Abstract
Type I interferons (IFNs) are multifunctional cytokines that regulate immune responses and cellular functions but also can have detrimental effects on human health. A tight regulatory network therefore controls IFN signaling, which in turn may interfere with medical interventions. The JAK–STAT signaling pathway transmits the IFN extracellular signal to the nucleus, thus resulting in alterations in gene expression. STAT2 is a well-known essential and specific positive effector of type I IFN signaling. Here, we report that STAT2 is also a previously unrecognized, crucial component of the USP18-mediated negative-feedback control in both human and mouse cells. We found that STAT2 recruits USP18 to the type I IFN receptor subunit IFNAR2 via its constitutive membrane-distal STAT2-binding site. This mechanistic coupling of effector and negative-feedback functions of STAT2 may provide novel strategies for treatment of IFN-signaling-related human diseases.
Similar content being viewed by others
References
Hertzog, P.J. & Williams, B.R. Fine tuning type I interferon responses. Cytokine Growth Factor Rev. 24, 217–225 (2013).
Schneider, W.M., Chevillotte, M.D. & Rice, C.M. Interferon-stimulated genes: a complex web of host defenses. Annu. Rev. Immunol. 32, 513–545 (2014).
Ivashkiv, L.B. & Donlin, L.T. Regulation of type I interferon responses. Nat. Rev. Immunol. 14, 36–49 (2014).
Zitvogel, L., Galluzzi, L., Kepp, O., Smyth, M.J. & Kroemer, G. Type I interferons in anticancer immunity. Nat. Rev. Immunol. 15, 405–414 (2015).
Borden, E.C. et al. Interferons at age 50: past, current and future impact on biomedicine. Nat. Rev. Drug Discov. 6, 975–990 (2007).
Sistigu, A. et al. Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy. Nat. Med. 20, 1301–1309 (2014).
Rieger, P.T. Interferon-alpha: a clinical update. Cancer Pract. 3, 356–365 (1995).
Dusheiko, G. Side effects of alpha interferon in chronic hepatitis C. Hepatology 26 (Suppl.1), S112–S121 (1997).
Crow, Y.J. Aicardi-Goutières syndrome. Handb. Clin. Neurol. 113, 1629–1635 (2013).
Meyer, O. Interferons and autoimmune disorders. Joint Bone Spine 76, 464–473 (2009).
Uzé, G., Lutfalla, G. & Mogensen, K.E. Alpha and beta interferons and their receptor and their friends and relations. J. Interferon Cytokine Res. 15, 3–26 (1995).
Li, X., Leung, S., Qureshi, S., Darnell, J.E. Jr. & Stark, G.R. Formation of STAT1-STAT2 heterodimers and their role in the activation of IRF-1 gene transcription by interferon-alpha. J. Biol. Chem. 271, 5790–5794 (1996).
Platanias, L.C. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat. Rev. Immunol. 5, 375–386 (2005).
van Boxel-Dezaire, A.H., Rani, M.R. & Stark, G.R. Complex modulation of cell type-specific signaling in response to type I interferons. Immunity 25, 361–372 (2006).
Porritt, R.A. & Hertzog, P.J. Dynamic control of type I IFN signalling by an integrated network of negative regulators. Trends Immunol. 36, 150–160 (2015).
Marchetti, M. et al. Stat-mediated signaling induced by type I and type II interferons (IFNs) is differentially controlled through lipid microdomain association and clathrin-dependent endocytosis of IFN receptors. Mol. Biol. Cell 17, 2896–2909 (2006).
Schreiber, G. & Piehler, J. The molecular basis for functional plasticity in type I interferon signaling. Trends Immunol. 36, 139–149 (2015).
Liu, L.Q. et al. A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation. Mol. Cell. Biol. 19, 3029–3038 (1999).
Schwer, H. et al. Cloning and characterization of a novel human ubiquitin-specific protease, a homologue of murine UBP43 (Usp18). Genomics 65, 44–52 (2000).
Malakhov, M.P., Malakhova, O.A., Kim, K.I., Ritchie, K.J. & Zhang, D.E. UBP43 (USP18) specifically removes ISG15 from conjugated proteins. J. Biol. Chem. 277, 9976–9981 (2002).
