Abstract
STING is essential for control of infections and for tumor immunosurveillance, but it can also drive pathological inflammation. STING resides on the endoplasmic reticulum (ER) and traffics following stimulation to the ERGIC/Golgi, where signaling occurs. Although STING ER exit is the rate-limiting step in STING signaling, the mechanism that drives this process is not understood. Here we identify STEEP as a positive regulator of STING signaling. STEEP was associated with STING and promoted trafficking from the ER. This was mediated through stimulation of phosphatidylinositol-3-phosphate (PtdIns(3)P) production and ER membrane curvature formation, thus inducing COPII-mediated ER-to-Golgi trafficking of STING. Depletion of STEEP impaired STING-driven gene expression in response to virus infection in brain tissue and in cells from patients with STING-associated diseases. Interestingly, STING gain-of-function mutants from patients interacted strongly with STEEP, leading to increased ER PtdIns(3)P levels and membrane curvature. Thus, STEEP enables STING signaling by promoting ER exit.
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Data availability
The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.
Change history
14 September 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41590-020-0803-5
References
Paludan, S. R. Activation and regulation of DNA-driven immune responses. Microbiol. Mol. Biol. Rev.79, 225–241 (2015).
Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science339, 786–791 (2013).
Wu, J. et al. Cyclic GMP–AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science339, 826–830 (2013).
Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature455, 674–678 (2008).
Shang, G., Zhang, C., Chen, Z. J., Bai, X. C. & Zhang, X. Cryo-EM structures of STING reveal its mechanism of activation by cyclic GMP–AMP. Nature567, 389–393 (2019).
Tanaka, Y. & Chen, Z. J. STING specifies IRF3 phosphorylation by TBK1 in the cytosolic DNA signaling pathway. Sci. Signal.5, ra20 (2012).
Liu, S. et al. Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF induces IRF3 activation. Science347, aaa2630 (2015).
Prabakaran, T. et al. Attenuation of cGAS–STING signaling is mediated by a p62/SQSTM1-dependent autophagy pathway activated by TBK1. EMBO J.37, e97858 (2018).
Gulen, M. F. et al. Signalling strength determines proapoptotic functions of STING. Nat. Commun.8, 427 (2017).
Reinert, L. S. et al. Sensing of HSV-1 by the cGAS–STING pathway in microglia orchestrates antiviral defense in the CNS. Nat. Commun.7, 13348 (2016).
Li, X. D. et al. Pivotal roles of cGAS–cGAMP signaling in antiviral defense and immune adjuvant effects. Science341, 1390–1394 (2013).
Woo, S. R. et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity41, 830–842 (2014).
Crow, Y. J. et al. Mutations in the gene encoding the 3′−5′ DNA exonuclease TREX1 cause Aicardi–Goutieres syndrome at the AGS1 locus. Nat. Genet.38, 917–920 (2006).
Liu, Y. et al. Activated STING in a vascular and pulmonary syndrome. N. Engl. J. Med.371, 507–518 (2014).
Fu, J. et al. STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade. Sci. Transl. Med.7, 283ra252 (2015).
Hanson, M. C. et al. Nanoparticulate STING agonists are potent lymph node–targeted vaccine adjuvants. J. Clin. Investig.125, 2532–2546 (2015).
Haag, S. M. et al. Targeting STING with covalent small-molecule inhibitors. Nature559, 269–273 (2018).
Saitoh, T. et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc. Natl Acad. Sci. USA106, 20842–20846 (2009).
Dobbs, N. et al. STING activation by translocation from the ER is associated with infection and autoinflammatory disease. Cell Host Microbe18, 157–168 (2015).
Srikanth, S. et al. The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum. Nat. Immunol.20, 152–162 (2019).
Konno, H., Konno, K. & Barber, G. N. Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell155, 688–698 (2013).
Luo, W. W. et al. iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING. Nat. Immunol.17, 1057–1066 (2016).
Wei, J. et al. SNX8 modulates innate immune response to DNA virus by mediating trafficking and activation of MITA. PLoS Pathog.14, e1007336 (2018).
Yang, L. et al. UBXN3B positively regulates STING-mediated antiviral immune responses. Nat. Commun.9, 2329 (2018).
Sun, M. S. et al. TMED2 potentiates cellular IFN responses to DNA viruses by reinforcing MITA dimerization and facilitating its trafficking. Cell Rep.25, 3086–3098 (2018).
