scholarly journals Implications of cholesterol and sphingomyelin in STING phosphorylation by TBK1

2021 ◽  
Author(s):  
Kanoko Takahashi ◽  
Takahiro Niki ◽  
Emari Ogawa ◽  
Kiku Fumika ◽  
Yu Nishioka ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Microsomal Membrane ◽  
Interferon Response ◽  
Type I ◽  
Phosphorylation Reaction ◽  
Reaction Proceeds ◽  
A Cell ◽  
Golgi Network ◽  
Microbial Dna

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3 (IRF3)-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout (KO) cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

scholarly journals A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kanoko Takahashi ◽  
Takahiro Niki ◽  
Emari Ogawa ◽  
Kiku Fumika ◽  
Yu Nishioka ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Microsomal Membrane ◽  
Interferon Response ◽  
Type I ◽  
Phosphorylation Reaction ◽  
A Cell ◽  
Golgi Network ◽  
Microbial Dna

AbstractStimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, “ER-to-Golgi” traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-β-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

scholarly journals Kinase activity of TBK1 is required for its binding to STING, but not for its recruitment to the Golgi

2021 ◽  
Author(s):  
Haruka Kemmoku ◽  
Yoshihiko Kuchitsu ◽  
Kojiro Mukai ◽  
Tomohiko Taguchi
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Cell System ◽  
Interferon Response ◽  
Type I ◽  
Double Knockout ◽  
Binding Interface ◽  
A Cell ◽  
Golgi Network

AbstractStimulator of interferon genes (STING) is an innate immune protein for DNA pathogens. In response to the emergence of DNA in the cytosol, STING relocates from the endoplasmic reticulum (ER) to the Golgi and induces the type I interferon response through cytosolic TANK-binding kinase 1 (TBK1). The molecular mechanism underlying TBK1 activation by STING remains poorly understood. Here we report a cell system by which STING and TBK1 are simultaneously monitored. The system utilizes STING/TBK1-double knockout (KO) cells, fluorescent protein-tagged TBK1 and STING, and super-resolution microscopy. After STING stimulation, TBK1 is directly recruited to the trans-Golgi network (TGN), not to the other parts of the Golgi. The recruitment of TBK1 does not require its kinase activity. C-terminal STING variants (ΔC9 and L373A), in which the TBK1-STING binding interface is mutated or deleted, induce the recruitment of TBK1. These results indicate that the kinase activity of TBK1 or the C-terminal motif of STING is not required for its recruitment to TGN, but rather for the formation of the stable STING signalling complex at TGN.

scholarly journals Development of Small-Molecule STING Activators for Cancer Immunotherapy

Biomedicines ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 33
Author(s):  
Hee Ra Jung ◽  
Seongman Jo ◽  
Min Jae Jeon ◽  
Hyelim Lee ◽  
Yeonjeong Chu ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
Cyclic Gmp ◽  
Therapeutic Potential ◽  
Interferon Response ◽  
Type I ◽  
Anti Cancer ◽  
Cancer Agent ◽  
Sting Pathway

In cancer immunotherapy, the cyclic GMP–AMP synthase–stimulator of interferon genes (STING) pathway is an attractive target for switching the tumor immunophenotype from ‘cold’ to ‘hot’ through the activation of the type I interferon response. To develop a new chemical entity for STING activator to improve cyclic GMP-AMP (cGAMP)-induced innate immune response, we identified KAS-08 via the structural modification of DW2282, which was previously reported as an anti-cancer agent with an unknown mechanism. Further investigation revealed that direct STING binding or the enhanced phosphorylation of STING and downstream effectors were responsible for DW2282-or KAS-08-mediated STING activity. Furthermore, KAS-08 was validated as an effective STING pathway activator in vitro and in vivo. The synergistic effect of cGAMP-mediated immunity and efficient anti-cancer effects successfully demonstrated the therapeutic potential of KAS-08 for combination therapy in cancer treatment.

scholarly journals Interferon Regulatory Factor IRF-7 Induces the Antiviral Alpha Interferon Response and Protects against Lethal West Nile Virus Infection

2008 ◽  
Vol 82 (17) ◽  
pp. 8465-8475 ◽  
Author(s):  
Stephane Daffis ◽  
Melanie A. Samuel ◽  
Mehul S. Suthar ◽  
Brian C. Keller ◽  
Michael Gale ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Cortical Neurons ◽  
Interferon Regulatory Factor ◽  
Interferon Response ◽  
Type I ◽  
Type I Ifn ◽  
Regulatory Factor ◽  
West Nile

