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The University of Manchester Research
Disease-associated mutations identify a novel region inhuman STING necessary for the control of type I interferonsignalingDOI:10.1016/j.jaci.2016.10.031
Document VersionAccepted author manuscript
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Citation for published version (APA):Melki, I., Rose, Y., Uggenti, C., Van Eyck, L., Frémond, M-L., Kitabayashi, N., Rice, G. I., Jenkinson, E. M., Boulai,A., Jeremiah, N., Gattorno, M., Volpi, S., Sacco, O., Terheggen-Lagro, S. W. J., Tiddens, H. A. W. M., Meyts, I.,Morren, M-A., De Haes, P., Wouters, C., ... Crow, Y. J. (2017). Disease-associated mutations identify a novelregion in human STING necessary for the control of type I interferon signaling. The Journal of allergy and clinicalimmunology. https://doi.org/10.1016/j.jaci.2016.10.031Published in:The Journal of allergy and clinical immunology
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https://doi.org/10.1016/j.jaci.2016.10.031https://www.research.manchester.ac.uk/portal/en/publications/diseaseassociated-mutations-identify-a-novel-region-in-human-sting-necessary-for-the-control-of-type-i-interferon-signaling(5bef9358-218d-4871-90bd-7be28bd825c4).html/portal/gillian.rice.html/portal/emma.jenkinson.htmlhttps://www.research.manchester.ac.uk/portal/en/publications/diseaseassociated-mutations-identify-a-novel-region-in-human-sting-necessary-for-the-control-of-type-i-interferon-signaling(5bef9358-218d-4871-90bd-7be28bd825c4).htmlhttps://www.research.manchester.ac.uk/portal/en/publications/diseaseassociated-mutations-identify-a-novel-region-in-human-sting-necessary-for-the-control-of-type-i-interferon-signaling(5bef9358-218d-4871-90bd-7be28bd825c4).htmlhttps://doi.org/10.1016/j.jaci.2016.10.031
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Disease-associated mutations identify a novel region in human STING necessary 1
for the control of type I interferon signaling 2
3
Isabelle Melki, MD,a,b,c,d Yoann Rose, BSc,a,b Carolina Uggenti, PhD,a,b Lien Van 4
Eyck, MD,a,b,l Marie-Louise Frémond, MD,a,b,d Naoki Kitabayashi, BSc,a,b Gillian I 5
Rice, PhD,e Emma M Jenkinson, PhD,e Anaïs Boulai, BSc,a,b Nadia Jeremiah, PhD,b,p 6
Marco Gattorno, MD,f Sefano Volpi, MD, PhD,f Olivero Sacco, MD,g Suzanne W.J. 7
Terheggen-Lagro, MD,h Harm A.W.M. Tiddens, MD, PhD,i,j Isabelle Meyts, MD, 8
PhD,k,l Marie-Anne Morren, MD,l,m Petra De Haes, MD, PhD,l,m Carine Wouters, 9
MD, PhD,d,k,l Eric Legius, MD,n,o Anniek Corveleyn, PhD,o Frederic Rieux-Laucat, 10
PhD,b,p Christine Bodemer, MD, PhD,b,q Isabelle Callebaut, PhD,r Mathieu P Rodero, 11
PhD,a,b Yanick J Crow, MD, PhD,a,b,e 12
13 aINSERM UMR 1163, Laboratory of Neurogenetics and Neuroinflammation, Paris, 14
France 15 bParis Descartes University, Sorbonne-Paris-Cité, Institut Imagine, Paris, France 16 cGeneral Pediatrics, Infectious Disease and Internal Medicine Department, Hôpital 17
Robert Debré, AP-HP, Paris, France 18 dPediatric Hematology-Immunology and Rheumatology Department, Hôpital Necker-19
Enfants Malades, AP-HP, Paris, France 20 eManchester Centre for Genomic Medicine, Institute of Human Development, Faculty 21
of Medical and Human Sciences, Manchester Academic Health Centre, Manchester, 22
United Kingdom 23 fUO Pediatria 2 Reumatologia, G. Gaslini, Genova, Italy 24 gUO Pneumologia, G. Gaslini, Genova, Italy 25 hPaediatric Pulmonologist, Department of Paediatric Pulmonology Emma Children’s 26
Hospital AMC DD, Amsterdam, Netherlands 27 iDepartment of Radiology, Erasmus Medical Center, Rotterdam, Netherlands 28 jDepartment of Paediatrics, Respiratory Medicine and Allergology,Erasmus Medical 29
Center-Sophia Children’s Hospital, Zuid-Holland, Netherlands 30 kDepartment of Pediatrics, University Hospitals Leuven, Leuven, Belgium 31 lDepartment of Immunology and Microbiology, Childhood Immunology, University 32
of Leuven, Leuven, Belgium 33 mDepartment of Dermatology, University Hospitals Leuven, Leuven, Belgium 34
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nDepartment of Human Genetics, KU Leuven, Leuven, Belgium 35 oClinical Department of Human Genetics, University Hospitals, Leuven, Belgium 36 pINSERM UMR 1163, Laboratory of Immunogenetics of Pediatric Autoimmunity, 37
Paris, France 38 qDermatology Department, National reference center for rare skin diseases MAGEC, 39
Hôpital Necker-Enfants Malades, Paris, France 40 rCNRS UMR7590, Sorbonne Université, Université Pierre et Marie Curie-Paris6–41
MNHN–IRD–IUC, Paris, France 42
43
Corresponding author: Yanick J Crow 44
INSERM UMR 1163, Laboratory of Neurogenetics and Neuroinflammation, Paris, 45
France, 24, boulevard du Montparnasse, 75015 Paris, France. Mail: 46
yanickcrow@mac.com, Phone: +331 42 75 44 51, Fax: +331 44 49 50 70 47
48
Sources of funding: This study was funded by the European Research Council, the 49
National Research Agency (France), the Imagine Institute (France), the Research 50
Foundation Flanders (FWO) and Telethon (Italy). 51
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Capsule summary 52
53
Three new mutations in TMEM173 are reported in patients presenting STING-54
associated autoinflammation. These mutations confer constitutive activation 55
independent of ligand stimulation, revealing a novel functionally important region of 56
the protein. 57
58
Key words 59
60
Stimulator of interferon genes (STING), TMEM173, interferon, type I 61
interferonopathy, STING associated vasculopathy with onset in infancy (SAVI) 62
