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Global proteomic analyses of STING-positive and -negative macrophages reveal STING and non-STING differentially regulated cellular and molecular pathways

ABSTRACT: The STING signaling pathway plays important roles in many cancers, viral and bacterial infections, and autoimmune diseases. Understanding how STING mediates expression of many proteins in the host and bacterial cells can provide important clues about the biochemical processes, opening the door for the development of new therapies. Using STING KO cells, we were able to identify many differentially expressed proteins between the wild type and STING KO RAW macrophage. We identified proteins that were exclusively detected only in the wild type or only in the STING KO, as well as those that were comply identified but showed different levels of expressions between the two cell lines. This included several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Our data broaden the knowledge of proteins that are related to STING signaling, and may have identified novel proteins that have therapeutic value. This will require rigorous validation in the future studies. Many protein biomarkers can be regulated by post-translational modifications and protein-protein interactions. While beyond the scope of the current study, we note that methods are available at the Purdue Proteomics Facility for mapping post-translational modifications and protein-protein interactions. We envision this to be an exciting line of enquiry for us in the future.

INSTRUMENT(S): Q Exactive Orbitrap

ORGANISM(S): Human Macrophage Cell Lines

SUBMITTER: Prof. Herman Sintim

PROVIDER: MSV000084058 | MassIVE | Wed Jul 03 16:57:00 BST 2019

REPOSITORIES: MassIVE

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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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Global proteomic analyses of STING-positive and -negative macrophages reveal STING and non-STING differentially regulated cellular and molecular pathways

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