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ESCRT regulates the termination of STING activity by mediating its degradation

ABSTRACT: 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.

ORGANISM(S): Mus Musculus

SUBMITTER: Yang Chen

PROVIDER: PXD043331 | iProX | Tue Jun 27 00:00:00 BST 2023

REPOSITORIES: iProX

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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.

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ESCRT regulates the termination of STING activity by mediating its degradation

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