ESCRT-dependent STING degradation curtails steady-state and cGAMP-induced signaling
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Steven A. Carr
PROVIDER: MSV000090900 | MassIVE | Wed Dec 14 08:53:00 GMT 2022
REPOSITORIES: MassIVE
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Project description:STING transmits signals downstream of the cytosolic DNA sensor cGAS, leading to transcriptional up-regulation of cytokines. However, components of the STING signaling pathway, such as IRF3 and IFNAR1, are not essential for autoinflammatory disease in STING gain-of-function (SAVI) mice. Recent discoveries revealed that STING also functions as a proton channel that deacidifies the Golgi. Since pH impacts Golgi enzyme activity, protein maturation, and trafficking, we hypothesized that STING proton channel activity influences multiple Golgi functions. Here, we show that STING-mediated proton efflux non-transcriptionally regulates Golgi trafficking of protein cargos. This process requires the Golgi-associated protein ArfGAP2, a cell type-specific dual regulator of STING-mediated proton efflux and signaling. Deletion of ArfGAP2 in hematopoietic and endothelial cells markedly reduces STING-mediated cytokine and chemokine secretion, immune cell activation, and autoinflammatory pathology in SAVI mice. Thus, ArfGAP2 facilitates STING-mediated signaling and cytokine release in hematopoietic cells, significantly contributing to autoinflammatory disease pathogenesis.
2024-12-31 | GSE283416 | GEO
Project description:STING transmits signals downstream of the cytosolic DNA sensor cGAS, leading to transcriptional up-regulation of cytokines. However, components of the STING signaling pathway, such as IRF3 and IFNAR1, are not essential for autoinflammatory disease in STING gain-of-function (SAVI) mice. Recent discoveries revealed that STING also functions as a proton channel that deacidifies the Golgi. Since pH impacts Golgi enzyme activity, protein maturation, and trafficking, we hypothesized that STING proton channel activity influences multiple Golgi functions. Here, we show that STING-mediated proton efflux non-transcriptionally regulates Golgi trafficking of protein cargos. This process requires the Golgi-associated protein ArfGAP2, a cell type-specific dual regulator of STING-mediated proton efflux and signaling. Deletion of ArfGAP2 in hematopoietic and endothelial cells markedly reduces STING-mediated cytokine and chemokine secretion, immune cell activation, and autoinflammatory pathology in SAVI mice. Thus, ArfGAP2 facilitates STING-mediated signaling and cytokine release in hematopoietic cells, significantly contributing to autoinflammatory disease pathogenesis.
2024-12-31 | GSE283415 | GEO
Project description:STING transmits signals downstream of the cytosolic DNA sensor cGAS, leading to transcriptional up-regulation of cytokines. However, components of the STING signaling pathway, such as IRF3 and IFNAR1, are not essential for autoinflammatory disease in STING gain-of-function (SAVI) mice. Recent findings indicate that STING can also function as a proton channel that deacidifies the Golgi. Since pH impacts Golgi enzyme activity, protein maturation, and trafficking, we hypothesized that STING proton channel activity influences multiple Golgi functions. Here, we show that STING-mediated proton efflux non-transcriptionally regulates Golgi trafficking of protein cargos. This process requires the Golgi-associated protein ArfGAP2, a cell type-specific dual regulator of STING-mediated proton efflux and signaling. Deletion of ArfGAP2 in hematopoietic and endothelial cells markedly reduces STING-mediated cytokine and chemokine secretion, immune cell activation, and autoinflammatory pathology in SAVI mice. Thus, ArfGAP2 facilitates STING-mediated signaling and cytokine release in hematopoietic cells, significantly contributing to autoinflammatory disease pathogenesis.
2024-11-30 | PXD058416 | Pride
Project description:The cGAS-STING pathway forms a major component of the innate immune system. cGAS-STING signalling is induced by detection of either foreign (i.e. pathogenic) or mislocalised host double stranded (ds)DNA present within the cytosol. STING acts as the major signalling hub, where it controls activation of transcription factors IRF3 and NF-B for expression of type I interferons and inflammatory cytokines, respectively. Under resting conditions STING resides on the ER membrane. However, following activation STING traffics to the Golgi to initiate downstream signalling, and subsequently to endolysosomal regions for degradation and termination of signalling. However, while STING is known to be degraded by lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a mass spectrometry approach to assess phosphorylation changes in primary macrophages following STING activation. This identified a large number of phosphorylation events in proteins involved in intracellular transport, including vesicular trafficking. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We observed that while macrophages exhibit rapid degradation of STING (i.e., 4-6 h), basal STING protein levels return slowly (i.e., >24 h). Despite STING protein recovery, ultimately macrophages remain unresponsive to re-challenge with STING ligands. Using a combination of imaging and biochemical approaches we subsequently identify that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on endosomes, which facilitates the degradation of STING. Disrupting ESCRT recognition of STING via knockdown of the ESCRT-0 component HRS or inhibiting ESCRT function via overexpression of a dominant negative form of VPS4a enhances STING signalling and cytokine production. Therefore, we have characterised the mechanisms that controls effective termination of STING signalling. Dysregulation of STING localisation or its degradation has been implicated in several diseases including autoimmune, autoinflammatory and neuroinflammatory diseases, hence a clearer understanding of STING degradation is imperative for a better understanding of STING-dependent disease pathologies.
