Dataset Information

Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling

ABSTRACT: Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 different stably expressed, full-length E3s. Bioinformatic analysis linked ER-E3s and their interactors to multiple homeostatic, regulatory, and metabolic pathways. Among these were four membrane-embedded interactors of RNF26, a polytopic E3 whose abundance is auto-regulated by ubiquitin-proteasome dependent degradation. RNF26 co-assembles with TMEM43, ENDOD1, TMEM33 and TMED1 to form a complex capable of modulating innate immune signalling through the cGAS-STING pathway. This RNF26 complex represents a new modulatory axis of STING and innate immune signalling at the ER membrane. Collectively, these data reveal the broad scope of regulation and differential functionalities mediated by ER-E3s for both membrane-tethered and cytoplasmic processes.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Philip Charles

LAB HEAD: John Christianson

PROVIDER: PXD019559 | Pride | 2020-07-06

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	1of2.mgf	Mgf
	20160126_020715_RFI_EmmaFenech_All_SwissProtUniProtSearch.xlsx	Xlsx
	20160126_HF0068_Emma_newrun_newREF_SwissProtUniprotSearch.xlsx	Xlsx
	2of2.mgf	Mgf
	Batch_effect-removed_Log2_Mean_Intensities.tsv	Tabular

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Publications

Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling.

Fenech Emma J EJ Lari Federica F Charles Philip D PD Fischer Roman R Laétitia-Thézénas Marie M Bagola Katrin K Paton Adrienne W AW Paton James C JC Gyrd-Hansen Mads M Kessler Benedikt M BM Christianson John C JC

eLife 20200702

Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow, we mapped over 450 protein-protein interactions for 21 stably expressed, full-length E3s. Bioinform ...[more]

PMID: 32614325

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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.