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Abstract 63
64
Gain-of-function mutations in TMEM173 encoding STING (stimulator of interferon 65
genes) underlie a recently described type I interferonopathy. We report three 66
individuals variably exhibiting the core features of STING-associated vasculopathy 67
with onset in infancy (SAVI) including systemic inflammation, destructive skin 68
lesions and interstitial lung disease. Molecular and in vitro data demonstrate that the 69
pathology in these patients is due to substitutions at positions 206, 281 and 284 of the 70
human protein. These mutations confer cGAMP-independent constitutive activation 71
of type I interferon signaling through TBK1 (TANK binding kinase). Structural 72
analysis indicates that these three amino acids lie in a discrete region of the protein, 73
thereby implicating a novel cluster of amino acids in STING as functionally important 74
in the regulation of type I interferon signaling. 75
76
Key Messages 77
78
• p.Cys206, p.Arg281 and p.Arg284 are new mutations in TMEM173 identified 79
in patients with STING-associated vasculopathy with onset in infancy (SAVI). 80
• These mutations confer constitutive activation independent of ligand 81
stimulation, thereby revealing a novel, functionally important region of the 82
protein. 83
84
Abbreviations 85
86
3D: Three-dimensional; AMP: Adenosine monophosphate; ANA: Antinuclear 87
antibodies; BSA: Bovine serum albumin; c: Complementary DNA; CBD: cGAMP 88
binding domain; cGAMP: 2'3'-cyclic GMP-AMP; cGAS: Cyclic GMP-AMP 89
synthase; cm: Centimeter; CT: Computed tomography; CTD: C-terminal cyclic 90
dinucleotide-binding domain; DNA: deoxyribonucleic acid; ER: Endoplasmic 91
reticulum; ERGIC: ER-Golgi intermediate compartment; ExAC: Exome Aggregation 92
Consortium; GMP: Cyclic guanosine monophosphate; HEK: Human embryonic 93
kidney; IFN: Interferon; IFNα: Interferon alpha; IFNAR: Interferon alpha receptor; 94
IFNß: Interferon beta; IRF3: Interferon regulatory factor 3; ISGs: Interferon-95
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stimulated genes; JAK1: Janus kinase 1; Kg: Kilogram; mAb: Monoclonal antibody; 96
MAFFT: Multiple alignment using fast fourier transform; P: Patient; pcDNA: plasmid 97
cDNA; PCR: Polymerase chain reaction; PDB: Protein Data Bank; RCSB: Research 98
Collaboratory for Structural Bioinformatics; RNA: Ribonucleic acid; p: Protein; 99
SAVI: STING associated vasculopathy with onset in infancy; SD: Standard deviation; 100
STAT1: Signal transducer and activator of transcription 1; STING: Stimulator of 101
interferon genes; TBK1: TANK binding kinase; TBS: Tris-buffered saline; Tm: 102
Melting temperature; TMEM173: Transmembrane protein 173; TNF: Tumor necrosis 103
factor; TYK2: Tyrosine kinase 2; WB: Western blot; WT: Wild type. 104
105
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Introduction 106
107
Gain-of-function mutations in TMEM173 encoding STING (stimulator of interferon 108
genes) have been described to cause an autoinflammatory syndrome1–8 belonging to a 109
recently defined class of disorders referred to as the type I interferonopathies9,10. This 110
phenotype, known as STING associated vasculopathy with onset in infancy (SAVI), 111
is characterized by early-onset systemic inflammation with fever, a severe skin 112
vasculopathy leading in some cases to extensive tissue loss, and interstitial lung 113
disease resulting in pulmonary fibrosis and end-stage respiratory failure1–5,7,8 114
115
To date, twenty-three patients from fifteen families have been reported with gain-of-116
function mutations in STING1–8, a key adaptor molecule in the cytosolic DNA-117
sensing pathway, expressed in various endothelial and epithelial cell types, as well as 118
in haematopoetic cells, such as T cells, macrophages and dendritic cells. Once 119
stimulated, STING induces the transcription of type I interferons (IFN) and the 120
expression of a set of IFN-stimulated genes (ISGs), thus establishing an antiviral 121
state11–13. In all of these published cases the heterozygous substitution involves one of 122
three amino acids at positions 147, 154, 155, and 166, being located either in the 123
linker connecting the transmembrane domain of STING to the cyclic dinucleotide 124
domain (CTD) (p.Val147), or within the N-terminal dimerization region of the CTD 125
(p.Asn154, p.Val155 and p.Gly166)14,15. It has been suggested that these disease-126
associated residues play non-redundant roles in retaining the protein on the 127
endoplasmic reticulum (ER), causing STING to constitutively localize to the ER-128
Golgi intermediate compartment (ERGIC) and activate downstream signaling through 129
the TANK binding kinase (TBK1) - Interferon regulatory factor 3 (IRF3) axis15,16. 130
131
We describe three individuals variably exhibiting the characteristic features of 132
STING-associated autoinflammation. However, in contrast to earlier reports, our data 133
show that the pathology in these patients is due to substitutions at positions 206, 281 134
and 284 of the human protein, thus implicating a novel region of STING as 135
functionally important in the regulation of type I IFN signaling. 136
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Methods 137
138
Patient and study approval 139
The study was approved by the Comité de Protection des Personnes (ID-140
RCB/EUDRACT: 2014-A01017-40) and undertaken with written informed parental 141
consent. 142
143
Genetic analysis 144