Malakhova, O.A. et al. UBP43 is a novel regulator of interferon signaling independent of its ISG15 isopeptidase activity. EMBO J. 25, 2358–2367 (2006).
Wilmes, S. et al. Receptor dimerization dynamics as a regulatory valve for plasticity of type I interferon signaling. J. Cell Biol. 209, 579–593 (2015).
François-Newton, V. et al. USP18-based negative feedback control is induced by type I and type III interferons and specifically inactivates interferon α response. PLoS One 6, e22200 (2011).
Zhang, X. et al. Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation. Nature 517, 89–93 (2015).
Meuwissen, M.E. et al. Human USP18 deficiency underlies type 1 interferonopathy leading to severe pseudo-TORCH syndrome. J. Exp. Med. 213, 1163–1174 (2016).
Ritchie, K.J. et al. Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection. Nat. Med. 10, 1374–1378 (2004).
Kim, K.I. et al. Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling. J. Immunol. 175, 847–854 (2005).
Honke, N. et al. Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus. Nat. Immunol. 13, 51–57 (2011).
Honke, N. et al. Usp18 driven enforced viral replication in dendritic cells contributes to break of immunological tolerance in autoimmune diabetes. PLoS Pathog. 9, e1003650 (2013).
Goldmann, T. et al. USP18 lack in microglia causes destructive interferonopathy of the mouse brain. EMBO J. 34, 1612–1629 (2015).
Ketscher, L. et al. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance. Proc. Natl. Acad. Sci. USA 112, 1577–1582 (2015).
Yim, H.Y. et al. Elevated response to type I IFN enhances RANKL-mediated osteoclastogenesis in Usp18-knockout mice. J. Immunol. 196, 3887–3895 (2016).
Löchte, S., Waichman, S., Beutel, O., You, C. & Piehler, J. Live cell micropatterning reveals the dynamics of signaling complexes at the plasma membrane. J. Cell Biol. 207, 407–418 (2014).
Levy, D.E. & Darnell, J.E. Jr. Stats: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol. 3, 651–662 (2002).
Ketscher, L. & Knobeloch, K.P. ISG15 uncut: dissecting enzymatic and non-enzymatic functions of USP18 in vivo. Cytokine 76, 569–571 (2015).
Pellegrini, S., John, J., Shearer, M., Kerr, I.M. & Stark, G.R. Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway. Mol. Cell. Biol. 9, 4605–4612 (1989).
Rani, M.R. et al. Characterization of beta-R1, a gene that is selectively induced by interferon beta (IFN-beta) compared with IFN-alpha. J. Biol. Chem. 271, 22878–22884 (1996).
Leung, S., Qureshi, S.A., Kerr, I.M., Darnell, J.E. Jr. & Stark, G.R. Role of STAT2 in the alpha interferon signaling pathway. Mol. Cell. Biol. 15, 1312–1317 (1995).
Hambleton, S. et al. STAT2 deficiency and susceptibility to viral illness in humans. Proc. Natl. Acad. Sci. USA 110, 3053–3058 (2013).
Yang, E., Wen, Z., Haspel, R.L., Zhang, J.J. & Darnell, J.E. Jr. The linker domain of Stat1 is required for gamma interferon-driven transcription. Mol. Cell. Biol. 19, 5106–5112 (1999).
Nguyen, V.P. et al. Stat2 binding to the interferon-alpha receptor 2 subunit is not required for interferon-alpha signaling. J. Biol. Chem. 277, 9713–9721 (2002).
Li, X., Leung, S., Kerr, I.M. & Stark, G.R. Functional subdomains of STAT2 required for preassociation with the alpha interferon receptor and for signaling. Mol. Cell. Biol. 17, 2048–2056 (1997).
Piehler, J., Roisman, L.C. & Schreiber, G. New structural and functional aspects of the type I interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface. J. Biol. Chem. 275, 40425–40433 (2000).
Improta, T. et al. Transcription factor ISGF-3 formation requires phosphorylated Stat91 protein, but Stat113 protein is phosphorylated independently of Stat91 protein. Proc. Natl. Acad. Sci. USA 91, 4776–4780 (1994).
Martinez-Moczygemba, M., Gutch, M.J., French, D.L. & Reich, N.C. Distinct STAT structure promotes interaction of STAT2 with the p48 subunit of the interferon-alpha-stimulated transcription factor ISGF3. J. Biol. Chem. 272, 20070–20076 (1997).
