A novel STING1 variant causes a recessive form of STING-associated vasculopathy with onset in infancy (SAVI)
Authors: Bin Lin, Roberta Berard, Abdulrahman Al Rasheed, Buthaina Aladba, Philip J. Kranzusch, Maggie Henderlight, Alexi Grom, Dana Kahle, Sofia Torreggiani, Alexander G. Aue, Jacob Mitchell, Adriana A. de Jesus, Grant S. Schulert, Raphaela Goldbach-Mansky
Journal: Journal of Allergy and Clinical Immunology (2020)

Stimulator of interferon response genes (STING) encoded by stimulator of interferon response cGAMP interactor 1 (STING1), previously known as transmembrane protein 173 (TMEM173) is an important pattern recognition receptor that detects microbial dinucleotides and functions as an adaptor molecule in the cytosolic DNA sensing pathway that binds 2'3'-cyclic GMP-AMP (cGAMP), which is generated when cytosolic DNA activates cyclic GMP-AMP synthase (cGAS).1Ishikawa H. Ma Z. Barber G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity.Nature. 2009; 461: 788-792Crossref PubMed Scopus (1674) Google Scholar,2Ablasser A. Chen Z.J. cGAS in action: Expanding roles in immunity and inflammation.Science. 2019; 363Crossref PubMed Scopus (352) Google Scholar STING activation stimulates the induction of type I interferons, which activate interferon responses. Gain-of-function (GOF) variants in STING1 lead to autoactivation without ligand binding and cause a rare autoinflammatory disease named STING-associated vasculopathy with onset in infancy (SAVI) (Online Mendelian Inheritance in Man catalog no. 615934).3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar,4Jeremiah N. Neven B. Gentili M. Callebaut I. Maschalidi S. Stolzenberg M.C. et al.Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations.J Clin Invest. 2014; 124: 5516-5520Crossref PubMed Scopus (337) Google Scholar Patients with SAVI present in infancy with the following symptoms: recurrent fevers; cold-induced skin vasculitis that can progress to tissue loss and amputation of fingers and toes; and/or interstitial lung disease, which is the main cause of the mortality that often occurs before patients reach adulthood. So far, all reported cases of SAVI have been caused by autosomal dominant variants, with most of them occurring de novo. We have identified 6 patients from 4 unrelated families, all of whom are of Arabic ethnicity and harbor pathogenic STING1 variants that are disease causing only in homozygosity. The patients had clinical disease suggestive of SAVI and were enrolled into institutional review board–approved protocols, including the National Institutes of Health natural history protocol (NCT02974595). Patient 1, the index patient, presented at 4 weeks of age with a cough and failure to thrive, as well as with a maculopapular violaceous rash with a livedoid appearance (Fig 1, A and B). A chest computed tomography scan showed diffuse bilateral parenchymal opacities. His lung disease progressed despite steroid therapy and short-term treatment with the JAK inhibitor tofacitinib, and he died of respiratory failure at 5 months of age. His older brother (patient 2) died at 18 months of age with chronic cough and failure to thrive. Although genetic testing was not performed, the clinical manifestations and similarities of patient 2 to those of his younger brother strongly suggest that he had SAVI. Patient 3 presented at 3 months of age with recurrent fever, erythematous rash, cough, and dyspnea, ultimately progressing to oxygen dependence. He had a chest computed tomography scan with results consistent with interstitial lung disease and a lung biopsy specimen that showed chronic interstitial pneumonitis with intraalveolar hemorrhage. He is currently taking the JAK inhibitor ruxolitinib. His brother, patient 4, had respiratory symptoms starting at the age of 6 months; he was diagnosed with SAVI at the age of 15 months and was initially treated with steroids, after which baricitinib (a selective JAK1/2 inhibitor) was added to his treatment regimen. Patient 5 presented at the age of 2 months with cough, tachypnea, and recurrent lung infections; she was diagnosed with chronic aspiration pneumonia and had a laryngeal cleft that was repaired at the age of 4.5 years. Because of her lung disease (Fig 1, E), she has required supplemental oxygen since the age of 7 months and bilevel positive airway pressure support while sleeping since the second year