DNA was extracted from whole blood samples using standard methods. Whole-145
exome sequencing was performed on genomic DNA from patient 2 (P2) and her 146
mother using SureSelect Human All Exon kit (Agilent Technologies) for targeted 147
enrichment and Illumina HiSeq2000 for sequencing. Sanger sequencing was 148
performed on DNA from patient 1 (P1), patient 3 (P3) and their parents, and from P2 149
and her mother to confirm the variant found by exome sequencing (Primers in Table 150
EII). Variant frequency data in controls was derived from the Exome Aggregation 151
Consortium (ExAC)17. 152
153
Structural analysis 154
The experimental 3D structures of STING were extracted from the Research 155
Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) and 156
analyzed using Chimera (https://www.cgl.ucsf.edu/chimera/). 157
158
Construct generation 159
Site-directed mutagenesis was performed to generate the desired TMEM173 variants 160
by using the Q5® Site-Directed Mutagenesis Kit (E0554S from NEB) following the 161
manufacturer’s instructions. pMSCV-hygro(+) (Addgene) carrying wild type (WT) 162
TMEM173 gene was used as a template for the PCR reactions18. This plasmid carries 163
the haplotype most frequently recorded in the general population (p.Arg71, p.Gly230, 164
p.Arg232, p.Arg293)19. Mutagenic primer sequences were designed using the NEBase 165
Changer software (http://nebasechanger.neb.com/). Cycling parameters are indicated 166
in Table EI. NEB 5-alpha Competent E. coli were transformed with the newly 167
synthesized pcDNA, and colonies were screened for the presence of the desired 168
variants (primers in Table EII). 169
170
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Cell culture procedures 171
Human embryonic kidney (HEK) 293T cells were grown in 96-well plates at 37°C in 172
5% CO2 in DMEM (GIBCO #31966-021) supplemented with 10% (v/v) fetal bovine 173
serum (GIBCO #10270), 100 U/ml penicillin and 100 mg/ml streptomycin (GIBCO 174
#15070-063). At 70% confluency, HEK293T cells were co-transfected with 60 ng of 175
vector pMSCV- hygro(+) either empty or encoding the TMEM173 variants, 40 ng of 176
IFNß promoter-driven firefly luciferase reporter plasmid (IFNß-pGL3) and 1.4 ng of 177
constitutively expressed renilla luciferase reporter plasmid (pRL-TK) by using 178
TransIT-293 (Mirus #MIR2700 from Euromedex). 24 hours later, cells were 179
stimulated by transfecting 1.3-12 µg/ml 2’3’-cGAMP_STING ligand (tlrl-nacga23, 180
InvivoGen) using lipofectamine 2000 (#11668027 from Thermo Fischer). 24 hours 181
after cGAMP stimulation, cells were lysed with passive lysis buffer 5X (#E194A 182
from Promega) containing protease and phosphatase inhibitors. One third of the lysate 183
for each condition was used for the luciferase assay, and the remaining material for 184
protein analysis by western blotting (WB). 185
186
Luciferase assay 187
The IFNß-pGL3 plasmid was used to measure IFNß promoter activity, and the renilla 188
reporter control plasmid to normalize for transfection efficiency. Luciferase assays 189
were performed using the Dual-Glo® Luciferase Assay System (E2940 from 190
Promega) following the manufacturer’s protocol. Luminescence was acquired on a 191
FLUOstar OPTIMA microplate reader (BMG LABTECH). Firefly luciferase activity 192
was normalized against renilla luciferase activity. 193
194
Western blot 195
Bolt LDS Sample Buffer (4X) (#B0008 Novex Life Technologies) and Bolt Sample 196
Reducing agent (10X) (#B0009 Novex Life Technologies) were added to protein 197
lysates, and samples were resolved on 4-12% Bis-Tris Plus gels (# NW04122BOX 198
Invitrogen) transferred to nitrocellulose membrane (#IB23002 Invitrogen). When the 199
phosphorylation status of the protein was investigated, membranes were blocked in 5 200
% BSA in TBS, and primary phospho-antibodies were incubated overnight in the 201
blocking solution. Otherwise, membranes were blocked with 5 % milk in TBS and 202
primary antibodies were incubated overnight in the blocking solution. Proteins were 203
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blotted with monoclonal anti-STING (R&D MAB7169), anti-Phospho IRF3 Rabbit 204
mAb (Ser396, #4947 Cell Signaling), anti-IRF3 Rabbit mAb (#11904 Cell Signaling) 205
and anti-cofilin Rabbit mAb (#5175 Cell Signaling). Primary antibodies were detected 206
with horseradish peroxidase–conjugated secondary antibody (#7074S and 7076S, Cell 207
Signaling). All western blot images were captured and quantified with a ChemiDoc 208
MP imager and Image Lab software (Bio-Rad Laboratories, Hercules, CA) after 209
adding Clarity Western ECL substrate (Biorad). 210
211
STAT1 phosphorylation assay staining 212
Ficolled PBMCs were treated or not with ruxolitinib 1 µM for 45 minutes at 37°C. 213
Cells were fixed and permeabilized according to PerFix EXPOSE Kit from Beckman 214
Coulter (#B26976) recommendations. Cells were stained for pSTAT1 PE-anti-STAT1 215
pY701, and cell surface marker (PE-CyTM 7-CD19, APC-CD3, BV421-CD8) for 1 216
hour at room temperature. Flow cytometry analysis was performed on a Gallios 217
Beckman Coulter flow cytometer. Results were analyzed using Kaluza software v1.3. 218
219
Results 220
221
Clinical presentation (Table I, Fig 1) 222
223
P1 (F1058) is a 7-year-old female born to non-consanguineous parents of white 224
European ethnicity. She was delivered at term weighing 3.2kg. She presented at the 225
age of 3 months with feeding difficulties and respiratory distress necessitating oxygen 226
