Mass spectrometry analysis of proteins interacting with STING mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Qi Wu
LAB HEAD: Søren Riis Paludan
PROVIDER: PXD038063 | Pride | 2024-02-19
REPOSITORIES: Pride
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Project description:STING gain-of-function (GOF) mutations lead to T cell lymphopenia, in the context of severe combined immunodeficiency (SCID) for STING GOF V154M mice. This T cell lymphopenia, which is of central origin, has been described as type I IFN independent and associated with dysfunctions of the rare mature T cells found in the periphery. To better describe the biological mechanisms of these dysfunctions, we performed a transcriptomic analysis by RNA-seq on sorted splenic CD4+ and CD8+ mature T cells from STING GOF mice. We highlighted an unexpected T cell exhaustion phenotype that could partly explain their dysfunctions. Acquired very early in life, but only once the peripheral environment is reached, the phenotype appeared to depend neither on type I IFNs, nor on the intrinsic activation of STING in T or stromal cells. Mechanistically, the few mature T cells reaching the periphery seem to be rapidly impacted by the lymphopenic environment through increased antigenic and IL-7 stimulations that could lead to their exhaustion. By using STING GOF long term-hematopoietic stem cells (LT-HSC) transplantations with supportive wild-type bone marrow (BM) cells, we prevented the T cell exhaustion of STING GOF T cells in the resulting non lymphopenic context. With the support of lymphopenic RAG1 hypomorphic mice developing the phenotype, our data uncover a lymphopenia-mediated T cell exhaustion mechanism in STING GOF mice, for which a synergistic effect of the mutation is also envisaged.
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Project description:BMDCs from WT, Fcgr2b-/- and Stinggt/gt were cultured, then stimulated with DMXAA for 3 and 6 hr. Cells were collected and lysed with 1 % IGEPAL CA-630, 0.5% TritonX-100, 150 mM NaCl, 50 mM Tris pH 7.4, 5% glycerol, 100 mM beta-Glycerophosphate, 2 mM Na3VO4 and 1X proteases inhibitor cocktail (Roche). First, antibody were mixed with the magnetic beads by adding 10 µg of STING antibody with 400 ug of SureBeads™ Protein A magnetic beads (Biorad, California, USA) and incubated for 1 hour at room temperature. Then, Protein lysates were added and incubated with antibody-conjugated beads for overnight at 4oC. After incubation, the beads were washed 3 times with wash buffer (150 mM NaCl and 50 mM Tris-HCL pH 7.4). Samples were eluted by adding 5X laemmli buffer and, boiled 950C for 10 minutes. The eluted protein samples were separated by 10 % SDS-PAGE gel. The STING interacting proteins from co-IP were analyzed by in-gel digestion followed by LC-MS/MS analysis.
2020-09-14 | PXD019239 | Pride
Project description:Stimulator of interferon genes (STING) is a central component of the cytosolic nucleic acids sensing pathway and as such master regulator of the type I interferon response. Due to its critical role in physiology and its’ involvement in a variety of diseases, STING has been a focus for drug discovery. Targeted protein degradation (TPD) has emerged as a promising pharmacology for targeting previously considered undruggable proteins by hijacking the cellular ubiquitin proteasome system (UPS) with small molecules. Here, we identify AK59 as a STING degrader leveraging HERC4, a HECT-domain E3 ligase. Additionally, our data reveals that AK59 is effective on the common pathological STING mutations, suggesting a potential clinical application of this mechanism. Thus, these findings introduce HERC4 to the field of TPD and a compound-induced degradation of STING, suggesting potential therapeutic applications.
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Project description:Chronic stimulation of innate immune pathways by microbial agents or damaged tissue is known to promote inflammation-driven tumorigenesis by unclarified mechanisms1-3. Here we demonstrate that mutagenic 7,12-dimethylbenz(a)anthracene (DMBA), etoposide or cisplatin induces nuclear DNA leakage into the cytosol to intrinsically activate STING (Stimulator of Interferon Genes) dependent cytokine production. Inflammatory cytokine levels were subsequently augmented in a STING-dependent extrinsic manner by infiltrating phagocytes purging dying cells. Consequently, STING-/- mice, or wild type mice adoptively transferred with STING-/- bone marrow, were almost completely resistant to DMBA-induced skin carcinogenesis compared to their wild type counterparts. Our data emphasizes, for the first time, a role for STING in the induction of cancer, sheds significant insight into the causes of inflammation-driven carcinogenesis, and may provide therapeutic strategies to help prevent malignant disease Total RNA obtained from wild type murine embryonic fibroblasts (WT MEFs), STING deficient MEFs (SKO), Trex1 deficient MEFs (TKO), and both STING and Trex1 deficient MEFs (STKO) treated with DMBA and examined cytokine production by these cells.
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