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• W2033702347 abstract "The innate immune system detects pathogen- and host-derived double-stranded DNA exposed to the cytosol and induces type I interferon (IFN) and other cytokines. Here, we identified interferon-inducible tripartite-motif (TRIM) 56 as a regulator of double-stranded DNA-mediated type I interferon induction. TRIM56 overexpression enhanced IFN-β promoter activation after double-stranded DNA stimulation whereas TRIM56 knockdown abrogated it. TRIM56 interacted with STING and targeted it for lysine 63-linked ubiquitination. This modification induced STING dimerization, which was a prerequisite for recruitment of the antiviral kinase TBK1 and subsequent induction of IFN-β. Taken together, these results indicate that TRIM56 is an interferon-inducible E3 ubiquitin ligase that modulates STING to confer double-stranded DNA-mediated innate immune responses. The innate immune system detects pathogen- and host-derived double-stranded DNA exposed to the cytosol and induces type I interferon (IFN) and other cytokines. Here, we identified interferon-inducible tripartite-motif (TRIM) 56 as a regulator of double-stranded DNA-mediated type I interferon induction. TRIM56 overexpression enhanced IFN-β promoter activation after double-stranded DNA stimulation whereas TRIM56 knockdown abrogated it. TRIM56 interacted with STING and targeted it for lysine 63-linked ubiquitination. This modification induced STING dimerization, which was a prerequisite for recruitment of the antiviral kinase TBK1 and subsequent induction of IFN-β. Taken together, these results indicate that TRIM56 is an interferon-inducible E3 ubiquitin ligase that modulates STING to confer double-stranded DNA-mediated innate immune responses. TRIM56 expression is induced by dsDNA and poly (I:C) stimulation TRIM56 enhanced IFN-β promoter activation after dsDNA stimulation TRIM56 promoted K63-linked ubiquitination and dimerization of STING These modifications are required to form a complex with TBK1 and induce IFN-β The innate immune system has evolved as the first line of host defense against a variety of invading pathogens. The initiation of innate immune responses relies on the recognition of pathogen components by germline-encoded pattern-recognition receptors (PRRs), which include membrane-bound Toll-like receptors (TLRs) and cytosolic RIG-I-like receptors (RLRs), NOD-like receptors (NLRs; also known as nucleotide-binding domain and leucine-rich repeat containing molecules), and unidentified DNA sensors (Baccala et al., 2009Baccala R. Gonzalez-Quintial R. Lawson B.R. Stern M.E. Kono D.H. Beutler B. Theofilopoulos A.N. Sensors of the innate immune system: Their mode of action.Nat. Rev. Rheumatol. 2009; 5: 448-456Crossref PubMed Scopus (88) Google Scholar, Medzhitov, 2007Medzhitov R. Recognition of microorganisms and activation of the immune response.Nature. 2007; 449: 819-826Crossref PubMed Scopus (1803) Google Scholar, Schroder and Tschopp, 2010Schroder K. Tschopp J. The inflammasomes.Cell. 2010; 140: 821-832Abstract Full Text Full Text PDF PubMed Scopus (3460) Google Scholar, Takeuchi and Akira, 2010Takeuchi O. Akira S. Pattern recognition receptors and inflammation.Cell. 2010; 140: 805-820Abstract Full Text Full Text PDF PubMed Scopus (4727) Google Scholar, Yoneyama and Fujita, 2009Yoneyama M. Fujita T. RNA recognition and signal transduction by RIG-I-like receptors.Immunol. Rev. 2009; 227: 54-65Crossref PubMed Scopus (432) Google Scholar). Upon recognition, they initiate signal transduction pathways that lead to the induction of type I interferon (IFN) and proinflammatory cytokines, which are required for innate immune responses as well as for shaping subsequent adaptive immune responses. DNA derived from DNA viruses and bacteria as well as damaged host cells or cells that have escaped from apoptosis are triggers for innate immune responses (Hornung and Latz, 2010Hornung V. Latz E. Intracellular DNA recognition.Nat. Rev. Immunol. 