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Termination of STING responses is mediated via ESCRT-dependent degradation

ABSTRACT: cGAS-STING signalling is induced by detection of foreign or mislocalised host double stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling, and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.

SUBMITTER: Katherine Balka

PROVIDER: S-SCDT-10_15252-EMBJ_2022112712 | biostudies-other |

REPOSITORIES: biostudies-other

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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.

Project description:UnlabelledSynaptic vesicle (SV) pools must maintain a functional repertoire of proteins to efficiently release neurotransmitter. The accumulation of old or damaged proteins on SV membranes is linked to synaptic dysfunction and neurodegeneration. However, despite the importance of SV protein turnover for neuronal health, the molecular mechanisms underlying this process are largely unknown. Here, we have used dissociated rat hippocampal neurons to investigate the pathway for SV protein degradation. We find that neuronal activity drives the degradation of a subset of SV proteins and that the endosomal sorting complex required for transport (ESCRT) machinery and SV-associated GTPase Rab35 are key elements of this use-dependent degradative pathway. Specifically, neuronal activity induces Rab35 activation and binding to the ESCRT-0 protein Hrs, which we have identified as a novel Rab35 effector. These actions recruit the downstream ESCRT machinery to SV pools, thereby initiating SV protein degradation via the ESCRT pathway. Our findings show that the Rab35/ESCRT pathway facilitates the activity-dependent removal of specific proteins from SV pools, thereby maintaining presynaptic protein homeostasis.Significance statementSynaptic transmission is mediated by the release of chemical neurotransmitters from synaptic vesicles (SVs). This tightly regulated process requires a functional pool of SVs, necessitating cellular mechanisms for removing old or damaged proteins that could impair SV cycling. Here, we show that a subset of SV proteins is degraded in an activity-dependent manner and that key steps in this degradative pathway are the activation of the small GTPase Rab35 and the subsequent recruitment of the endosomal sorting complex required for transport (ESCRT) machinery to SV pools. Further, we demonstrate that ESCRT-0 component Hrs is an effector of Rab35, thus providing novel mechanistic insight into the coupling of neuronal activity with SV protein degradation and the maintenance of functional SV pools.

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Termination of STING responses is mediated via ESCRT-dependent degradation

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