therapy (Table I). Computed tomography (CT) scan of the lungs at age 1.5 years 227
revealed extensive ground glass abnormalities, and there was interstitial fibrosis on 228
lung biopsy without signs of vasculitis (Fig 1, A). Telangiectatic skin lesions on the 229
cheeks and nose were noted from the age of 4 months (Fig 1, B). She has not 230
experienced any involvement of the digits. Limited skin biopsy did not reveal any 231
sign of vasculitis. Elevated transaminases and hepatomegaly prompted a liver biopsy 232
at age 2 years, when the presence of necrotizing granulomatous hepatitis was 233
suggested (intralobular granulomas characterized by necrotizing zones with mixed 234
inflammatory infiltrates). She has demonstrated no further evidence of liver disease. 235
She had a gastrostomy fitted at age 2 years because of poor weight gain, and 236
subsequently suffered recurrent skin infections with poor healing around the entry 237
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site. In light of a continued oxygen requirement and raised inflammatory markers she 238
was treated with immunosuppressants (steroids, methotrexate and anti-TNFα therapy) 239
starting at age 23 months, but with limited efficacy. She remains oxygen dependent 240
with a severe deficit of lung function and markedly limited exercise tolerance. 241
242
P2 (F1125) is a 25-year-old female born to non-consanguineous parents of white 243
European ethnicity. There is no family history of note. She demonstrated intra-uterine 244
growth retardation (birth weight at term -3 SD), with subsequent failure to thrive 245
(adult weight / height of 143 cm and 34 kg) requiring a gastrostomy to be placed by 246
age 1 year until puberty. She suffered recurrent bacterial infections of the upper 247
respiratory tract, leading to a severe septicemia at the age of five months (due to 248
Pseudomonas aeruginosa) and palatal and nasal septum necrosis. Screening for 249
primary immune deficiencies was negative. A widespread livedo was noted from 250
birth, which worsened gradually with an impressive purpuric aspect in patches on 251
both cheeks and buttocks, and a marked dark purple marbling on the limbs (Fig 1, C 252
and D). When last seen at the age of 25 years she had lost a large part of the external 253
nares, and demonstrated extreme acrocyanosis of the hands and feet. She has never 254
experienced fevers, and systemic markers of inflammation and autoantibody titers 255
have been consistently normal until recent antinuclear antibodies (ANA) were 256
detected at a low level (1/160). Lung function is normal. She is not currently treated. 257
258
P3 (F1802) is a 15-year-old male born to non-consanguineous parents of white 259
European ethnicity. A fixed erythema of the cheeks was noted shortly after birth. 260
Telangiectasia of the cheeks, buttocks and limbs were observed for the first time at 261
age 10 years. From the age of 14 years he has suffered recurrent acrocyanosis 262
moderately influenced by cold temperature, with painful acral erosions that heal as 263
atrophic scars on the ear helices and digits. Lesional gluteal skin biopsies revealed 264
dilated capillaries without obvious vasculitis or thrombosis. He has been treated with 265
nifedipin, pentoxyfillin and acetyl salicylic acid, with limited efficacy. He has never 266
experienced fevers or other features of systemic disease. Blood analysis, including 267
markers of inflammation and autoantibody profile, has been consistently normal. 268
Recent chest CT revealed no evidence of interstitial lung disease. Lung function 269
testing and a six minute exercise tolerance test were normal. 270
271
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Identification of three new variants in STING 272
273
A summary of the molecular data relating to these three patients is given in Figure 2 274
and Table I. A multiple sequence alignment of STING proteins from various species 275
is provided in Supplementary Figure E1. 276
277
P1 (F1058) was observed to carry a c.842G>A (p.Arg281Gln) variant in exon 7 of 278
TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 279
alleles on the ExAC database. Both parents were WT at this nucleotide, and 280
microsatellite testing confirmed that the substitution had arisen de novo. The arginine 281
at position 281 is conserved in mouse, but is an alanine in Xenopus tropicalis and a 282
glutamate in Danio rerio. We note a similarly limited degree of conservation of the 283
valine at position 147, substitution of which for a leucine has been previously shown 284
to act as a gain-of-function mutation1. 285
286
P2 (F1125) was observed to carry a c.850A>G (p.Arg284Gly) variant in exon 7 of 287
TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 288
alleles on the ExAC database. The mother was WT for this variant. DNA was not 289
available from the father. The arginine is conserved to Nematostella vectensis. 290
291
P3 (F1802) was observed to carry a c.617G>A (p.Cys206Tyr) variant in exon 6 of 292
TMEM173 (Fig 2, A) which has not been recorded on more than 120,000 control 293
alleles on the ExAC database. Both parents were WT at this nucleotide, and 294
microsatellite testing confirmed that the substitution had arisen de novo. The cysteine 295
at this position is generally well conserved, except in Danio rerio where it is replaced 296
by an alanine. 297
298
3D structural analysis reveals a new mutation hotspot in STING 299
300
In order to gain further insight into the effect of the three STING substitutions that we 301