2010; 10: 123-130Crossref PubMed Scopus (253) Google Scholar, Ishii and Akira, 2006Ishii K.J. Akira S. Innate immune recognition of, and regulation by, DNA.Trends Immunol. 2006; 27: 525-532Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, Nagata et al., 2010Nagata S. Hanayama R. Kawane K. Autoimmunity and the clearance of dead cells.Cell. 2010; 140: 619-630Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, Yanai et al., 2009Yanai H. Savitsky D. Tamura T. Taniguchi T. Regulation of the cytosolic DNA-sensing system in innate immunity: A current view.Curr. Opin. Immunol. 2009; 21: 17-22Crossref PubMed Scopus (45) Google Scholar). Although TLR9 is known to recognize CpG DNA derived from viruses and bacteria in the endolysosome, there is evidence that double-stranded (ds) DNA delivered to the cytoplasm is recognized by one or more cytosolic sensors (Ishii et al., 2006Ishii K.J. Coban C. Kato H. Takahashi K. Torii Y. Takeshita F. Ludwig H. Sutter G. Suzuki K. Hemmi H. et al.A Toll-like receptor-independent antiviral response induced by double-stranded B-form DNA.Nat. Immunol. 2006; 7: 40-48Crossref PubMed Scopus (618) Google Scholar, Stetson and Medzhitov, 2006Stetson D.B. Medzhitov R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response.Immunity. 2006; 24: 93-103Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, Yoshida et al., 2005Yoshida H. Okabe Y. Kawane K. Fukuyama H. Nagata S. Lethal anemia caused by interferon-beta produced in mouse embryos carrying undigested DNA.Nat. Immunol. 2005; 6: 49-56Crossref PubMed Scopus (269) Google Scholar). DNA-dependent activator of IFN-regulatory factors (DAI) (also known as ZBP1 or DLM-1) was identified as an intracellular dsDNA sensor that binds to dsDNA and increases type I IFN induction (Takaoka et al., 2007Takaoka A. Wang Z. Choi M.K. Yanai H. Negishi H. Ban T. Lu Y. Miyagishi M. Kodama T. Honda K. et al.DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response.Nature. 2007; 448: 501-505Crossref PubMed Scopus (1170) Google Scholar). However, a dispensable role of DAI in dsDNA-elicited innate and adaptive immune responses in vivo was revealed by a study on DAI-deficient mice (Ishii et al., 2008Ishii K.J. Kawagoe T. Koyama S. Matsui K. Kumar H. Kawai T. Uematsu S. Takeuchi O. Takeshita F. Coban C. Akira S. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines.Nature. 2008; 451: 725-729Crossref PubMed Scopus (461) Google Scholar). It was also suggested that AT-rich dsDNA serves as a template for transcription to dsRNA by RNA polymerase III, which is recognized by the RLR member RIG-I, suggesting that RIG-I recognizes intermediates of dsDNA rather than dsDNA itself (Ablasser et al., 2009Ablasser A. Bauernfeind F. Hartmann G. Latz E. Fitzgerald K.A. Hornung V. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate.Nat. Immunol. 2009; 10: 1065-1072Crossref PubMed Scopus (602) Google Scholar, Chiu et al., 2009Chiu Y.H. Macmillan J.B. Chen Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway.Cell. 2009; 138: 576-591Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar). IFN-inducible absent in melanoma 2 (AIM2), which contains a pyrin and a HIN-200 DNA-binding domain, was identified to bind to dsDNA and form an ASC-containing inflammasome, which triggers caspase-1-dependent IL-1β production rather than type I IFN (Bürckstümmer et al., 2009Bürckstümmer T. Baumann C. Blüml S. Dixit E. Dürnberger G. Jahn H. Planyavsky M. Bilban M. Colinge J. Bennett K.L. Superti-Furga G. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome.Nat. Immunol. 2009; 10: 266-272Crossref PubMed Scopus (753) Google Scholar, Fernandes-Alnemri et al., 2009Fernandes-Alnemri T. Yu J.W. Datta P. Wu J. Alnemri E.S. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA.Nature. 2009; 458: 509-513Crossref PubMed Scopus (1189) Google Scholar, Hornung et al., 2009Hornung V. Ablasser A. Charrel-Dennis M. Bauernfeind F. Horvath G. Caffrey D.R. Latz E. Fitzgerald K.A. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC.Nature. 2009; 458: 514-518Crossref PubMed Scopus (1572) Google Scholar, Roberts et al., 2009Roberts T.L. Idris A. Dunn J.A. Kelly G.M. Burnton C.M. Hodgson S. Hardy L.L. Garceau V. Sweet M.J. Ross I.L. et al.HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.Science. 