ABSTRACT Type I interferon (IFN-α/β) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7−/− mice. Compared to congenic wild-type mice, IRF-7−/− mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-α gene expression and protein production were reduced and viral titers were increased in IRF-7−/− primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-β response remained largely intact. Our data suggest that the early protective IFN-α response against WNV occurs through an IRF-7-dependent transcriptional signal.

scholarly journals CX-5461 Treatment Leads to Cytosolic DNA-Mediated STING Activation in Ovarian Cancer

Cancers ◽  
2021 ◽  
Vol 13 (20) ◽  
pp. 5056
Author(s):  
Robert Cornelison ◽  
Kuntal Biswas ◽  
Danielle C. Llaneza ◽  
Alexandra R. Harris ◽  
Nisha G. Sosale ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Time Frame ◽  
Interferon Response ◽  
Type I ◽  
Pol I ◽  
Downstream Effects ◽  
Immune Pathway ◽  
Polymerase I

Epithelial ovarian cancer (EOC) is the deadliest of the gynecologic malignancies, with an overall survival rate of <30%. Recent research has suggested that targeting RNA polymerase I (POL I) with small-molecule inhibitors may be a viable therapeutic approach to combating EOC, even when chemoresistance is present. CX-5461 is one of the most promising POL I inhibitors currently being investigated, and previous reports have shown that CX-5461 treatment induces DNA damage response (DDR) through ATM/ATR kinase. Investigation into downstream effects of CX-5461 led us to uncovering a previously unreported phenotype. Treatment with CX-5461 induces a rapid accumulation of cytosolic DNA. This accumulation leads to transcriptional upregulation of ‘STimulator of Interferon Genes’ (STING) in the same time frame, phosphorylation of IRF3, and activation of type I interferon response both in vitro and in vivo. This activation is mediated and dependent on cyclic GMP–AMP synthase (cGAS). Here, we show THAT CX-5461 leads to an accumulation of cytosolic dsDNA and thereby activates the cGAS–STING–TBK1–IRF3 innate immune pathway, which induces type I IFN. CX-5461 treatment-mediated immune activation may be a powerful mechanism of action to exploit, leading to novel drug combinations with a chance of increasing immunotherapy efficacy, possibly with some cancer specificity limiting deleterious toxicities.

scholarly journals Targeted Delivery of Chloroquine to Plasmacytoid Dendritic Cells Enhances Inhibition of the Type I Interferon Response

2021 ◽  
Author(s):  
Marilyn E Allen ◽  
Amit Golding ◽  
Violeta Rus ◽  
Nicholas B Karabin ◽  
Sophia Li ◽  
...  
Keyword(s):  
Lupus Erythematosus ◽  
Targeted Delivery ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Interferon Response ◽  
Type I ◽  
Healthy Human ◽  
Biodistribution Studies

Systemic lupus erythematosus (SLE) causes damaging inflammation in multiple organs via the accumulation of immune complexes. These complexes activate plasmacytoid DCs (pDCs) via TLR7 and TLR9, contributing to disease pathogenesis by driving secretion of inflammatory type I IFNs. Antimalarial drugs, such as chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE. However, they require ~3 months of continuous use before achieving therapeutic efficacy and can accumulate in the retinal pigment epithelium with chronic use resulting in retinopathy. We hypothesized that poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) filamentous nanocarriers, filomicelles (FMs) could improve drug activity and reduce toxicity by directly delivering CQ to pDCs via passive, morphology-based targeting. Healthy human PBMCs were treated with soluble CQ or CQ-loaded FMs, stimulated with TLR agonists or SLE patient sera, and type I IFN secretion was quantified via multi-subtype IFN-α ELISA and MX1 gene expression using real-time RT-qPCR. Our results showed that 50 µg CQ/mg FM decreased MX1 expression and IFN-α production after TLR activation with either synthetic nucleic acid agonists or immune complex rich sera from SLE patients. Cellular uptake and biodistribution studies showed that FMs preferentially accumulate in human pDCs in vitro and in tissues frequently damaged in SLE patients (i.e., liver and kidneys) while sparing the eye in vivo. These results showed that nanocarrier morphology enables drug delivery, and CQ-FMs may be equally effective and more targeted than soluble CQ at inhibiting SLE-relevant pathways.

scholarly journals Pro-apoptotic caspase deficiency reveals a cell-extrinsic mechanism of NK cell regulation