of life, when digital clubbing was noticed. She developed pulmonary hypertension by the age of 4 years. At the age of 5 she developed polyarthritis, an erythematous rash over the soles of her feet and clubbing. She began taking baricitinib at the age of 7 years; there was clinical improvement, but her oxygen dependence continued. Patient 6 presented at the age of 8 months with a history of persistent tachypnea and failure to thrive, polyarthritis, intermittent vasculitic rashes, and clubbing (Fig 1, C and D). All of the parents and siblings were asymptomatic with normal inflammatory markers. Additional clinical and laboratory information is available in Table E1 (in this article's Online Repository at www.jacionline.org). Patients 1, 3, and 4 underwent targeted sequencing of STING1, whereas whole exome sequencing was performed on families 3 and 4. Sequencing of all patients revealed a homozygous STING1 (NM_198282) variant c.841C>T, p.Arg281Trp, p.R281W (Fig 1, F). This variant was present in heterozygosity in only 2 of 282,822 alleles reported in gnomAD (gnomad.broadinstitute.org) and is predicted to be damaging by PolyPhen-2 and deleterious by the scale-invariant feature transform algorithm (SIFT), and it has a Combined Annotation-Dependent Depletion (CADD)-PHRED-scaled score of 26.4. This variant has not been reported previously in patients with SAVI (https://infevers.umai-montpellier.fr). All of the parents, as well as several of the unaffected siblings, were heterozygous carriers of this variant (Fig 1, F). A variant affecting the same amino acid residue but mutated to a glutamine (c.842G>A, p.Arg281Gln, p.R281Q) has previously been described to cause an autosomal dominant form of SAVI5Melki I. Rose Y. Uggenti C. Van Eyck L. Fremond M.L. Kitabayashi N. et al.Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.J Allergy Clin Immunol. 2017; 140: 543-552.e5Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar (Fig 1, G). A strikingly high interferon response gene signature and systemic inflammation in patients with SAVI has led to their designation as having an autoinflammatory interferonopathy.6Kim H. de Jesus A.A. Brooks S.R. Liu Y. Huang Y. VanTries R. et al.Development of a validated interferon score using nanostring technology.J Interferon Cytokine Res. 2018; 38: 171-185Crossref PubMed Scopus (76) Google Scholar A standardized interferon response gene score was elevated only in patients who were homozygous for the p.R281W variant and who had clinical disease, and not in the siblings and parents who were heterozygous carriers of the variant or in healthy controls (Fig 1, H). Transfection of HEK293T cells with the STING1 construct containing the R281W mutation led to activation of IFNB1 luciferase reporter without ligand binding (see Fig E1 in this article's Online Repository at www.jacionline.org), indicating that p.R281W is a pathogenic GOF variant. The mutant remained responsive to cGAMP (see Fig E1), suggesting no effect on ligand binding. Disease-causing SAVI variants3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar cluster in 2 areas. The cryoelectron microscopy structure model of STING7Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (69) Google Scholar has mapped the class 1 variants, p.N154S, p.V155M, and p.V147L to the connector helix loop (Fig 1, G) which controls a cGAMP ligand–induced 180o rotation of the ligand-binding area of the STING dimer that induces polymerization and activation. The class 3 variants, including p.R281Q, p.R284G5Melki I. Rose Y. Uggenti C. Van Eyck L. Fremond M.L. Kitabayashi N. et al.Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.J Allergy Clin Immunol. 2017; 140: 543-552.e5Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar and p.R284S,8Konno H. Chinn I.K. Hong D. Orange J.S. Lupski J.R. Mendoza A. et al.Pro-inflammation associated with a gain-of-function mutation (R284S) in the innate immune sensor STING.Cell Rep. 2018; 23: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar are located at the polymerization interface7Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (69) Google Scholar (Fig 1, G) and have been postulated to bind a C-terminal tail that might prevent polymerization and autoactivation.9Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Concordant with previous reports, we hypothesized that the p.R281W variant confers a GOF