2009; 323: 1057-1060Crossref PubMed Scopus (623) Google Scholar). Collectively, these findings suggest the existence of unknown cytosolic PRRs that sense dsDNA and induce type I IFN production. Stimulator of interferon genes (STING) (also known as MITA, ERIS, or MPYS) was identified as a molecule that activates the IFN-β promoter after overexpression and is a transmembrane protein that resides in the ER and/or mitochondria (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1545) Google Scholar, Jin et al., 2008Jin L. Waterman P.M. Jonscher K.R. Short C.M. Reisdorph N.A. Cambier J.C. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals.Mol. Cell. Biol. 2008; 28: 5014-5026Crossref PubMed Scopus (262) Google Scholar, Sun et al., 2009Sun W. Li Y. Chen L. Chen H. You F. Zhou X. Zhou Y. Zhai Z. Chen D. Jiang Z. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.Proc. Natl. Acad. Sci. USA. 2009; 106: 8653-8658Crossref PubMed Scopus (453) Google Scholar, Zhong et al., 2008Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. Shu H.B. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). Cells derived from STING-deficient mice fail to induce type I IFN production in response to dsDNA and infection with herpes simplex virus 1 (HSV-1) and Listeria monocytogenes (L. monocytogenes) that deliver DNA to the host cytosol. STING-deficient mice are also susceptible to HSV-1 infection (Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1354) Google Scholar). In addition, STING-deficient mice show a remarkable reduction in cytotoxic T cell responses after plasmid DNA vaccination (Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1354) Google Scholar), indicating that STING is an essential component of dsDNA-mediated immune responses in vivo. Furthermore, STING-deficient cells display a considerable reduction in type I IFN production after infection with the negative single-stranded RNA virus vesicular stomatitis virus (VSV) that is recognized by RIG-I (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1545) Google Scholar). STING interacts with IPS-1 (also known as MAVS, CARDIF, or VISA), an essential adaptor for RLRs, at mitochondria (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1545) Google Scholar, Zhong et al., 2008Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. Shu H.B. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). Thus, in addition to its pivotal role in the responses to cytosolic DNA, STING plays a pivotal role in RIG-I-mediated antiviral innate immune responses. In response to dsDNA stimulation, STING becomes relocalized to punctate structures, where the kinase TBK1 is recruited (Ishikawa and Barber, 2008Ishikawa H. Barber G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling.Nature. 2008; 455: 674-678Crossref PubMed Scopus (1545) Google Scholar, Saitoh et al., 2009Saitoh T. Fujita N. Hayashi T. Takahara K. Satoh T. Lee H. Matsunaga K. Kageyama S. Omori H. Noda T. et al.Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response.Proc. Natl. Acad. Sci. USA. 2009; 106: 20842-20846Crossref PubMed Scopus (487) Google Scholar, Zhong et al., 2008Zhong B. Yang Y. Li S. Wang Y.Y. Li Y. Diao F. Lei C. He X. Zhang L. Tien P. Shu H.B. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation.Immunity. 2008; 29: 538-550Abstract Full Text Full Text PDF PubMed Scopus (844) Google Scholar). It is likely that this recruitment induces TBK1 activation that results in phosphorylation of the transcription factor IRF3 and transcription of type I IFN genes during dsDNA-driven signaling. However, it remains to be defined how STING activation is controlled. In the present study, we performed a functional screening and identified tripartite-motif (TRIM) 56. TRIM56 was induced by type I IFN and its overexpression enhanced dsDNA-mediated type I IFN induction. TRIM56 interacted with STING and promoted STING ubiquitination, which was a prerequisite for TBK1 recruitment and type I IFN induction. To search for molecules involved