Lee, M. N. et al. Identification of regulators of the innate immune response to cytosolic DNA and retroviral infection by an integrative approach. Nat. Immunol.14, 179–185 (2013).
Szappanos, D. et al. The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity. PLoS Pathog.14, e1007397 (2018).
Liu, Y. P. et al. Endoplasmic reticulum stress regulates the innate immunity critical transcription factor IRF3. J. Immunol.189, 4630–4639 (2012).
Paludan, S. R., Reinert, L. S. & Hornung, V. DNA-stimulated cell death: implications for host defence, inflammatory diseases and cancer. Nat. Rev. Immunol.19, 151–153 (2019).
Ni, G., Konno, H. & Barber, G. N. Ubiquitination of STING at lysine 224 controls IRF3 activation. Sci. Immunol.2, eaah7119 (2017).
Ge, L., Melville, D., Zhang, M. & Schekman, R. The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis. Elife2, e00947 (2013).
Ge, L., Zhang, M. & Schekman, R. Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER–Golgi intermediate compartment. Elife3, e04135 (2014).
Ogawa, E., Mukai, K., Saito, K., Arai, H. & Taguchi, T. The binding of TBK1 to STING requires exocytic membrane traffic from the ER. Biochem. Biophys. Res. Commun.503, 138–145 (2018).
Gui, X. et al. Autophagy induction via STING trafficking is a primordial function of the cGAS pathway. Nature567, 262–266 (2019).
Hanna, M. G. T. et al. Sar1 GTPase activity is regulated by membrane curvature. J. Biol. Chem.291, 1014–1027 (2016).
Balch, W. E., McCaffery, J. M., Plutner, H. & Farquhar, M. G. Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum. Cell76, 841–852 (1994).
Long, K. R. et al. Sar1 assembly regulates membrane constriction and ER export. J. Cell Biol.190, 115–128 (2010).
Shibata, Y., Voeltz, G. K. & Rapoport, T. A. Rough sheets and smooth tubules. Cell126, 435–439 (2006).
He, S. et al. PtdIns(3)P-bound UVRAG coordinates Golgi–ER retrograde and Atg9 transport by differential interactions with the ER tether and the beclin 1 complex. Nat. Cell Biol.15, 1206–1219 (2013).
Hirama, T. et al. Membrane curvature induced by proximity of anionic phospholipids can initiate endocytosis. Nat. Commun.8, 1393 (2017).
Kihara, A., Noda, T., Ishihara, N. & Ohsumi, Y. Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae. J. Cell Biol.152, 519–530 (2001).
An, J. et al. Expression of cyclic GMP–AMP synthase in patients with systemic lupus erythematosus. Arthritis Rheumatol.69, 800–807 (2017).
Mukai, K. et al. Activation of STING requires palmitoylation at the Golgi. Nat. Commun.7, 11932 (2016).
Kawasaki, T., Takemura, N., Standley, D. M., Akira, S. & Kawai, T. The second messenger phosphatidylinositol-5-phosphate facilitates antiviral innate immune signaling. Cell Host Microbe14, 148–158 (2013).
Brandizzi, F. & Barlowe, C. Organization of the ER–Golgi interface for membrane traffic control. Nat. Rev. Mol. Cell Biol.14, 382–392 (2013).
Jongsma, M. L. et al. An ER-associated pathway defines endosomal architecture for controlled cargo transport. Cell166, 152–166 (2016).
Raiborg, C. et al. Repeated ER-endosome contacts promote endosome translocation and neurite outgrowth. Nature520, 234–238 (2015).
Rasmussen, S. B. et al. Activation of autophagy by alpha-herpesviruses in myeloid cells is mediated by cytoplasmic viral DNA through a mechanism dependent on stimulator of IFN genes. J. Immunol.187, 5268–5276 (2011).
Lee, C. C., Avalos, A. M. & Ploegh, H. L. Accessory molecules for Toll-like receptors and their function. Nat. Rev. Immunol.12, 168–179 (2012).
Chan, Y. K. & Gack, M. U. Viral evasion of intracellular DNA and RNA sensing. Nat. Rev. Microbiol.14, 360–373 (2016).
Hendel, A. et al. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. Nat. Biotechnol.33, 985–989 (2015).
Wang, Q. et al. The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING. Immunity41, 919–933 (2014).