Brierley, M.M. & Fish, E.N. Functional relevance of the conserved DNA-binding domain of STAT2. J. Biol. Chem. 280, 13029–13036 (2005).
Melen, K., Kinnunen, L. & Julkunen, I. Arginine/lysine-rich structural element is involved in interferon-induced nuclear import of STATs. J. Biol. Chem. 276, 16447–16455 (2001).
Chawla-Sarkar, M., Leaman, D.W. & Borden, E.C. Preferential induction of apoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation with TRAIL/Apo2L induction in melanoma cell lines. Clin. Cancer Res. 7, 1821–1831 (2001).
González-Navajas, J.M., Lee, J., David, M. & Raz, E. Immunomodulatory functions of type I interferons. Nat. Rev. Immunol. 12, 125–135 (2012).
Chen, H.M. et al. Critical role for constitutive type I interferon signaling in the prevention of cellular transformation. Cancer Sci. 100, 449–456 (2009).
Zheng, H., Qian, J., Baker, D.P. & Fuchs, S.Y. Tyrosine phosphorylation of protein kinase D2 mediates ligand-inducible elimination of the Type 1 interferon receptor. J. Biol. Chem. 286, 35733–35741 (2011).
Yoshimura, A., Naka, T. & Kubo, M. SOCS proteins, cytokine signalling and immune regulation. Nat. Rev. Immunol. 7, 454–465 (2007).
Sarasin-Filipowicz, M. et al. Alpha interferon induces long-lasting refractoriness of JAK-STAT signaling in the mouse liver through induction of USP18/UBP43. Mol. Cell. Biol. 29, 4841–4851 (2009).
Zou, W. et al. Microarray analysis reveals that Type I interferon strongly increases the expression of immune-response related genes in Ubp43 (Usp18) deficient macrophages. Biochem. Biophys. Res. Commun. 356, 193–199 (2007).
Kotenko, S.V. et al. IFN-ës mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4, 69–77 (2003).
Brierley, M.M., Marchington, K.L., Jurisica, I. & Fish, E.N. Identification of GAS-dependent interferon-sensitive target genes whose transcription is STAT2-dependent but ISGF3-independent. FEBS J. 273, 1569–1581 (2006).
Au-Yeung, N., Mandhana, R. & Horvath, C.M. Transcriptional regulation by STAT1 and STAT2 in the interferon JAK-STAT pathway. JAK-STAT 2, e23931 (2013).
Park, C., Li, S., Cha, E. & Schindler, C. Immune response in Stat2 knockout mice. Immunity 13, 795–804 (2000).
London, N., Raveh, B. & Schueler-Furman, O. Druggable protein-protein interactions: from hot spots to hot segments. Curr. Opin. Chem. Biol. 17, 952–959 (2013).
Burkart, C. et al. Usp18 deficient mammary epithelial cells create an antitumour environment driven by hypersensitivity to IFN-λ and elevated secretion of Cxcl10. EMBO Mol. Med. 5, 1035–1050 (2013).
Colland, F. et al. Functional proteomics mapping of a human signaling pathway. Genome Res. 14, 1324–1332 (2004).
James, P., Halladay, J. & Craig, E.A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436 (1996).
Urbé, S. et al. Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions. Mol. Biol. Cell 23, 1095–1103 (2012).
Roder, F., Birkholz, O., Beutel, O., Paterok, D. & Piehler, J. Spatial organization of lipid phases in micropatterned polymer-supported membranes. J. Am. Chem. Soc. 135, 1189–1192 (2013).
Subach, O.M. et al. Conversion of red fluorescent protein into a bright blue probe. Chem. Biol. 15, 1116–1124 (2008).
Ritchie, K.J. et al. Dysregulation of protein modification by ISG15 results in brain cell injury. Genes Dev. 16, 2207–2212 (2002).
Boussif, O. et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297–7301 (1995).
Waichman, S. et al. Functional immobilization and patterning of proteins by an enzymatic transfer reaction. Anal. Chem. 82, 1478–1485 (2010).
Wedeking, T. et al. Spatiotemporally controlled reorganization of signaling complexes in the plasma membrane of living cells. Small 11, 5912–5918 (2015).