in the innate immune responses to cytosolic DNA, we carried out an expression screening of a mouse carcinoma cDNA library. HEK293 cells were transiently transfected with a plasmid derived from the library along with an IFN-β promoter-luciferase reporter plasmid. The cells were then stimulated with dsDNA [poly (dA:dT)-poly (dT:dA)], herein referred to as poly (dA:dT), or dsRNA [poly (I:C)] by transfection, followed by measurement of the luciferase expression by a reporter assay. In a primary screening of around 1000 clones, we obtained one clone that displayed a marked increase in luciferase expression compared with the control plasmid-transfected cells (Figure 1A ). This clone contained an open reading frame for TRIM56, a member of the TRIM family proteins (accession numbers BC045615, BG976873) (Figure 1B). Mouse TRIM56 contains an open reading frame of 2205 bases, encoding 734 amino acids. TRIM56 contains a RING domain, B-box domain, and coiled-coil domain in the N-terminal region (Figure 1B). The C-terminal region of TRIM56 did not share considerable homology with the C-terminal regions of other TRIMs. Expression of TRIM56 was upregulated after stimulation with poly (I:C) and poly (dA:dT) as evaluated by RT-PCR analysis (Figure 1C), consistent with a previous report showing that TRIM56 is IFN inducible (Carthagena et al., 2009Carthagena L. Bergamaschi A. Luna J.M. David A. Uchil P.D. Margottin-Goguet F. Mothes W. Hazan U. Transy C. Pancino G. Nisole S. Human TRIM gene expression in response to interferons.PLoS ONE. 2009; 4: e4894Crossref PubMed Scopus (182) Google Scholar). We constructed an expression plasmid encoding FLAG-tagged TRIM56 to confirm its capability of increasing the IFN-β promoter activity. Overexpression of FLAG-TRIM56 alone into primary mouse embryonic fibroblast (MEF) cells resulted in 8-fold increases in the reporter gene expression (Figure 1D). Furthermore, FLAG-TRIM56 overexpression enhanced poly (dA:dT)- and poly (I:C)-stimulated IFN-β promoter activation (Figure 1D). Similarly, enhanced IFN-β promoter activation by TRIM56 was also observed in HEK293 cells (Figure S1A available online). Moreover, TRIM56 overexpression also increased the IFN-β promoter activation induced by 5,6-dimethylxanthenone-4-acetic acid (DMXAA), an antitumor agent that induces type I IFN via TBK1 and IRF3 (Figure S1B; Roberts et al., 2007Roberts Z.J. Goutagny N. Perera P.Y. Kato H. Kumar H. Kawai T. Akira S. Savan R. van Echo D. Fitzgerald K.A. et al.The chemotherapeutic agent DMXAA potently and specifically activates the TBK1-IRF-3 signaling axis.J. Exp. Med. 2007; 204: 1559-1569Crossref PubMed Scopus (110) Google Scholar). Next, we transduced a retroviral vector encoding TRIM56 into MEF cells to investigate whether TRIM56 regulates innate immune responses to poly (dA:dT). We infected MEF cells derived from TBK1-deficient mice with retrovirus encoding TBK1 and found that TBK1 transduction restored poly (dA:dT) stimulation-induced production of IP-10, an IFN-inducible chemokine (Figure 1E). Notably, cotransduction of TBK1 together with TRIM56 markedly enhanced IP-10 production in response to poly (dA:dT) stimulation (Figure 1E). These findings suggested that TRIM56 positively regulates dsDNA-mediated IFN-β induction. To address the physiological function of TRIM56 in dsDNA-induced responses, we employed a knockdown strategy involving two distinct siRNAs targeting the human TRIM56 gene (Tr56-1 and Tr56-2). HEK293 cells treated with these siRNAs displayed a considerable reduction in TRIM56 expression, as confirmed by RT-PCR (Figure 2A , top) and immunoblot (Figure 2A, bottom) analyses. These siRNAs also decreased TRIM56 expression in HeLa cells (data not shown). In TRIM56 knockdown cells, IFN-β and IFN-stimulation responsive element (ISRE) promoter activation was significantly reduced after poly (dA:dT) transfection (Figure 2B). IFN-β promoter activation induced by poly (I:C) was also reduced by TRIM56 knockdown (Figure 2C). Moreover, poly (dA:dT)-induced upregulation of IFNB1 and CXCL10 was reduced in TRIM56-knockdown HEK293 cells (Figure 2D). CXCL10 mRNA induction after Listeria monocytogenes infection was also reduced in TRIM56-knockdown HeLa cells (Figure 2E). Furthermore, TRIM56 knockdown in HEK293 cells resulted in impaired IRF3 nuclear translocation by poly (dA:dT) (Figure 2F). These results strongly suggest that TRIM56 is involved in poly (dA:dT) and poly (I:C)-induced type I IFN production. We next investigated whether TRIM56 is required for poly (dA:dT)-mediated responses in normal human cells. We nucleofected normal human lung fibroblasts with control siRNA or siRNA for TRIM56 (Tr56-1) and found that the amount of TRIM56 mRNA was considerably reduced in Tr56-1-nucleofected cells compared with control siRNA-nucleofected cells (Figure 2G, top). TRIM56 knockdown abrogated IFNB1 and IL-6 mRNA induction by poly (dA:dT) (Figure 2G, top) and CXCL10 and CCL5 (RANTES) induction after infection with Newcastle disease virus (NDV) that stimulates the RIG-I pathway (Figure 2G, bottom). We then examined an involvement of TRIM56 in poly (dA:dT)- and poly (I:C)-induced IFN-β promoter activation by a reporter assay. In TRIM56 knocked down cells, poly (dA:dT) and poly (I:C) stimulation failed to enhance IFN-β promoter activity (Figure 2H). Thus, TRIM56 is required for poly (dA:dT)- and poly (I:C)-mediated type I IFN induction in human primary cells. To determine the cellular localization of TRIM56, we expressed RFP-TRIM56 in HeLa cells via a retrovirus system. We found that RFP-TRIM56 was localized to the cytoplasm in the unstimulated condition (Figure 3A ). In response to poly (dA:dT) and DMXAA stimulation, RFP-TRIM56 displayed a punctate pattern in the cytoplasm (Figure 3A). We found that TRIM56 was not localized at the ER, endosome, peroxisome, or mitochondria in unstimulated and poly (dA:dT)-stimulated cells (Figures S2A–S2D). The findings that TRIM56 is an IFN-inducible gene and controls the poly (dA:dT)-mediated pathway suggest that TRIM56 may serve as an intracellular sensor for dsDNA. We therefore examined whether or not TRIM56 colocalizes with poly (dA:dT). We coexpressed RFP-TRIM56 along with FITC-labeled poly (dA:dT) into HeLa cells and examined their localization. There was no colocalization between RFP-TRIM56 and FITC-labeled poly (dA:dT) in HeLa cells (Figure 3B). We next tested interaction between TRIM56 and poly (dA:dT). To this end, we precipitated cell lysates prepared from HEK293 cells overexpressing FLAG-human TRIM56, FLAG-mouse TRIM56, FLAG-p202, or FLAG-IPS-1 with biotin-conjugated poly (dA:dT) plus streptavidin agarose, and blotted with FLAG antibody. FLAG-p202 was used as a positive control because p202 (also known as IFI202b) was identified to bind to dsDNA (Roberts et al., 2009Roberts T.L. Idris A. Dunn J.A. Kelly G.M. Burnton C.M. Hodgson S. Hardy L.L. Garceau V. Sweet M.J. Ross I.L. et al.HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA.Science. 2009; 323: 1057-1060Crossref PubMed Scopus (623) Google Scholar). We found that both human and mouse TRIM56 did not associate with poly (dA:dT) (Figure 3C). Collectively, these findings suggest that TRIM56 is unlikely to be a DNA sensor. Next, we addressed the issue of whether TRIM56 is involved in STING- and IPS-1-mediated IFN-β induction. IFN-β promoter activation induced by STING and IPS-1 overexpression was significantly reduced in TRIM56 knockdown cells (Figure 4A ). In contrast, IFN-β promoter activation induced by IRF3 overexpression was unimpaired in TRIM56 knockdown cells (Figure 4A), suggesting that TRIM56 is required for the function of STING and IPS-1. IFN-β promoter activation induced by STING overexpression was increased by coexpression of TRIM56 but not TRIM29 (Figure 4B). Furthermore, IPS-1-mediated IFN-β promoter activation was also increased by TRIM56 overexpression (Figure 4B). In contrast, TRIM56 did not enhance TRIF-mediated IFN-β promoter activation (Figure 4B). We further evaluated the interaction between TRIM56 and STING by coimmunoprecipitation assays. FLAG-STING was coprecipitated with TRIM56 antibody but not with two control antibodies (Figure 4C), indicating that