observed in the patients described above, we examined the experimental 3D structures 302
of the CTD of STING (Fig 2, B). p.Cys206 is buried within the 3D structure, whilst 303
p.Arg281 and p.Arg284 lie on the surface of the protein. However, these three amino 304
acids are in close proximity and are aligned, with p.Arg284 located in between 305
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p.Cys206 and p.Arg281 (Fig 2, C and D). The substitution of the cysteine for a 306
tyrosine is predicted to induce a steric clash, which might locally destabilize the 3D 307
structure of the helix bundle and affect the positioning of the 281 / 284 arginine 308
residues. 309
310
p.Cys206, p.Arg281 and p.Arg284 mutations in STING induce over-activation of 311
IFNß transcription 312
313
In order to determine if these mutations were gain-of-function, we used a luciferase-314
based IFNß transcription reporter assay to measure their impact on type I IFN 315
induction1,15,16. As previously shown, the p.Val155Met and p.Asn154Ser mutants 316
induce reporter activity in the absence of ligand (Fig 2, E)1,2,15. We observed a 317
similarly robust activation with patient-associated substitutions at p.Cys206Tyr, 318
p.Arg281Gln and p.Arg284Gly (Fig 2, E), supporting the hypothesis that these 319
variants are constitutively active. 320
321
To investigate whether these three variants lie within a novel functional cluster in 322
STING, we went on to mutate other residues spatially close to the amino acids at 206, 323
281 and 284. Specifically, we changed p.Asp205 to p.Asp205Tyr and p.Asp205His, 324
p.Leu265 to p.Leu265Tyr, p.Phe269 to p.Phe269Tyr, p.Gln273 to p.Gln273Ala and 325
p.Gln273Leu, and p.Glu282 to p.Glu282Ala and p.Glu282Asp (Fig 3, A and B). All 326
these amino acids are exposed at the surface of the protein, except p.Leu265 which is 327
buried and in contact with p.Cys206. In contrast to our patient-associated variants, 328
substitution of any of these residues, except for the aspartate at position 205, behaved 329
in a manner similar to WT (Fig 3, C and Table EIII). In contrast, substitution of the 330
p.Asp205 resulted in an upregulation of IFNß reporter activity as for the gain-of-331
function variants (Fig 3, C and Table EIII). 332
333
Altogether, these results suggest the identification of a novel region implicated in 334
STING regulation, involving at least four amino acids. The fact that all of the 335
different substitutions tested at these residues (two, four, three and three at p.Asp205, 336
p.Cys206, p.Arg281 and p.Arg284 respectively) result in increased IFNß activity 337
indicates that subtle conformational changes are sufficient to disrupt the normal 338
control of the pathway. The specificity of this effect was emphasized by the fact that 339
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four other amino acids, p.Leu265, p.Phe269, p.Gln273 and p.Glu282, located in close 340
proximity in the same tertiary structure did not induce a gain-of-function when 341
substituted. 342
343
STING gain-of-function induced by p.Cys206Tyr, p.Arg281Gln and 344
p.Arg284Gly is independent of cGAMP binding 345
346
While all previously described STING-associated mutations are located close to the 347
cGAMP binding site, the three novel mutations described here lie on the surface of 348
the protein, without direct contact with this binding site (Fig 2, B). In order to 349
determine if the gain of function observed in association with our new mutations 350
resulted from ligand independent constitutive activation, or reflected an increased 351
sensitivity to low levels of endogenous cGAMP, we generated double mutants 352
involving the arginine at 232 which is essential for cGAMP binding14,15. For every 353
previously and newly described mutation we observed a similarly robust upregulation 354
of signaling, suggesting that despite clearly distinct localization in the tertiary 355
structure they all confer ligand-independent constitutive activation (Fig 4, A). 356
357
Furthermore, using an antibody against phosphorylated residue p.Ser396 of IRF3, we 358
recorded increased phosphorylation of IRF3 in cells expressing the three patient-359
associated mutations, comparable with the previously described p.Val155Met. 360
Consistent with our luciferase results, this increased IRF3 phosphorylation was not 361
impaired by introducing a second mutation at p.Arg232Ala, which abrogates cGAMP 362
binding and IRF3 phosphorylation in the WT state (Fig 4, B and E2, A and D). 363
364
STING gain-of-function induced by p.Cys206Tyr, p.Arg281Gln and 365
p.Arg284Gly is independent of the membrane fusion pathway 366
367
Recently, it has been reported that a STING-dependent but cGAS-independent 368
pathway can induce type I IFN expression in response to membrane fusion of 369
enveloped RNA viruses20–22. In this model, the arginine at 169 in human STING was 370
shown to be important for stimulation of IFN expression by liposomes but not 371
cGAMP, demonstrating that activation by these two stimuli can be mechanistically 372
separated. p.Arg169 is surface exposed, located just outside the STING dimerization 373
-
domain and facing away from the cGAMP-binding pocket. To determine if the 374
arginine at position 169 was crucial in the constitutive activation that we observed 375
with our patient-associated mutations, we substituted an alanine for the wild-type 376
arginine at 169 (p.Arg169Ala) and expressed the double-mutants in our in vitro 377