that is weaker than the heterozygous SAVI variants. To test this possibility, we transfected HEK293T cells with either wild-type or mutant STING constructs including R281W at increasing concentrations and assessed IFNB1 reporter activity (Fig 2, A). At equal transfection efficiency, consistent with the homozygosity requirement at this location, R281W mutant constructs exhibited lower autoactivation than R281Q constructs did but were similar to mutant constructs with the pathogenic V155M variant. This suggests differential autoactivation thresholds for disease-causing class 1 variants at the connector helix loop (ie, V155M) versus class 3 variants at the polymer interface (ie, R281 and R284). In line with this hypothesis, pathogenic variants at the class 3 residues that cause disease in heterozygosity, including R281Q, R284S, and R284G, are all more autoactivating than V155M in the transfection model (Fig 2, A and B), with R284S, R284G, and G158A constituting the top 3 autoactivating SAVI variants (Fig 2, B). Although class 1 disease-causing SAVI variants are GOF variants, mutating these residues into charged amino acids (eg, V155R3Liu Y. Jesus A.A. Marrero B. Yang D. Ramsey S.E. Sanchez G.A.M. et al.Activated STING in a vascular and pulmonary syndrome.N Engl J Med. 2014; 371: 507-518Crossref PubMed Scopus (817) Google Scholar and G158E8Konno H. Chinn I.K. Hong D. Orange J.S. Lupski J.R. Mendoza A. et al.Pro-inflammation associated with a gain-of-function mutation (R284S) in the innate immune sensor STING.Cell Rep. 2018; 23: 1112-1123Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) leads to loss of function, likely through destabilizing the STING dimer structure. In contrast, none of the 6 different amino acid mutations at position R281 caused loss of function (Fig 2, B [green labels]). In fact, all of the mutations except for R281A, which is similar to the wild type, led to autoactivation. Maintenance of a positive charge at residue R281 (by mutating into lysine [K] or histidine [H]) was insufficient to keep STING inactive at the basal state. Moreover, mutations into hydrophobic amino acids such as tryptophan (W), leucine (L), or methionine (M), or into negatively charged amino acids (including glutamic acid [E]), cause autoactivation (Fig 2, A and B). The autoactivation was not due to differential protein expression in the transfection assay (see Fig E2 in this article's Online Repository at www.jacionline.org). The R281 and R284 mutations that were assessed remained responsive to cGAMP stimulation (Fig 2, B and see Fig E1), indicating no effect on ligand binding. The broad autoactivation of the R281 and R284 mutations was consistent with the model of polymer interface binding to an inhibitor to suppress STING autoactivation.9Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar Although this potential inhibitor is not addressed in the 180o rotation model of the connector helix loop mutations, it is quite likely that the 180o rotation also leads to loss of inhibitor binding on the polymer interface, thus allowing side-by-side packing. Our data thus suggest a possible common mechanism to reconcile the 2 previously reported structural models (Fig 2, C and see also the Discussion section for detailed description of the model shown in Fig E3 in this article's Online Repository at www.jacionline.org), which is supported by similar superactivating mutations in both the connector helix loop (G158A) and the polymer interface (R281E, R284G, and R284S). In summary, we have reported 6 patients from 4 unrelated families with recessively inherited STING1 variants and characteristic clinical features of SAVI. Our data support the notion that the disease-causing homozygous p.R281W variant causes GOF but is weaker in activating STING than the previously reported heterozygous variants, and it requires biallelic mutations to constitutively activate STING. We have demonstrated a critical role of residue R281 in maintaining STING in an inactive state, likely without affecting dimer conformation. Our data unveil limitations of the current structural models that cannot explain the superactivating potential of the R281 and R284 mutations and raise questions as to whether class 3 residues represent an autoinhibitory domain or a binding site for an external inhibitor of STING. Considering the critical function of R281 and the nearby