endogenous TRIM56 forms a complex with FLAG-STING. We then searched for the region of TRIM56 that was responsible for the interaction with STING by constructing several deletion mutants and found that the STING C-terminal region was coprecipitated with Myc-TRIM56 FL (encoding full-length TRIM56), Myc-TRIM56 ΔR (lacking the RING finger domain), and Myc-TRIM56 ΔN (lacking the RING-finger and B-box domains) but not with Myc-TRIM56 ΔC (encoding the N-terminal RING-finger and B-box domains) (Figure 4D), indicating that the C-terminal region of TRIM56 is necessary for the interaction with the C-terminal region of STING. Unlike STING, we were unable to detect an interaction between TRIM56 and IPS-1 (data not shown). STING was previously shown to display punctate structures in response to poly (dA:dT) stimulation. This finding prompted us to investigate whether TRIM56 colocalizes with STING. To this end, we coexpressed GFP-STING and RFP-TRIM56 and determined their cellular localizations after poly (dA:dT) stimulation. GFP-STING displayed punctate structures, and several dots were merged with those of RFP-TRIM56 (Figure S3A). It was reported that STING becomes relocalized from the ER to perinuclear vesicles containing exocyst components (Ishikawa et al., 2009Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1354) Google Scholar, Saitoh et al., 2009Saitoh T. Fujita N. Hayashi T. Takahara K. Satoh T. Lee H. Matsunaga K. Kageyama S. Omori H. Noda T. et al.Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response.Proc. Natl. Acad. Sci. USA. 2009; 106: 20842-20846Crossref PubMed Scopus (487) Google Scholar). However, colocalization between TRIM56 and exocyst complex component 2 (EXOC2) (also known as SEC5) was not observed (Figure S3B). These findings suggest that TRIM56 is localized with STING after poly (dA:dT) stimulation and rapidly removed before STING relocalizes to exocyst. In HEK293 cells transiently transfected with FLAG-STING, immnoblotting with anti-FLAG showed two bands with sizes of approximately 40 and 80 kDa. The 80 kDa band disappeared when anti-FLAG immunoprecipitates were treated with 8M urea, indicating that the 80 kDa band corresponds to a STING dimer, which was consistent with a previous report (Figure S4A; Sun et al., 2009Sun W. Li Y. Chen L. Chen H. You F. Zhou X. Zhou Y. Zhai Z. Chen D. Jiang Z. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization.Proc. Natl. Acad. Sci. USA. 2009; 106: 8653-8658Crossref PubMed Scopus (453) Google Scholar). Notably, the 80 kDa band was increased by poly (dA:dT) stimulation and slightly increased by poly (I:C) stimulation (Figure 5A ). Moreover, the anti-FLAG detected smeared bands over the 80 kDa band region in the poly (dA:dT)-stimulated cells (Figure 5A). These results suggest that poly (dA:dT) stimulation induces STING dimerization as well as modifications such as ubiquitination. Immunoblot analysis with ubiquitin antibody indicated that poly (dA:dT) stimulation increased the ubiquitination of anti-FLAG-STING immunoprecipitates, which was impaired by TRIM56 knockdown (Figure 5B, top). Furthermore, TRIM56 knockdown abrogated poly (dA:dT)-induced smeared bands over the 80 kDa band region as detected by anti-FLAG (Figure 5B, bottom). These results suggest that poly (dA:dT) stimulation triggered ubiquitination of STING or STING-associated proteins in a manner dependent on TRIM56. We overexpressed FLAG-STING along with or without Myc-TRIM56, and STING ubiquitination was examined by immunoblot analysis via ubiquitin antibody. Ubiquitination of FLAG-STING was induced in cells expressing TRIM56 (Figure 5C, top). Moreover, an amount of the 80 kDa band corresponding to the dimer of FLAG-STING in TRIM56-expressing cells was increased compared with that in control cells (Figure 5C, middle). Immunoblot analysis via ubiquitin lysine (K) 63 antibody indicated that TRIM56 overexpression increased the STING ubiquitination linked to K63 (Figure 5D). By contrast, TRIM56 overexpression did not affect the K48-linked ubiquitination of STING as determined by immunoblot analysis via ubiquitin