Arimoto, K. et al. Plakophilin-2 promotes tumor development by enhancing ligand-dependent and -independent epidermal growth factor receptor dimerization and activation. Mol. Cell. Biol. 34, 3843–3854 (2014).
Muster, B. et al. Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells. PLoS One 5, e11910 (2010).
Sprague, B.L., Pego, R.L., Stavreva, D.A. & McNally, J.G. Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys. J. 86, 3473–3495 (2004).
Sprague, B.L. & McNally, J.G. FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15, 84–91 (2005).
Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Edn Engl. 47, 5465–5469 (2008).
VandeVondele, S., Vörös, J. & Hubbell, J.A. RGD-grafted poly-L-lysine-graft-(polyethylene glycol) copolymers block non-specific protein adsorption while promoting cell adhesion. Biotechnol. Bioeng. 82, 784–790 (2003).
Sergé, A., Bertaux, N., Rigneault, H. & Marguet, D. Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes. Nat. Methods 5, 687–694 (2008).
Goldman, L.A. et al. Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis. J Interferon Cytokine Res. 19, 15–26 (1999).
Acknowledgements
We thank A. Garcia-Sastre (Icahn School of Medicine at Mount Sinai) for Stat2−/− MEFs, D. Cheresh (Moores UCSD Cancer Center) for MDA-MB-231, G. Stark (Cleveland Clinic) for sharing U-series cell lines, S. Fujita (Ehime University School of Medicine) for KT-1 cells, R. Xiang (The Scripps Research Institute) for WEHI-3B cells, S. Urbe (University of Liverpool) for GFP-fusion STAT2 and USP18 constructs, V. Verkhusha (Albert Einstein College of Medicine) for the mTag-BFP construct, T. Akagi (KAN Research Institute) for providing pCX4-series vectors, S. Kotenko (Rutgers New Jersey Medical School) for the pcDEF-hIFNAR2 DNA construct, D. Baker (Biogen Idec) for supplying recombinant human IFNβ and anti–human IFNAR1 antibody, the staff of Hybrigenics for their contribution, G. Hikade for technical support, and R. Kurre for advice on fluorescence microscopy. This study was supported by NIH R01HL091549 and R01CA177305 to D.-E.Z. and SFB 944 from the DFG to J.P. and J.J.H.
Author information
Authors and Affiliations
Contributions
K.A., S.L., and S.A.S. designed, performed, and analyzed experiments, and wrote the manuscript; C.B., Y.Z., S.M., J.-B.F., S.W., J.J.H., Z.L., M.Y., S.P., and F.C. performed experiments or provided critical information; J.P. and D.-E.Z. conceived the project, designed experiments, analyzed experimental data, and wrote the manuscript.
Corresponding authors
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Cell micropatterning for probing the interaction of cytosolic effector proteins at the plasma membranes of living cells.
(a) Binary surface patterning by microcontact printing of poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) (structure in the center), which was either chemically coupled to RGD-peptide (I), (RGD peptide drawn in violet) or coupled to the HaloTag ligand (II), (HaloTag ligand (HTL) drawn in red) (left image). Concept of spatial organization of IFN receptor subunits in the plasma membrane of living cells cultured on the surface of a micropatterned coverslide. IFNAR2 (blue) fused to the HaloTag is captured into HTL-functionalized areas while cell attachment via focal adhesions is mediated by RGD-functionalized areas. Effector proteins such as STAT2 and USP18 are locally recruited to the receptor (right image). (b) HeLa cells transfected with HaloTag-mTagBFP-IFNAR2 (blue channel) and mEGFP (green channel) and TagRFP-T (orange channel) as a negative control. Scale bar: 10 μm. Representative images of 17 cells analyzed in two independent experiments. (c) Constitutive binding of STAT2 and USP18 to micropatterned IFNAR2. HeLa cells transfected with HaloTag-mTagBFP-IFNAR2 (blue channel), STAT2-TagRFP-T (orange channel) and mEGFP-USP18 (green channel). Scale bar: 10 μm. Representative images of 26 cells analyzed in two independent experiments. (d) Intensity profiles of STAT2-TagRFP-T, mEGFP-USP18, TagRFP-T, mEGFP and mTagBFP-IFNAR2 within the yellow ROI depicted in the images projected along the direction of the line pattern. (e) Dynamics of the STAT2 and USP18 interaction with micropatterned IFNAR2 in HeLa cells probed by FRAP and monoexponential fit of the recovery curves (representative of 5 cells analyzed). These time constants describe the exchange kinetics of prey proteins interacting with the immobilized bait and thus are a measure of complex stability, which can be considered rate-limiting because the high expression levels of unbound prey protein ensure fast association.