Pettitt, T. R., Dove, S. K., Lubben, A., Calaminus, S. D. & Wakelam, M. J. Analysis of intact phosphoinositides in biological samples. J. Lipid Res.47, 1588–1596 (2006).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol.11, R106 (2010).
Acknowledgements
The technical assistance of K. S. Petersen, the AU FACS Core Facility and the AU Health Bioimaging Core Facility is greatly appreciated. We acknowledge T. Melia, Yale University, for critical reading of the manuscript. This work was funded by the European Research Council (ERC-AdG ENVISION; 786602), the Novo Nordisk Foundation (NNF18OC0030274) and the Lundbeck Foundation (R198-2015-171; R268-2016-3927); B.-c.Z. is funded by a postdoctoral grant from the Danish Council for Independent Research, Medical Sciences (5053-00083B); the postdoctoral salary to S.A. was funded by the European Union under the Horizon 2020 Research and Innovation Program and Marie Skłodowska-Curie Actions (MSCA) – international fellowship (PathAutoBio 796840); the PhD scholarship to S.J.W. was funded by the European Union under the Horizon 2020 Research and Innovation Program and the MSCA-Innovative Training Networks Programme MSCA-ITN (EDGE, 675278); the postdoctoral grant to A.T. was funded by the Lundbeck Foundation (R264-2017-3344).
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Contributions
B.-c.Z. and S.R.P. conceived the idea and designed the experiments. B.-c.Z. unraveled the mechanism. S.B.J. identified STEEP in the MS screen. B.-c.Z., R.Nandakumar, L.S.R., J.H., A.L., Z.-l.G., C.-l.S., S.B.J., S.A., M.F.B., C.S., Y.Z., S.J.W., D.O., T.P., C.B., R.Narita, Y.C. and C.-g.Z. performed the experiments. A.T., H.S., C.M.D., T.N. and R.G.-M. provided materials. Z.G., R.H., Z.J.C., J.J.E., R.O.B., M.K.T. and S.R.P. supervised experiments. B.-c.Z. and S.R.P. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Basic characteristics and properties of CxORF56/STEEP.
a, Comparison of protein sequence identify between STING and STEEP in different species. b, mRNA Expression of STEEP in human tissues. The data are from proteinatlas.org. c, Illustration of the three known isoforms of STEEP. Source, uniprot.org. d, Predicted nuclear localization and nuclear exit signals in STEEP, based on sequence analyses by cNLS mapper and LocNES, respectively. e, Whole cell lysates, cytoplasmic fractions, and nuclear fractions from PMA-differnetiated THP-1 cells were immunoplotted for STEEP and β-actin. A representative blot is shown from n = 3 biologically independent experiments with similar results. f, Predicted transmembrane regions in STEEP and STING. Based on sequence analysis by TMHMM.
Extended Data Fig. 2 STEEP is a positive regulator in STING pathway.
a, Reporter gene assay for HEK-293T cells transfected with 50 ng STEEP or empty vector, IFNB1 promoter luciferase reporter, β-actin Renilla reporter, and MAVS or TRIF as indicated for 24 h (n = 3) (nsP = 0.86, nsP = 0.23, lift to right).b, Immunoprecipitation of Flag-tagged STING and STEEP (n = 3 biologically independent experiments). c, Confocal microscopy of HeLa cells transfected with HA-STEEP and FLAG-STING for 24 h followed by mock treatment or cGAMP stimulation. Representative data from one experiment are shown (n = 3 biologically independent experiments). d, Alignment of STEEP/CxORF56 from the indicated species, and highlight of residues mutated to alanine in Mut 1–5. Data in panel a are shown as means of biological triplicates +/- st.dev. Statistical analysis of data shown in panel a was performed using two-tailed Student’s t-test.
Extended Data Fig. 3 Targeting of STEEP by CRISPR.
a, Illustration of gRNAs sequence and location used to target human STEEP. b, Immunoblot for STEEP in THP-1-derived clones transduced with STING-targeting gRNAs. c, STEEP KO THP-1 cells were transfected with mRNA encoding WT STEEP. 24 h later, the cells were lysed, and immunoblotted as shown. For comparison, lysates from WT THP-1 cells were included. d-f, Efficiency of STEEP gene targeting in primary human fibroblasts d, primary human macrophage e, and SAVI patient fibroblasts f. g, gRNAs used to target STEEP in mice. h-i, Efficiency of STEEP targeting in MEF cells. The KO scores were calculated based on the ICE Analysis tool from Synthego. In panels b and c, data shown are representative blots from three biologically independent experiments with similar results.