region, novel therapeutic options may arise from high-throughput screens of drugs that bind to this area and inhibit polymerization. We thank the patients and their families for participating in this study. All patients were enrolled into the institutional review board–approved National Institutes of Health natural history protocol Studies of the Natural History, Pathogenesis, and Outcome of Autoinflammatory Diseases (NOMID/CAPS, DIRA, CANDLE, SAVI, NLRC4-MAS, Still's-like Diseases, and Other Undifferentiated Autoinflammatory Diseases) (17-I-0016; NCT02974595). Parents gave written informed consent for their children in accordance with the Declaration of Helsinki. HEK293T cells were cotransfected with STING constructs and an IFNB1 firefly luciferase reporter construct with Lipofectamine 3000 reagent (ThermoFisher Scientific, Waltham, Mass; catalog no. L3000015) in 96-well plates (black wall with clear bottom [BD Falcon, Frankin Lakes, NJ; catalog no. 353219]) with a reverse transfection protocol. Briefly, for 1 well of 96-well plate, 0.5 μL of IFNB1 firefly luciferase reporter construct (100 ng/μL) was mixed with 5 μL of Opti-MEM, 0.2 μL of P300 reagent, and various amounts of STING constructs (6.25-400 pg in a volume of 0.25-0.4 μL), as indicated. The vector dilutions were then mixed with the Lipofectamine 3000 dilutions containing 5 μL of Opti-MEM and 0.3 μL of Lipofectamine 3000 and subsequently incubated at room temperature for 10 to 30 minutes. During the incubation period, HEK293T cells were harvested by trypsin digestion and diluted in complete Dulbecco modified Eagle medium to a density of 400,000 cells/mL. The transfection mix was applied to the wells following the incubation, after which 75 μL of diluted HEK293T cells (30,000 cells) was added to each well. To mix, the plate was gently tapped and then rocked back and forth to allow even distribution of the cells, followed by 10 minutes of incubation at room temperature to allow the cells to settle. The plates were then returned to 37°C in a humidified atmosphere with 5% CO2 for 24 hours. The luciferase assay was carried out by using the ONE-Glo EX Luciferase Assay System (Promega, Madison, Wis; catalog no. E8120), with 50 μL of luciferase reagent per well. For cGAMP stimulation, 1.8 μL of cGAMP (1 μg/μL) was diluted with 3.2 μL of complete Dulbecco modified Eagle medium and added to 1 well (of a 96-well plate) containing 85 μL of cultured HEK 293 T cells at 6 hours after transfection for a final concentration of 20 μg/mL. Transfection was carried out as already described except that 24-well plates were used (Corning Costar, Corning, NY; catalog no. 3524) and reagents and cells were added in quantities 4 times larger than in the 96-well plate. Briefly, 800 pg of STING construct and 200 ng of IFNB1 firefly luciferase reporter construct were cotransfected into 120,000 HEK293T cells in each well. Cells were harvested at 24 hours after transfection and were first washed with 1 mL of PBS, after which 50 μL of complete radioimmunoprecipitation assay (RIPA) buffer (composed of 9 mL of distilled water and 1 mL of 10× RIPA buffer [Cell Signaling Technology, Danvers, Mass; catalog no. 9086]; 1 tablet of cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail [Roche Applied Science, Penzberg, Germany; catalog no. 11836170001]; 1 tablet of PhosSTOP phosphatase inhibitor [Roche Applied Science, catalog no. 4906837001]; and 0.125 g of N-ethylmaleimide [Sigma, St Louis, Mo; catalog no. E3876]) was added. The mixture was placed on ice for 10 minutes to lyse the cells. The lysate was collected into a 1.5-mL tube, vortexed for 15 seconds, and then spun at 17,000 g at 4°C for 10 minutes. The supernatant was transferred to a new tube. Protein concentrations were measured by using a DC protein assay kit (BIO-RAD, Hercules, Calif; catalog no. 5000116) with BSA as a standard. All samples were diluted to the same concentration with complete RIPA buffer, then mixed with NuPAGE Sample Reducing Agent (10X) (Invitrogen, Carlsbad, Calif; catalog no. NP0004) and XT Sample Buffer (4X) (BIO-RAD, catalog no. 1610791), and then heated at 70°C for 10 minutes. For each sample, 12 μg of protein was loaded in 10% Criterion XT Bis-Tris Protein Gel (BIO-RAD, catalog no. 3450112) and electrophoresis was carried out in 3-(N-morpholino)propanesulfonic acid buffer (BIO-RAD, catalog no.1610793) by