Supplementary Figure 2 Cooperation of STAT2 and USP18 in regulating ISG expression.
Stat2-/- MEFs or Stat2-expressing Stat2-/- MEFs were infected with vector control (-) or FLAG-Usp18 (+) retrovirus. These cells were treated with murine IFNβ (100 U/ml) for 0, 6 or 12 hours as indicated. Relative mRNA expression of the indicated IFN-inducible genes (a) Igtp, Ifit1, Gbp1, and Isg15 (b) Irf9, and Cxcl9 were examined by qRT-PCR analysis. Data are presented as mean ± S.D. of two independent experiments. Data are normalized to Gapdh was used as a reference gene.
Supplementary Figure 3 The N- and C-terminal regions of USP18 bind STAT2 and are important for inhibiting the IFN response.
(a) A schematic drawing of USP18 and its deletion mutants used in this study. The ability of a given deletion mutant to interact with STAT2 (+ or - binding) is indicated to the right.. (b) Both N-terminal and C-terminal regions of USP18 interact with STAT2. IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding STAT2-Myc and either FLAG-USP18 or the indicated deletion mutant. (c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding STAT2-FLAG and GFP or USP18 303-312 GFP as indicated. (d) MIP empty vector (-), MIP-FLAG-USP18 (USP18), or MIP-FLAG-USP18 303-312 deletion mutant (USP18∆303-312) expressing 2fTGH cells were treated with IFNα (1000 U/ml) for 15minutes. Cell lysates were analyzed by Western blotting with indicated antibodies. (e) MIP vector, USP18, or USP18 303-312 aa deletion mutant expressing KT-1 cells were treated with IFNα (1000 U/ml) for 30 minutes. Cell lysates were analyzed by Western blotting with indicated antibodies.
Supplementary Figure 4 The N- and C-terminal regions of USP18 bind IFNAR2 and are important for inhibiting the IFN response.
(a) A schematic drawing of USP18 and its deletion mutants used in this study. The ability of a given deletion mutant to interact with IFNAR2 (+ or - binding) is indicated to the right. (b) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GST-IFNAR2 ICD and mock, or FLAG-USP18 and its deletion mutants. (c) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding IFNAR2-FLAG and GFP or USP18 36-51 GFP. (d) IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after co-transfection with plasmids encoding GST-IFNAR2 ICD and mock, or FLAG-USP18 36-242aa, or FLAG-USP18 51-242aa. (e) 293T cells were co-transfected with plasmids encoding FLAG-STAT1 and FLAG-USP18 or its mutants as indicated. Twenty-four hours after transfection, cells were treated with IFNα (1000 U/ml) for 15minutes. Cell lysates were analyzed with indicated antibodies.
Supplementary Figure 5 Role of STAT2 in the recruitment of USP18 to IFNAR2, as probed by cell micropatterning.
(a) mEGFP-USP18 binding to micropatterned full-length HaloTag-IFNAR2 in HeLa cells. Cartoon of the assays (left) and total internal reflection fluorescence microscopy (TIRFM) image (right). The contrast within the highlighted area is shown below the TIRFM image. The red channel corresponds to fluorescent ATTO655IFNα2 for staining of IFNAR2. Representative image of 15-20 cells analyzed in two independent experiments. Scale bar: 10 μm. (b) mEGFP-USP18 (left) and JAK1-mEGFP (right) binding to micropatterned IFNAR2 truncated after residue 346, i.e. downstream of the JAK1 binding site. Representative images of 21 cells analyzed in two independent experiments. Scale bar: 10 μm. (c) USP18-mEGFP binding to micropatterned IFNAR2 truncated after residue 375. Representative images of 16 cells analyzed in two independent experiments. Scale bar: 10 μm. (d) Cell micropatterning assays with fragments of the intracellular IFNAR2 domain (aa 265-515) for probing binding of STAT2-TagRFP and mEGFP-USP18, respectively. The fragments used as bait are depicted at the top. (e) Contrast observed for STAT2 and USP18 binding to micropatterned IFNAR2 or IFNAR2 fragments fused to a transmembrane domain (TMD) as depicted in a and d, respectively. STAT2 and USP18 were either separately expressed (blue boxes) or co-expressed (orange and green boxes, respectively) in Hela cells. For statistical evaluation, 14-29 cells were analyzed in two independent experiments. n indicates number of cells used in each experiment. Significance was quantified using the two-sample Kolmogorov-Smirnov test. *** P ˂ 0.001, n.s. not significant. (f) Representative TIRFM images of 29 cells analyzed in two independent experiments obtained for a fragment comprising the minimum constitutive STAT2 binding site of IFNAR2. Scale bar: 10 μm.