K48 antibody (data not shown). These results suggest that TRIM56 preferentially catalyzes K63-linked ubiquitination of STING. We denatured anti-FLAG immunoprecipitates of cell lysates prepared from cells transfected with FLAG-STING and Myc-TRIM56 by boiling in a buffer containing 1% SDS, followed by reimmunoprecipitated with anti-FLAG and analyzed by immunoblotting with antiubiquitin. In this condition, ubiquitination was still observed (Figure S4B), which strongly suggests that ubiquitin chains are covalently attached to STING. It was recently demonstrated that RIG-I binds to unanchored K63-linked polyubiquitin chains that are likely to be synthesized by TRIM25, activating downstream signaling (Zeng et al., 2010Zeng W. Sun L. Jiang X. Chen X. Hou F. Adhikari A. Xu M. Chen Z.J. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity.Cell. 2010; 141: 315-330Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). This suggests the possibility that in addition to covalent attachment of ubiquitin, STING is also associated with unanchored ubiquitin chains. In this regard, we lysed cells transfected with FLAG-STING and Myc-TRIM56 in the regular lysis buffer that did not contain SDS, treated anti-FLAG immunoprecipitates with Isopeptidase T (Iso T; also known as USP5) that removes unanchored ubiquitin chains, and blotted with anti-ubiquitin. Iso T treatment decreased the amount of ubiquitination (Figure S4C), suggesting that STING is associated with unanchored ubiquitin chains or their associated proteins such as RIG-I under nondenatured condition. To determine the lysine residue(s) in STING that are critical for TRIM56-mediated ubiquitination, we constructed a series of FLAG-STING mutants in which individual lysine residues were substituted with an arginine residue (K20R, K137R, and K150R). HA-ubiquitin and Myc-TRIM56 were coexpressed with FLAG-STING or STING mutants in HEK293 cells, and anti-FLAG immunoprecipitates were immunoblotted with HA antibody. The amount of ubiquitination was unaffected by mutation of lysine 20 or 137, but was severely abrogated by mutation of lysine 150 (Figure 6A ). Notably, immunoblot analysis with anti-FLAG demonstrated that STING K150R failed to form a dimer (Figure 6A). Moreover, poly (dA:dT) stimulation resulted in increased dimer formation as well as ubiquitination of STING, and these increases were abrogated by mutation of lysine 150 (Figure 6B). We next performed coimmunoprecipitation assay to examine whether STING lysine 150 is required for the dimer formation. When FLAG-STING and Myc-STING were expressed together in HEK293 cells, FLAG-STING was coprecipitated with Myc antibody, indicating that FLAG-STING interacts with Myc-STING. In contrast, interaction between FLAG-STING K150R and Myc-STING K150R was reduced (Figure 6C). Furthermore, poly (dA:dT) stimulation failed to induce the interaction between FLAG-STING K150R and Myc-STING K150R (data not shown). These results strongly suggest that STING lysine 150 is the major acceptor site for the TRIM56-mediated K63-linked ubiquitin chains and that this lysine residue is required for the dimer formation. Next, we addressed whether STING ubiquitination is linked to IFN-β induction. Overexpression of STING K150R failed to activate the IFN-β promoter whereas the activation induced by STING, STING K20R, and STING K137R were comparable (Figure 6D). Moreover, STING K150R remained unable to enhance IFN-β promoter activation even when coexpressed with TRIM56 (Figure 6E), and it acted as a dominant-negative for" @default.
• W2033702347 created "2016-06-24" @default.
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• W2033702347 creator A5003936795 @default.
• W2033702347 creator A5013773246 @default.
• W2033702347 creator A5018553332 @default.
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• W2033702347 date "2010-11-01" @default.
• W2033702347 modified "2023-10-16" @default.
• W2033702347 title "The Ubiquitin Ligase TRIM56 Regulates Innate Immune Responses to Intracellular Double-Stranded DNA" @default.
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