Extended Data Fig. 4 Effect of STEEP KO on STING signaling.
a, STEEP KO THP-1 cells electroporated with GFP mRNA, WT STEEP mRNA or mut5 STEEP mRNA were stimulated with cGAMP (150 nM, 1 h). Levels of pSTING and pTBK1 were monitored by Immunoblotting. (n = 3 biologically independent experiments). b-c, Immunoblot analysis of the indicated proteins from whole cell lysates of WT or STEEP KO Hela cells b, and human foreskin fibroblasts (c) after stimulation with dsDNA for the indicated time intervals. (n = 3 biologically independent experiments). All data shown in this figure are representative blots from three biologically independent experiments.
Extended Data Fig. 5 Impact of STEEP on STING trafficking.
a, The in vitro membrane budding reaction illustrated in Fig. 4a was performed with material from wild-type THP-1 cells in the presence or absence of ATP. M, membranes; P, post 20.000 g pellet; BV, budding vesicles. Representative blots from three biologically independent experiments with similar results are shown. b, Confocal microscopy of WT Hela and STEEP KO Hela cells stimulated with/without cGAMP for 0.5 h. The cells were immunostained with anti-STING (red), anti-calreticulin (green), anti-GM130 (purple). Sections were counterstained with DAPI to visualize nuclei. (n = 3 biologically independent experiments). c, ImageStream analysis of Sar1-ER colocation. FLAG-tagged Sar1 was transfected into HEK-293T with STING expression cells for 24 h, and stimulated with/without cGAMP (100 nM) for 30 min. After fixation and permeabilization, the cells were incubated with rabbit anti-calreticulin (ER marker) and mouse anti-FLAG. (n = 3) (**P = 0.00042). d, Immunoblot analysis of the indicated proteins from the whole Hela cell lysates transfected with SAR1 or SAR1-H79G. (n = 3 biologically independent experiments). e, f, Imagestream analysis of STING trafficking from ER (e) to Golgi (f) after transfection of SAR1 or SAR1-H79G for 24 h in HEK-293T with STING expression cells (e, **P = 0.00035; f, **P = 0.00010). g, Endogenous Sec24 was immunoprecipitated from THP-1 cell lysates isolated after +/-BFA treatment and cGAMP (100 nM) stimulation for the indicated time. Precipitates and lysates were immunoblotted with antibodies against Sec24A/B, STING, and vinculin (n = 3 biologically independent experiments). h, Confocal microscopy analysis of Sec24 foci in STEEP KO THP-1 cells rescued with WT or mut5 STEEP mRNA and stimulated with cGAMP for 10 min. Nuclei were stained with DAPI. For quantification of Sec24 foci, 12 cells per group were counted in a blinded fashion. Representative data from one experiment are shown (n = 3 biologically independent experiments). Each data point represents one cell and data are shown as means +/- st.dev. Statistical analysis was performed by a two-tailed unpaired t test with welch’s correction (*P = 0.016, nsP = 0.51, *P = 0.045, left to right). i, Budding vesicle analysis (as illustrated in Fig. 4a) for mCherry-VSVG and STING on WT and STEEP KO Hela cells transfected with mCherry-VSVG. The data shown is representative from two biologically independent experiments with similar results. j, Confocal microscopy imaging of SAR1-Flag (green) and calreticulin (red, ER marker) in WT and STEEP KO Hela cells stimulated with cGAMP (100 nM) for 20 min. Sections were counterstained with DAPI to visualize nuclei. (n = 3 biologically independent experiments). k, Imagestream analysis of ER membrane curvature in Flag-STING-transfected HEK-293T cells probed with GFP133 with/without 100 nM cGAMP stimulation for 20 min. (n = 3) (**P = 0.000011). l, Confocal microscopy analysis Hela cells transfected with Flag-tagged STING and GFP133 and treated with 100 nM cGAMP stimulation for 20 min. Cells were probed with antibodies against of STING (Red) and Calreticulin (ER marker, Purple). GFP133 (green) is an ER membrane curvature probe. (n = 3 biologically independent experiments). m, Imagestream analysis of ER membrane curvature and STING colocation in HEK-293T cells with/without 100 nM cGAMP stimulation for 20 min. (n = 3) (**P = 0.000044). (n) WT and STEEP KO THP-1 cells were stimulated with cGAMP for 10 min, fixed and stained with anti-Clim63, anti-RTN4, and anti-STING. Nuclei were stained with DAPI. For quantification of RTN4:Climp63 ratio, 10 cells per group were counted in a blinded fashion. Representative data from one experiment are shown (n = 3 biologically independent experiments). Each data point represents one cell and data are shown as means +/- st.dev. Statistical analysis was performed by a two-tailed unpaired t test with welch’s correction (nsP = 0.64, **P = 0.0046, **P = 0.0060, nsP = 0.071). o, Analysis of protein synthesis in WT and STEEP KO THP-1 cells using the Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit (n = 6) (nsP = 0.62, nsP = 0.10, left to right). p, ER- and Golgi-enriched pellets from lysates of STEEP-deficient Hela cells transfected with empty vector, HA-STEEP WT, or HA-STEEP mut5 were fractionated by gradient centrifugation. The collected fractions were immunoblotted with anti-STING, anti-HA, anti-GM130 (Golgi), and anti-Sec61B (ER). Representative blots from one experiment are shown (n = 3). For data from ImageStream analysis (panel c, e, f, k, and m), each data point represents the percent of positive cells from one representative sample and are shown as means +/- st.dev. Statistical analysis of data in panels c, e, f, k, m, and o was performed using two-tailed Student’s t-test.