2023-05-05 | PXD037108 | Pride
Project description:STING is a key signaling hub in pathogen defense response. The understanding of STING turnover in lysosomes remains superficial. Here, combining APEX- and Tandem Affinity Purification-based unbiased mass spectrometry screens, we identified ESCRT proteins that interact with STING. Further experimental characterization shows that these ESCRT machineries are required for both termination of STING activity and STING degradation in both its activated and steady states, and that the required ESCRT proteins differ between mice and humans. Upon activation, STING interacts with ESCRT proteins, leading to its lysosomal degradation. Furthermore, STING degradation is ubiquitin-independent, and requires binding between the STING cytoplasmic loop region and the TSG101 SB domain. Mutations in this loop disrupt their interaction, inhibiting STING degradation and consequently maintaining STING activation. In vitro synthesized peptides mimicking the cytoplasmic loop can disrupt STING-TSG101 interaction, inhibiting its STING degradation and activity termination. Notably, pre-treatment with loop mimic peptides can also attenuate HSV-1 proliferation in vitro. This study describes a previously unrecognized ESCRT-dependent, ubiquitin-independent mechanism underlying STING degradation and activity termination and suggests the further exploration of synthetic peptides that mediate targeted disruption of ESCRT-STING interactions as a therapeutic strategy for treating infectious diseases via prolonged STING activation.
2023-06-27 | PXD043331 |
Project description:STING-mediated activation of TAK1 at the ER, which in turn induces STING phosphorylation and initiates STEEP-mediated STING trafficking. These findings shed light on an early signaling event in the regulation of STING exit from ER.
2023-09-05 | PXD045096 |
Project description:Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.
2023-01-24 | PXD039411 | Pride
Project description:Programmed cell suicide of infected bacteria, known as abortive infection (Abi), serves as a central immune defense strategy to prevent the spread of bacteriophage viruses and other invasive genetic elements across a population. Many Abi systems utilize bespoke cyclic nucleotide immune messengers generated upon infection to rapidly mobilize cognate death effectors. Here, we identify a large family of bacteriophage nucleotidyltransferases (NTases) which synthesize competitor cyclic dinucleotide (CDN) ligands and inhibit NAD-depleting TIR effectors activated through a linked STING CDN sensor domain (TIR-STING). Through a functional screen of NTase-adjacent phage genes, we uncover candidate inhibitors of host TIR-STING suicide signaling. Among these, we demonstrate that a virus MazG-like nucleotide pyrophosphatase, Atd1, depletes the starvation alarmone (p)ppGpp, revealing a role for the alarmone-activated host toxin MazF as a key executioner of TIR-driven abortive infection. Phage NTases and counter-defenses like Atd1 preserve host viability to ensure virus propagation, and may be exploited as tools to modulate TIR and STING immune responses.
2023-03-27 | GSE222071 | GEO
Project description:Activation of the STING (Stimulator of Interferon Genes) pathway by microbial or self-DNA, as well as cyclic di nucleotides (CDN), results in the induction of numerous genes that suppress pathogen replication and facilitate adaptive immunity. However, sustained gene transcription is rigidly prevented to avoid lethal STING-dependent pro-inflammatory disease by mechanisms that remain unknown. We demonstrate here that after autophagy-dependent STING delivery of TBK1 (TANK-binding kinase 1) to endosomal/lysosomal compartments and activation of transcription factors IRF3 (interferon regulatory factors 3) and NF-κB (nuclear factor kappa beta), that STING is subsequently phosphorylated by serine/threonine UNC-51-like kinase (ULK1/ATG1) and IRF3 function is suppressed. ULK1 activation occurred following disassociation from its repressor adenine monophosphate activated protein kinase (AMPK), and was elicited by CDN’S generated by the cGAMP synthase, cGAS. Thus, while CDN’s may initially facilitate STING function, they subsequently trigger negative-feedback control of STING activity, thus preventing the persistent transcription of innate immune genes. Total RNA obtained from primary STING deficient mouse embryonic fibroblast reconstituted with mSTING (W), S365A variant (A), or S365D variant (D). These cells were transfected with dsDNA (ISD) for 3 hours.
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Project description:STING is an innate immune sensor for immune surveillance of viral or bacterial infection and maintenance of an immune-friendly microenvironment to unfavored tumorigenesis. Whether and how STING exerts any innate immunity independent function remains elusive. Here, we report that STING expression is increased in RCC patients and STING governs RCC growth through non-canonical innate immune signaling by maintaining mitochondrial ROS and calcium homeostasis. Mechanistically, we identify mitochondrial calcium transporter VDAC2 as a new STING binding partner and downstream effector, through suppression of which STING controls proper mitochondrial ROS/calcium levels. We also find palmitoylation of STING-C88/C91 residues is critical to mediate STING interaction with VDAC2 and inhibiting STING palmitoyl-transferases ZDHHCs by 2-BP significantly impedes RCC cell growth. Together, our studies reveal an innate immunity-independent function of STING in regulating RCC mitochondrial function and growth. We hope our study provides a rationale to apply palmitoylation inhibitors to treating RCC.
2022-11-29 | GSE190816 | GEO