system. We observed no effect of the 169 substitution with mutations at 206, 281 and 378
284, or with the previously described mutations at 154 and 155, indicating that human 379
disease-associated constitutive activation of STING is not dependent on this 380
alternative pathway (Fig 5, A). A similar result was obtained with the double mutant 381
p.Asp205His and p.Arg169Ala (Fig E2, B and E). Consistent with these data, 382
constitutively activated constructs bearing the second mutation p.Arg169Ala 383
demonstrated phosphorylated IRF3 at p.Ser396 in the basal state (Fig 5, B and E2, B 384
and E). 385
386
Constitutive activation of newly identified disease-causing STING mutants is 387
dependent on IRF3 phosphorylation 388
389
Phosphorylation of STING at p.Ser366 by TBK1 is critical for the recruitment and 390
activation of IRF3 by STING and the subsequent activation of IFN signaling23,24. To 391
investigate whether SAVI-associated variants were dependent on TBK1-mediated 392
STING phosphorylation and IRF3 recruitment, we introduced a mutation of serine to 393
alanine at position 366 (p.Ser366Ala) into our mutant constructs. In all cases, this 394
substitution led to an abolition of IFNß activation despite similar expression of single 395
and double STING mutants (Fig 6, A and B). This was associated with loss of IRF3 396
phosphorylation, indicating that patient-associated mutations are dependent on 397
phosphorylation at residue 366 for subsequent downstream activation of IRF3 (Fig 6, 398
B and Fig E2, C and D) and transcription of ISGs. 399
400
STING constitutive activation leads to increased phosphorylation of STAT1 in T 401
and B cells of P3 and normalize after ruxolitinib treatment in vitro 402
403
Type I interferons bind to a specific IFNα receptor (IFNAR), thereby activating Janus 404
kinase 1 (JAK1) and Tyrosine kinase 2 (TYK2), which then induce phosphorylation 405
of Signal transducer and activator of transcription 1 (STAT1). We investigated 406
STING constitutive activation in CD4, CD8 and CD19 cells from P3, and observed 407
-
increased levels of STAT1 phosphorylation compared to a control (Fig 7). Consistent 408
with published data, this upregulation normalized after 45 minutes of treatment with 409
ruxolitinib in vitro (Fig 7)1,5. 410
411
Discussion 412
413
Since its identification in 2008 as an adaptor molecule in the cytosolic DNA-sensing 414
pathway, STING has emerged as a central player in antiviral immunity, 415
autoinflammation and cancer11,13. Thus, an understanding of the mechanism of action 416
and control of STING in health and disease is of considerable scientific and medical 417
importance. 418
419
Here we describe three novel variants seen in association with a phenotype consistent 420
with previously described cases of STING-associated autoinflammation. In two of 421
these patients we were able to show that the amino acid substitutions arose de novo, 422
whilst in the third the mother was WT but DNA was unavailable from the father. 423
These substitutions, involving evolutionarily well-conserved residues, have not been 424
described in publically available databases comprising more than 120,000 alleles. As 425
for other molecularly confirmed cases of STING-associated autoinflammation, all 426
three substitutions were associated with the in vitro induction of IFNß reporter 427
activity. Considering these observations, it is very likely that the variants we describe 428
represent pathogenic gain-of-function mutations. 429
430
In contrast to all previously published cases of type I interferonopathy related to 431
mutations in STING, the three variants reported here do not lie in the linker region 432
connecting the N-terminal transmembrane domain of STING to the CTD or in the 433
first a-helix (a5) of the CTD involved in dimerization. The function of the arginine 434
residues at 281 and 284 is currently undefined. However, these residues are exposed 435
at the surface of the molecule, so that they are unlikely to involve the intramolecular 436
interaction of the STING dimer. A recent survey of STING mutations in the COSMIC 437
database led to the demonstration that a substitution of the arginine at 284 to 438
methionine (p.Arg284Met) was able to induce an IFNß reporter to a significantly 439
greater extent than WT STING25. Alternative substitutions of arginine 284 for lysine 440
or threonine (p.Arg284Lys, p.Arg284Thr) also represented hyperactive mutants. The 441
-
results of these experiments were further supported by demonstrating increased IFNß 442
in tissue culture supernatants, and an enhanced ability to inhibit viral replication. Of 443
note, the mutation identified in P2 affects this same residue, substituting the arginine 444
for a glycine. Thus, multiple types of amino acid substitution at 284 confer a gain-of-445
function, indicating a high degree of specificity for the function of an arginine at this 446
position. 447
448
Similar to the arginine at 284, we found that different substitutions of the arginine at 449
281 and the cysteine at 206 also resulted in constitutive activation. It is of note that 450
these three amino acids are aligned at the 3D level, with the arginine at 284 located in 451
between the cysteine at 206 and the arginine at 281. The substitution of the cysteine 452
for a tyrosine at 206 is predicted to lead to steric hindrance and locally disturb the 453
fold, which might affect the positioning of the arginine at 284. However, substituting 454