Extended Data Fig. 6 STEEP promotes PI3P production and ER membrane curvature.
a, b, Confocal microscopy analysis of PI3P level on ER. PI3P was probed with anti-PI3P antibody in THP-1 cells (a), or FYVE-GFP in Hela cells (b) or with/without 100 nM cGAMP stimulation for 20min. ER was stained and visualized using rabbit anti-Calreticulin (ER marker, Red) and mouse anti-flag, and relevant secondary antibodies. c, PIP Strip membranes blotted with various lipids were incubated sequentially with Flag-tagged WT, 139-C, or V155M STING or with empty vector (EV). An anti-Flag antibody conjugated with HRP was used to visualize the binding. Phospholipids with clear positive binding to WT STING are highlighted in red on the left PIP Strip illustration. d, Confocal analysis for the colocation of anti-STING and anti-PI3P antibodies in THP-1 cells with/without 100 nM cGAMP stimulation for 15 min. e, Immunoblot analysis of the indicated proteins from the whole cell lysates of Hela cells transfected MTMR3/MTMR3-C143S, and stimulated for 1h with cGAMP. (n = 3 biologically independent experiments). f, g, Imagestream analysis of STING trafficking from ER (f) to Golgi (g). HEK-293T cells were transfected by MTMR3/MTMR3-C143S and Flag-tagged STING for 24 h and stimulated cGAMP (150 nM) for 30 min. After fixation and permeabilization, flag and ER were labelled using rabbit anti-calreticulin (ER marker) and mouse anti-flag, and relevant secondary antibodies. (n = 3) (f, **P = 0.0020; g, **P = 0.00088). (h) Imagestream analysis of SAR1-ER colocation. Hela cells were transfected by MTMR3/MTMR3-C143S and Flag-tagged SAR1 for 24 h and stimulated cGAMP (100 nM) for 30 min. After fixation and pre-permeabilization, ER and Flag were labelled using rabbit anti-calreticulin (ER marker) and mouse anti-flag, and relevant secondary antibodies. (n = 3) (**P = 0.0014). (i) Imagestream analysis of ER membrane curvature. HEK cells were co-transfected by MTMR3/MTMR3-C143S, Flag-tagged STING and GFP133 for 24 h and stimulated with/without cGAMP for 30 min. After fixation and pre-permeabilization, the cells were stained with anti-calreticulin and relevant secondary antibodies. (n = 3) (nsP = 0.26, *P = 0.012, **P = 4.3E-05, **P = 1.0E-06, left to right). Panels a-e, representative images from n = 3 biologically independent experiments with at least 2 times similar results are shown. For data from ImageStream analysis (panel f, g, h, i), each data point represents the percent of positive cells from one representative sample and are shown as means +/- st.dev. Statistical analysis of data in panels f-h was performed using two-tailed Student’s t-test, and in panel i was performed using two-tailed one-way ANOVA test.