using Criterion Cell (BIO-RAD, catalog no. 1656001) at 200 V constant voltage for about 60 minutes. Next, 150 μL of NuPAGE Antioxidant (Invitrogen, catalog no. NP0005) was applied to the upper chamber buffer before the electrophoresis. The proteins were transferred to polyvinylidene difluoride membranes (Trans-Blot Turbo Midi PVDF Transfer Packs, BIO-RAD, catalog no. 1704157) with the Trans-Blot Turbo Transfer System (BIO-RAD, catalog no.1704150) under the preprogrammed protocol for One Midi Format gel with a mixed molecular weight (2.5 A and ≤25 V for 7 minutes). The blots were then incubated and washed with gentle agitation (50 rpm) with Labnet Orbit low speed lab shaker (Labnet International, Edison, NJ; catalog no. S2030-LS-B) as detailed here. First, the blots were incubated in 20 mL of blocking buffer (5% nonfat dry milk, 1× Tris-buffered saline [TBS], and 0.1% Tween-20) at room temperature for 1 hour. Then they were washed for 5 minutes 3 times with about 20 mL of washing buffer (1× TBS and 0.1% Tween-20); followed by incubation in 0.2 μg/mL of anti-STING antibody (R&D Systems, Minneapolis, Minn; catalog no. MAB7169-SP) diluted in 12 mL of primary antibody dilution buffer (5% BSA, 1× TBS, and 0.1% Tween-20) at 4°C overnight with gentle agitation. The blot was then washed with washing buffer for 10 minutes 3 times, followed by 1 hour of incubation at room temperature in 12 mL of blocking buffer containing 6 μL of secondary antibody (Amersham ECL Sheep anti-Mouse IgG, Horseradish Peroxidase–Linked Whole Antibody, catalog no. NA931-1ML) and 12 μL of hFAB rhodamine anti-actin primary antibody (BIO-RAD, catalog no. 12004163). To remove any unbound antibodies, the membranes were washed for 10 minutes 3 times, after which SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, catalog no. 34076) was applied. Images for STING1 and ACTB were captured with BIO-RAD ChemiDoc. STING detects microbial dinucleotides and functions as an adaptor molecule in the cytosolic DNA-sensing pathway that binds cGAMP, which is generated when cytosolic DNA activates cyclic cGAS.E2Sun 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.Science. 2013; 339: 786-791Crossref PubMed Scopus (2532) Google Scholar, E3Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. et al.Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, E4Diner E.J. Burdette D.L. Wilson S.C. Monroe K.M. Kellenberger C.A. Hyodo M. et al.The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.Cell Rep. 2013; 3: 1355-1361Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, E5Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Rohl I. et al.cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.Nature. 2013; 498: 380-384Crossref PubMed Scopus (932) Google Scholar, E6Zhang X. Shi H. Wu J. Zhang X. Sun L. Chen C. et al.Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.Mol Cell. 2013; 51: 226-235Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar The observed GOF of the STING autoactivating and disease-causing mutations was recently "explained" on the basis of 2 structural models.E1Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (247) Google Scholar,E7Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar The cryoelectron microscopy structure model suggests that cGAMP binding to the STING dimer causes a 180° rotation of the ligand-binding domain along a connector helix loop. This flattens the polymerization interfaces and enables side-by-side packing of STING dimers into polymersE1Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (247) Google Scholar that serve as a platform for TBK1 cross-phosphorylation and subsequent signaling activation.E8Zhang C. Shang G. Gui X. Zhang X. Bai X.C. Chen Z.J. Structural basis of STING binding with and phosphorylation by TBK1.Nature. 