other less bulky amino acids at 206 had the same effect, highlighting the specificity of 455
the WT cysteine at this position. 456
457
In order to define the extent of a putative functionally important region involving the 458
amino acids that we identified through human disease genetics, we went on to mutate 459
a selection of other residues predicted to lie in close proximity with these disease-460
associated mutations - specifically, p.Leu265, p.Phe269, p.Gln273 and p.Glu282. We 461
also derived a number of conservative and non-conservative substitutions at Asp205, 462
which lies in contact with the p.Arg284. Substitutions at these residues behaved in a 463
similar manner to WT protein, except for those at position 205, where we observed 464
enhanced signaling in our in vitro assay similar to the patient-associated mutations. Of 465
note, constitutive activation has been described previously at the equivalent residue in 466
murine STING12,26. 467
468
In regards of cGAMP-dependency, the involvement of a cGAMP-independent 469
signaling pathway and TBK1 downstream signaling, our data did not suggest any 470
difference in the behavior of variants at 206, 281 and 284 compared to previously 471
described disease-associated mutations at 147, 154 and 155. Recently, Dobbs et al. 472
suggested that the latter mutations may disrupt the retention of STING in the ER, 473
resulting in translocation to the ERGIC and activation of TBK1 / IRF3 in the absence 474
of cGAMP binding15. Furthermore, these mutations appeared to result in continued 475
-
activation, presumed due to a failure of STING degradation. Our data clearly 476
implicate a novel region of STING as important in type I IFN signaling, leading us to 477
speculate that the surface-exposed arginine residues at 281 and 284 may also be 478
involved in ER retention, or in binding to a negative regulator of STING25. 479
480
Our results indicate that mutations at residues 206, 281 and 284 of human STING are 481
responsible for the disabling autoinflammation observed in the three patients that we 482
describe. Given the favorable effects seen with JAK1/2 inhibition in other patients 483
with STING gain-of-function mutations, these patients may similarly benefit from 484
such a therapeutic approach5. Alternative potential treatment strategies might include 485
ER exit blocking agents, palmitoylation inhibitors or TBK1 antagonists15,27–29. 486
Considering that a p.Ser366Ala substitution abrogated the constitutive activation seen 487
with every patient-associated mutation tested in our in vitro model, TBK1 blockade 488
might be relevant to all SAVI patients. 489
490
A major aspect of the pathology due to mutations in TMEM173 appears to relate to a 491
dysfunction of endothelium consequent upon an upregulation of type I IFN signaling. 492
The explanation for variable expression observed between, and indeed within 493
families, will require further study, with such observations likely reflecting 494
differential environmental exposures and / or modifying genetic factors. Indeed, 495
whilst the cGAS-STING cytosolic DNA signaling pathway is essential for the 496
induction of an effective anti-viral response, human genetics is teaching us about the 497
risk of inflammatory disease associated with variation in this system. Polymorphisms 498
in STING, and other molecules involved in cytosolic nucleic acid recognition, can 499
confer differences in signaling to a type I IFN response - a non-binary situation where 500
a balance is struck between anti-viral priming and the risk of autoinflammation19,30. 501
Elucidation of the mechanisms of STING regulation is thus of great importance in 502
understanding both microbial pathogenesis and inflammation. The findings presented 503
here suggest a previously unappreciated aspect of the control of STING in this 504
context. 505
-
Acknowledgements 506
Y.J.C. acknowledges the European Research Council (GA 309449: Fellowship), and a 507
state subsidy managed by the National Research Agency (France) under the 508
"Investments for the Future" program bearing the reference ANR-10-IAHU-01. FRL 509
acknowledges a state subsidy managed by the National Research agency referenced as 510
Lumugene (ANR-14-CE14-0026-01). IM acknowledges the Programme Santé-511
Sciences MD/PhD of Imagine Institute. S.V. and M.G. gratefully acknowledge the 512
financial support of Telethon, Italy (Grant no. # GGP15241A). L.V.E is funded by a 513
research grant from the Research Foundation Flanders (FWO). We thank Nicolas 514
Manel for WT STING, p.Val155Met STING and IFNß-pGL3 plasmids. The authors 515
would like to thank the Exome Aggregation Consortium and the groups that provided 516
exome variant data for comparison. A full list of contributing groups can be found at 517
http://exac.broadinstitute.org/about. 518
519
Conflict of interest 520
The authors declare no conflict of interest. 521
-
References 522
523
1. Liu, Y. et al. Activated STING in a vascular and pulmonary syndrome. N. 524
Engl. J. Med. 371, 507–518 (2014). 525
2. Jeremiah, N. et al. Inherited STING-activating mutation underlies a familial 526
inflammatory syndrome with lupus-like manifestations. J. Clin. Invest. 124, 5516–527
5520 (2014). 528
3. Munoz, J. et al. Stimulator of Interferon Genes-Associated Vasculopathy With 529
Onset in Infancy: A Mimic of Childhood Granulomatosis With Polyangiitis. JAMA 530
Dermatol. 151, 872–877 (2015). 531
4. Omoyinmi, E. et al. Stimulator of interferon genes-associated vasculitis of 532