Extended Data Fig. 7 Vps34 Complex I promotes STING activation.
a, Immunoblot analysis of the indicated proteins from the cell lysates of THP-1 cells pre-treated with DMSO, 10 uM VPS34 inhibitor (VPS34-IN1) or 5 mM 3-MA and then stimulated with 150 nM cGAMP for the indicated time intervals. (n = 3 biologically independent experiments). b, Immunoblot analysis of the indicated proteins from the cell lysates of HaCaT cells transfected with siRNA-control or siRNA-vps34 for 36 h and then stimulated with cGAMP for the indicated times. (n = 3 biologically independent experiments). c, Reporter gene assay. HEK-293T with STING stable expression cells were transfected with 50 ng VPS34, Beclin1, ATG14, UVRAG or empty vector, IFNB1 promoter luciferase reporter, and β-actin Renilla reporter. After transfection for 24 h, the cells were treated with/without BafA1 and then stimulated with 200 nM cGAMP for 6 h (n = 3). Statistical analysis of data in panel c was performed using two-tailed one-way ANOVA test (**P = 1.87E-06, nsP = 0.88, **P = 2.46E-09, **P = 0.0063, **P = 0.00053, nsP = 0.10, **P = 1.74E-06, nsP = 0.99, left to right). d, The isolation of THP-1 cell fractions were obtained by Opti-prep gradient. After collecting the different layers, immunoblot analysis of the indicated proteins was carried out to probe for organelle enrichment of each fraction. The input was from whole cell lysate. Representative blots from two biologically independent experiments with similar results are shown. Data in panel c are shown as means of biological sample triplicates +/- st.dev.
Extended Data Fig. 8 Role for STEEP in host defense and inflammation.
a, The workflow for examination of HSV1 replication in genome-edited brain slices from Cas9lsl mice. b, To evaluate expression from the Cas9 and GFP-containing locus, emission of fluorescence from AAV9-treated Cas9lsl brain slices was monitored. The images represent brain slices 6 days after treatment. Representative images from two biologically independent experiments with similar results are shown. c-d, Levels of type I IFN bioactivity and CXCL10 protein in serum from five SLE patients and two healthy donors. e-g, Box plots of TCGA RNA expression profiles in Bladder Urothelial Carcinoma (BLCA, e), Cervical Kidney renal papillary cell carcinoma (KIRP, f), and Liver Hepatocellular Carcinoma (LIHC, g). Samples with the highest 25% and the lowest 25% of STEEP (upper panels) and STING (lower panels) expression were selected and grouped. CXCL10, MX2, Viperin (RSAD2), STAT1 and GAPDH gene expression levels were compared between STEEP/STING-high and STEEP/STING-low groups. The upper and lower ends of the boxes represent the upper and lower quartiles, and the horizontal line inside the box is median of the dataset. The bar extending parallel from the boxes is the “whiskers”, indicating upper and lower extreme of the dataset. Statistical significance was evaluated using two-tailed Mann-Whitney test (nsP > 0.01, *P < 0.01, **P < 0.001). h, Imagestream analysis of ER membrane curvature. HEK-293T cells were co-transfected by MTMR3/MTMR3-C143S, STING/ STING SAVI mutant and GFP133 for 24 h. After fixation and pre-permeabilization, the cells were stained with anti-calreticulin and relevant secondary antibodies. (n = 3) (nsP = 0.15, nsP = 0.10, *P = 0.038, *P = 0.029, left to right). For data from ImageStream analysis (panel h), each data point represents the percent of positive cells from one representative sample and are shown as means +/- st.dev. Statistical analysis of data panel h was performed using two-tailed Student’s t-test.
Extended Data Fig. 9 Proposed model for action of STEEP in STING signaling.
Interaction between STEEP and STING is observed in the resting state, but this is augmented following STING activation. This may involve the conformational change imposed on STING following cGAMP binding or mediated with low threshold in SAVI gain-of-function STING mutants. The STEEP-STING association increases recruitment of the VPS34 complex I to produce PI3P on the ER. The PI3P accumulation in turn induces ER membrane curvature in the STING containing areas, thus recruiting the COPII complex factors SAR1 and SEC24 to ER to form the ER exit site. This finally sorts STING into COPII vesicles for delivery to ERGIC/Golgi to enable antiviral and inflammatory responses.
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Zhang, Bc., Nandakumar, R., Reinert, L.S. et al. STEEP mediates STING ER exit and activation of signaling. Nat Immunol 21, 868–879 (2020). https://doi.org/10.1038/s41590-020-0730-5
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DOI: https://doi.org/10.1038/s41590-020-0730-5
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