2019; 567: 394-398Crossref PubMed Scopus (326) Google Scholar Mutations of residues in the connector helix loop are referred to as class 1 SAVI mutations and include p.N154S and p.V155M (see Fig 1, G in the print text). These mutations favor a remodeling of the STING dimer structure into an autoactivated conformation. This model, however, cannot explain the mechanism of autoactivation of class 3 mutations in the polymer interface, including p.R281Q, p.R284G, and p.R284S, which are not predicted to induce remodeling of the STING dimer. Another structural model proposes that residues in the polymer interface bind a C-terminal tail, which blocks STING polymerization.E7Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar The class 3 SAVI mutations might therefore lose the ability to bind the C-terminal tail or an inhibitor (the latter is shown in the model) and cause autoactivation. Despite the differences between the 2 models, both converge at a conformational change in the polymer interface and STING activation. Our data in fact suggest a possible common mechanism to reconcile the 2 previously reported structural models. The broad autoactivation of R281 and R284 mutations strongly suggests that the polymer interface binds to an inhibitor to suppress STING autoactivation. On the basis of this reasoning, cGAMP binding to STING may not simply "flatten" the polymer interface but may also lead to loss of inhibitor binding capability as a result of the structural change in the polymerization interface (in the top panel Fig E3,E1Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (247) Google Scholar, E2Sun 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.Science. 2013; 339: 786-791Crossref PubMed Scopus (2532) Google Scholar, E3Gao P. Ascano M. Wu Y. Barchet W. Gaffney B.L. Zillinger T. et al.Cyclic [G(2',5')pA(3',5')p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase.Cell. 2013; 153: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar, E4Diner E.J. Burdette D.L. Wilson S.C. Monroe K.M. Kellenberger C.A. Hyodo M. et al.The innate immune DNA sensor cGAS produces a noncanonical cyclic dinucleotide that activates human STING.Cell Rep. 2013; 3: 1355-1361Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, E5Ablasser A. Goldeck M. Cavlar T. Deimling T. Witte G. Rohl I. et al.cGAS produces a 2'-5'-linked cyclic dinucleotide second messenger that activates STING.Nature. 2013; 498: 380-384Crossref PubMed Scopus (932) Google Scholar, E6Zhang X. Shi H. Wu J. Zhang X. Sun L. Chen C. et al.Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING.Mol Cell. 2013; 51: 226-235Abstract Full Text Full Text PDF PubMed Scopus (634) Google Scholar, E7Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar the inhibitors [which bind to the 2 dimer subunits on the front and back, respectively] are shown in black). This allows 180o rotation and STING polymerization. Similarly, STING dimers with class 1 mutations mimic the cGAMP-bound STING structure and lost inhibitor binding, which leads to activation (in the middle panel of Fig E3, the location of connector helix loop is indicated by a red box). Our hypothetical model likely explains the higher autoactivation threshold for class 3 mutations than for class 1 mutations. On the basis of the current structural model, V155 is located in the closely packed dimer interface, and mutations into a methionine (M) change the molecular interactions inside the dimer, favoring an active dimer conformation.E1Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (247) Google Scholar This conformational change can even happen in the wild-type/mutant heterodimers, which is supported by the fact that V155M mutations are disease causing in heterozygosity and show a lower autoactivation threshold than class 3 mutations, including R281Q, R284S, and R284G (see also Fig 2, A and B). In contrast, for class 3 mutations in the polymer interface (indicated by a red box in the bottom panel of Fig E3), only the mutant-mutant homodimers are able to fully resolve the inhibitor blocking on both sides of the STING dimer (Fig E3, bottom panel). Consistent with this hypothesis, the class 3 mutations either require homozygosity to be disease causing (eg, p.R281W) or they need to be superautoactivating to cause SAVI in heterozygosity (eg, R281Q, R284S, and R284G). Further structural studies of the class 3 superautoactivating mutations are required to test this hypothesis and enhance our understanding of STING activation. Considering the reports of the STING C-terminal tail functioning as an autoinhibitory suppressorE7Ergun S.L. Fernandez D. Weiss T.M. Li L. STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition.Cell. 2019; 178: 290-301.e10Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar and discoveries of other STING binding partners, including TOLLIP, IRE1α,E9Pokatayev V. Yang K. Tu X. Dobbs N. Wu J. Kalb R.G. et al.Homeostatic regulation of STING protein at the resting state by stabilizer TOLLIP.Nat Immunol. 