathway by inhibiting the abnormal activation of this pathway.
| S-EPMC11325629 | biostudies-literature
Project description:Chromosomal instability (CIN) in cancer cells has been reported to activate the cGAS-STING innate immunity pathway via micronuclei formation, thus affecting tumor immunity and tumor progression. However, adverse effects of the cGAS/STING pathway as they relate to CIN have not yet been investigated. We addressed this issue using knockdown and add-back approaches to analyze each component of the cGAS/STING/TBK1/IRF3 pathway, and we monitored the extent of CIN by measuring micronuclei formation after release from nocodazole-induced mitotic arrest. Interestingly, knockdown of cGAS (cyclic GMP-AMP synthase) along with induction of mitotic arrest in HeLa and U2OS cancer cells clearly resulted in increased micronuclei formation and chromosome missegregation. Knockdown of STING (stimulator of interferon genes), TBK1 (TANK-binding kinase-1), or IRF3 (interferon regulatory factor-3) also resulted in increased micronuclei formation. Moreover, transfection with cGAMP, the product of cGAS enzymatic activity, as well as add-back of cGAS WT (but not catalytic-dead mutant cGAS), or WT or constitutively active STING (but not an inactive STING mutant) rescued the micronuclei phenotype, demonstrating that all components of the cGAS/STING/TBK1/IRF3 pathway play a role in preventing CIN. Moreover, p21 levels were decreased in cGAS-, STING-, TBK1-, and IRF3-knockdown cells, which was accompanied by the precocious G2/M transition of cells and the enhanced micronuclei phenotype. Overexpression of p21 or inhibition of CDK1 in cGAS-depleted cells reduced micronuclei formation and abrogated the precocious G2/M transition, indicating that the decrease in p21 and the subsequent precocious G2/M transition is the main mechanism underlying the induction of CIN through disruption of cGAS/STING signaling.
| S-EPMC7210884 | biostudies-literature
Project description:TANK-binding kinase 1 (TBK1) is a key kinase in regulating antiviral innate immune responses. While the oligomerization of TBK1 is critical for its full activation, the molecular mechanism of how TBK1 forms oligomers remains unclear. Here, we show that protein tyrosine kinase 2 beta (PTK2B) acts as a TBK1-interacting protein and regulates TBK1 oligomerization. Functional assays reveal that PTK2B depletion reduces antiviral signaling in mouse embryonic fibroblasts, macrophages and dendritic cells, and genetic experiments show that Ptk2b-deficient mice are more susceptible to viral infection than control mice. Mechanistically, we demonstrate that PTK2B directly phosphorylates residue Tyr591 of TBK1, which increases TBK1 oligomerization and activation. In addition, we find that PTK2B also interacts with the stimulator of interferon genes (STING) and can promote its oligomerization in a kinase-independent manner. Collectively, PTK2B enhances the oligomerization of TBK1 and STING via different mechanisms, subsequently regulating STING-TBK1 activation to ensure efficient antiviral innate immune responses.
| S-EPMC10663505 | biostudies-literature
Project description:Type I interferons (IFN-I), such as IFN-? and IFN-? are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-? than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-? in macrophages. Both bacteria induced IFN-? via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-? activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-? impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.
| S-EPMC5711412 | biostudies-literature
Project description:The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ∆CTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ∆CTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.
| S-EPMC8040795 | biostudies-literature
Project description:TANK-binding kinase 1 (TBK1) is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs) in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-? signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I) and mitochondria antiviral-signaling protein (MAVS). However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s) exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.
| S-EPMC5797597 | biostudies-literature
Project description:Viral infections trigger host innate immune responses, characterized by the production of type-I interferons (IFN) including IFN?. IFN? induces cellular antiviral defense mechanisms and thereby contributes to pathogen clearance. Accumulating evidence suggests that mitochondria constitute a crucial platform for the induction of antiviral immunity. Here we demonstrate that the mitochondrial protein phosphoglycerate mutase family member 5 (PGAM5) is important for the antiviral cellular response. Following challenge of HeLa cells with the dsRNA-analog poly(I:C), PGAM5 oligomers and high levels of PGAM5 were found in mitochondrial aggregates. Using immunoprecipitation, a direct interaction of PGAM5 with the mitochondrial antiviral-signaling protein (MAVS) was demonstrated. In addition, PGAM5 deficient cells showed diminished expression of IFN? and IFN? target genes as compared to WT cells. Moreover, PGAM5 deficient mouse embryonic fibroblasts (MEFs) exhibited decreased phosphorylation levels of IRF3 and TBK1 when challenged with poly(I:C) intracellularly. Finally, PGAM5 deficient MEFs, upon infection with vesicular stomatitis virus (VSV), revealed diminished IFN? expression and increased VSV replication. Collectively, our study highlights PGAM5 as an important regulator for IFN? production mediated via the TBK1/IRF3 signaling pathway in response to viral infection.
| S-EPMC7239892 | biostudies-literature