Supplementary Figure 6 Binding and functionality of FITC-labeled IFNα.
(a) FITC-labeled IFNα was titrated in order to determine the optimal staining concentration for cell-binding assays. Mean fluorescence intensity (MFI) of FITC-labeled IFNα, at the indicated concentrations, bound to the surface of 2fTGH cells was analyzed by flow cytometry. For all additional experiments FITC-labeled IFNα was used at a saturating concentration of 20 nM. (b) Relative amount of FITC-labeled IFNα bound to the surface of the indicated cell lines was examined by flow cytometry. Data are normalized to the mean fluorescence intensity of 2fTGH cells. Data are presented as mean ± S.E.M. for 3 independent experiments. ****P < 0.0001. (c) FITC-labeled IFNα retains an equivalent biological effect to unlabeled IFNα. 2fTGH cells were treated with 20 nM IFNα or FITC-labeled IFNα for the indicated time and cell lysates were collected and analyzed by Western blotting with the indicated antibodies. (d) Interaction between USP18 and STAT2 WT or STAT2 Y690F. IB analysis of WCL and anti-FLAG IP derived from U6A cells 24 hours after transfection with STAT2-Myc or STAT2 Y690F-Myc and mock or FLAG-USP18.
Supplementary Figure 7 Analysis of the inhibitor peptides between STAT2 and USP18.
(a) Identification of STAT2 mutations that disrupt STAT2-IRF9 interaction. IB analysis of WCL and anti-FLAG IP derived from 293T cells 24 hours after transfection with FLAG-IRF9 and STAT2 CC/DB Myc and its mutants as indicated. (b) Cells indicated in Figure 7B were treated with IFNα (1000 U/ml) for 12 hours and then expression of IFIT1 was analyzed by q-PCR. Data represents mean ± S.D. for 2 independent experiments. (c) MIP or MIP-STAT2 CC/DB 3A expressing THP-1 cells were treated with IFNβ (1000 U/ml) for 48 hours and then Annexin V positive cells were analyzed by flow cytometry. Data represents mean ± S.E.M. for 3 independently generated stable cell lines. *P < 0.05. (d) THP-1 and KT-1 cells were treated with TAT or USP18 aa 302-313 TAT peptide. Two hours after peptide treatment, IFNα (1000 U/ml) was added for 12 hours and then expression of GBP1 was analyzed by q-PCR. Data represents mean ± S.D. for 2 independent experiments.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–7 (PDF 1126 kb)
Supplementary Data Set 1
Raw gel images for all western blot data in main figures. (PDF 725 kb)
Rights and permissions
About this article
Cite this article
Arimoto, Ki., Löchte, S., Stoner, S. et al. STAT2 is an essential adaptor in USP18-mediated suppression of type I interferon signaling. Nat Struct Mol Biol 24, 279–289 (2017). https://doi.org/10.1038/nsmb.3378
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3378
- Springer Nature America, Inc.
This article is cited by
-
Drug resistance mechanisms and treatment strategies mediated by Ubiquitin-Specific Proteases (USPs) in cancers: new directions and therapeutic options
Molecular Cancer (2024)
-
Novel role of bone morphogenetic protein 9 in innate host responses to HCMV infection
EMBO Reports (2024)
-
Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2
Nature Communications (2024)
-
Brain –cyst-driven genes expression in Toxoplasma Gondii Tehran strain: a parasitic-immunogenicity assessment by dint of RNA-Seq
Veterinary Research Communications (2024)
-
Tumour cells can escape antiproliferative pressure by interferon-β through immunoediting of interferon receptor expression
Cancer Cell International (2023)