2020; 21: 158-167Crossref PubMed Scopus (46) Google Scholar and STIM1,E10Srikanth S. Woo J.S. Wu B. El-Sherbiny Y.M. Leung J. Chupradit K. et al.The Ca(2+) sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.Nat Immunol. 2019; 20: 152-162Crossref PubMed Scopus (161) Google Scholar it remains to be determined whether these STING-binding proteins/domains represent various STING inhibitors or form a single suppressor complex.Fig E2Protein expression of transfected constructs in HEK293T cells by Western blot. STING1-expressing constructs (800 pg) were transfected into 120,000 HEK293T cells, and STING1 and ACTB were detected in chemiluminescent and rhodamine channels, respectively, in the same blot.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Proposed disease models for class 1 and class 3 mutations. The 2 subunits of the STING dimer are shown in yellow and green. Sites of SAVI-causing mutation are indicated by red boxes. Inhibitors are shown as a gray ellipse. The model assumes that a putative inhibitor is bound to the polymer interface of the wild-type STING dimer, which blocks the polymer formation. cGAMP-binding to the binding domain of STING (top panel) or mutations in the connector region (middle panel) cause a 180° rotation of the ligand-binding domain along the connector helix loop, which leads to the release of the inhibitor. This allows polymer formation via side-by-side packing and STING activation. Mutations in the polymer interface (bottom panel) directly lead to inability of inhibitor binding, which causes STING autoactivation. WT, Wild-type.Modified with permission from Shang et al.E1Shang 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.Nature. 2019; 567: 389-393Crossref PubMed Scopus (247) Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical and laboratory findings and outcomes of patients with homozygous STING1 c.841C>T, p.R281WPatient no.Age at presentationNationalityCutaneous featuresPulmo-nary involvementChest CT findingsOther clinical featuresAutoantibodiesOther laboratory findingsCurrent treatment/status14 wkSyrianYesYesDiffuse bilateral parenchymal opacitiesFailure to thriveANA positiveElevated CRP level, ESR, anemiaDied at age 5 mo2UnknownSyrianUnknownYesNDUnknownUnknownUnknownDied at age 18 mo33 moSaudiYesYesInterstitial lung disease, enlarged mediastinal lymph nodesGERD, vomiting, diarrhea, failure to thrive, clubbingANA positive, ANCA positive (anti-PR3)Elevated CRP level; ESR; and IgG, IgA, and IgM levelsTreated with ruxolitinib with a favorable response46 moSaudiYes (facial rash)YesInterstitial lung disease and enlarged mediastinal lymph nodesDelayed gross motor development, failure to thrive, short stature, clubbingANA positive, anti-dsDNA–positive, ANCA positive (MPO)Elevated CRP level, ESR, and IgG levelTreated with baricitinib with a favorable response52 moQatarYesYesReticulonodular densities throughout, pulmonary hypertension, and enlarged hilar and mediastinal lymph nodesPolyarthritis, clubbing, pulmonary hypertensionANA positive, c-ANCA–positiveElevated CRP level, ESR, and IgG level; anemia; leukopeniaTreated with baricitinib with a favorable response68 moSyrianYesYesCystic lesions, RUL fibrosisPolyarthritis, clubbing, failure to thrive, hair lossANA positive, c-ANCA positiveElevated CRP level, ESR, elevated IgG and IgM levels; anemia; additional genetic finding∗Patient 5 had a heterozygous mutation in ADA2/CECR1, but with normal enzymatic levels.Treated with baricitinib with a favorable responseANA, Antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; anti-PR3, anti-proteinase 3 antibody; c-ANCA, cytoplasmic antineutrophil cytoplasmic antibody; CRP, C-reactive protein; CT, computed tomography; dsDNA, double-stranded DNA; ESR, erythrocyte sedimentation rate; GERD, gastroesophageal reflux disease; MPO, myeloperoxidase; ND, not done; RUL, right upper lobe.∗ Patient 5 had a heterozygous mutation in ADA2/CECR1, but with normal enzymatic levels. Open table in a new tab ANA, Antinuclear antibody; ANCA, antineutrophil cytoplasmic antibody; anti-PR3, anti-proteinase 3 antibody; c-ANCA, cytoplasmic antineutrophil cytoplasmic antibody; CRP, C-reactive protein; CT, computed tomography; dsDNA, double-stranded DNA; ESR, erythrocyte sedimentation rate; GERD, gastroesophageal reflux disease; MPO, myeloperoxidase; ND, not done; RUL, right upper lobe.