2021 ◽  
Author(s):  
Tayla M. Olsen ◽  
Wei Hong Tan ◽  
Arne C. Knudsen ◽  
Anthony Rongvaux
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Nk Cell ◽  
Apoptotic Cell ◽  
Type I ◽  
Dependent Manner ◽  
Apoptosis Pathway ◽  
A Cell

AbstractRegulated cell death is essential for the maintenance of cellular and tissue homeostasis. In the hematopoietic system, genetic defects in apoptotic cell death generally produce the accumulation of immune cells, inflammation and autoimmunity. In contrast, we found that genetic deletion of caspases of the mitochondrial apoptosis pathway reduces natural killer (NK) cell numbers and makes NK cells functionally defective in vivo and in vitro. Caspase deficiency results in constitutive activation of a type I interferon (IFN) response, due to leakage of mitochondrial DNA and activation of the cGAS/STING pathway. The NK cell defect in caspase-deficient mice is independent of the type I IFN response, but the phenotype is partially rescued by cGAS or STING deficiency. Finally, caspase deficiency alters NK cells in a cell-extrinsic manner. Type I IFNs and NK cells are two essential effectors of antiviral immunity, and our results demonstrate that they are both regulated in a caspase-dependent manner. Beyond caspase-deficient animals, our observations may have implications in infections that trigger mitochondrial stress and caspase-dependent cell death.

In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer and promotes vector clearance

Blood ◽  
2006 ◽  
Vol 109 (7) ◽  
pp. 2797-2805 ◽  
Author(s):  
Brian D. Brown ◽  
Giovanni Sitia ◽  
Andrea Annoni ◽  
Ehud Hauben ◽  
Lucia Sergi Sergi ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Transgene Expression ◽  
Lentiviral Vectors ◽  
Interferon Response ◽  
Type I ◽  
Nonparenchymal Cells ◽  
Antiviral Responses ◽  
Liver Gene

AbstractLiver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFNαβ response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFNαβ, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a cell-type–specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected.

scholarly journals Integrated Transcriptomic and Proteomic Analysis of Red Blood Cells from Rainbow Trout Challenged with VHSV Point Towards Novel Immunomodulant Targets

Vaccines ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 63 ◽  
Author(s):  
Nombela ◽  
Lopez-Lorigados ◽  
Salvador-Mira ◽  
Puente-Marin ◽  
Chico ◽  
...  
Keyword(s):  
Immune Response ◽  
Rainbow Trout ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Antigen Processing ◽  
Head Kidney ◽  
Interferon Response ◽  
Type I ◽  
Fish Viruses

Teleost red blood cells (RBCs) are nucleated and therefore can propagate cellular responses to exogenous stimuli. RBCs can mount an immune response against a variety of fish viruses, including the viral septicemia hemorrhagic virus (VHSV), which is one of the most prevalent fish viruses resulting in aquaculture losses. In this work, RBCs from blood and head kidney samples of rainbow trout challenged with VHSV were analyzed via transcriptomic and proteomic analyses. We detected an overrepresentation of differentially expressed genes (DEGs) related to the type I interferon response and signaling in RBCs from the head kidney and related to complement activation in RBCs from blood. Antigen processing and presentation of peptide antigen was overrepresented in RBCs from both tissues. DEGs shared by both tissues showed an opposite expression profile. In summary, this work has demonstrated that teleost RBCs can modulate the immune response during an in vivo viral infection, thus implicating RBCs as cell targets for the development of novel immunomodulants.

scholarly journals A Recombinant Adenovirus Expressing Ovine Interferon Tau Prevents Influenza Virus-Induced Lethality in Mice

2016 ◽  
Vol 90 (7) ◽  
pp. 3783-3788 ◽  
Author(s):  
V. Martín ◽  
E. Pascual ◽  
M. Avia ◽  
G. Rangel ◽  
A. de Molina ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Recombinant Adenovirus ◽  
Biologically Active ◽  
Interferon Response ◽  
Broad Host Range ◽  
Type I ◽  
Interferon Tau ◽  
Unique Type ◽  
Low Toxicity

Ovine interferon tau (IFN-τ) is a unique type I interferon with low toxicity and a broad host rangein vivo. We report the generation of a nonreplicative recombinant adenovirus expressing biologically active IFN-τ. Using the B6.A2G-Mx1 mouse model, we showed that single-dose intranasal administration of recombinant Ad5-IFN-τ can effectively prevent lethality and disease induced by highly virulent hv-PR8 influenza virus by activating the interferon response and preventing viral replication.

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