infancy. Arthritis Rheumatol. Hoboken NJ 67, 808 (2015). 533
5. Frémond, M.-L. et al. Efficacy of the Janus Kinase 1/2 Inhibitor Ruxolitinib in 534
the Treatment of Vasculopathy Associated with TMEM173-Activating Mutations in 535
three children. J. Allergy Clin. Immunol. (2016). doi:10.1016/j.jaci.2016.07.015 536
6. König, N. et al. Familial chilblain lupus due to a gain-of-function mutation in 537
STING. Ann. Rheum. Dis. (2016). doi:10.1136/annrheumdis-2016-209841 538
7. Picard, C. et al. Severe Pulmonary Fibrosis as the First Manifestation of 539
Interferonopathy (TMEM173 Mutation). Chest 150, e65-71 (2016). 540
8. Clarke, S. L. N. et al. Interstitial Lung Disease Caused by STING-associated 541
Vasculopathy with Onset in Infancy. Am. J. Respir. Crit. Care Med. 194, 639–642 542
(2016). 543
9. Crow, Y. J. Type I interferonopathies: a novel set of inborn errors of 544
immunity. Ann. N. Y. Acad. Sci. 1238, 91–98 (2011). 545
10. Crow, Y. J. & Casanova, J.-L. STING-associated vasculopathy with onset in 546
infancy--a new interferonopathy. N. Engl. J. Med. 371, 568–571 (2014). 547
11. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that 548
facilitates innate immune signalling. Nature 455, 674–678 (2008). 549
12. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-550
GMP. Nature 478, 515–518 (2011). 551
13. Barber, G. N. STING: infection, inflammation and cancer. Nat. Rev. Immunol. 552
15, 760–770 (2015). 553
14. Zhang, X. et al. Cyclic GMP-AMP containing mixed phosphodiester linkages 554
is an endogenous high-affinity ligand for STING. Mol. Cell 51, 226–235 (2013). 555
-
15. Dobbs, N. et al. STING Activation by Translocation from the ER Is 556
Associated with Infection and Autoinflammatory Disease. Cell Host Microbe 18, 557
157–168 (2015). 558
16. Liu, S. et al. Phosphorylation of innate immune adaptor proteins MAVS, 559
STING, and TRIF induces IRF3 activation. Science 347, aaa2630 (2015). 560
17. Consortium, E. A. et al. Analysis of protein-coding genetic variation in 60,706 561
humans. bioRxiv 30338 (2015). doi:10.1101/030338 562
18. Gentili, M. et al. Transmission of innate immune signaling by packaging of 563
cGAMP in viral particles. Science 349, 1232–1236 (2015). 564
19. Yi, G. et al. Single nucleotide polymorphisms of human STING can affect 565
innate immune response to cyclic dinucleotides. PloS One 8, e77846 (2013). 566
20. Holm, C. K. et al. Virus-cell fusion as a trigger of innate immunity dependent 567
on the adaptor STING. Nat. Immunol. 13, 737–743 (2012). 568
21. Olagnier, D. & Hiscott, J. Breaking the barrier: membrane fusion triggers 569
innate antiviral immunity. Nat. Immunol. 13, 713–715 (2012). 570
22. Holm, C. K. et al. Influenza A virus targets a cGAS-independent STING 571
pathway that controls enveloped RNA viruses. Nat. Commun. 7, 10680 (2016). 572
23. Tanaka, Y. & Chen, Z. J. STING Specifies IRF3 Phosphorylation by TBK1 in 573
the Cytosolic DNA Signaling Pathway. Sci Signal 5, ra20-ra20 (2012). 574
24. Konno, H., Konno, K. & Barber, G. N. Cyclic dinucleotides trigger ULK1 575
(ATG1) phosphorylation of STING to prevent sustained innate immune signaling. 576
Cell 155, 688–698 (2013). 577
25. Tang, E. D. & Wang, C.-Y. Single amino acid change in STING leads to 578
constitutive active signaling. PloS One 10, e0120090 (2015). 579
26. Shu, C., Yi, G., Watts, T., Kao, C. C. & Li, P. Structure of STING bound to 580
cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the 581
immune system. Nat. Struct. Mol. Biol. 19, 722–724 (2012). 582
27. Mukai, K. et al. Activation of STING requires palmitoylation at the Golgi. 583
Nat. Commun. 7, 11932 (2016). 584
28. Hasan, M. et al. Cutting Edge: Inhibiting TBK1 by Compound II Ameliorates 585
Autoimmune Disease in Mice. J. Immunol. Baltim. Md 1950 195, 4573–4577 (2015). 586
29. Hasan, M. & Yan, N. Therapeutic potential of targeting TBK1 in autoimmune 587
diseases and interferonopathies. Pharmacol. Res. (2016). 588
doi:10.1016/j.phrs.2016.04.008 589
-
30. Shigemoto, T. et al. Identification of loss of function mutations in human 590
genes encoding RIG-I and MDA5: implications for resistance to type I diabetes. J. 591
Biol. Chem. 284, 13348–13354 (2009). 592
31. Ouyang, S. et al. Structural analysis of the STING adaptor protein reveals a 593
hydrophobic dimer interface and mode of cyclic di-GMP binding. Immunity 36, 594
1073–1086 (2012). 595
32. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software 596
version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 597
(2013). 598
33. Robert, X. & Gouet, P. Deciphering key features in protein structures with the 599
new ENDscript server. Nucleic Acids Res. 42, W320-324 (2014). 600
601
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Figure Legends 602
603
TABLE I. Clinical and molecular data. 604
ANA: Antinuclear Antibodies; ANCA: Anti-neutrophilic cytoplasmic antibodies; F: 605
Female; M: Male 606
607
FIG 1. Clinical and radiological phenotypes. A. High resolution chest CT scan of 608
P1 demonstrating evolution of bilateral alveolar and interstitial disease with ground-609
glass lesions and interlobular septa thickening (at ages 3, 5 and 8). Improvement of 610
interstitial lung disease between ages 3 and 5 years was observed after oral steroid 611
treatment. B. Telangiectatic skin lesions on the cheeks and nose of P1. C. Purpuric 612
aspect to patches on both cheeks of P2 at age 1.5 years. D. Widespread livedo and 613
marked dark purple marbling on the limbs of P2 at age 1.5 years. y: years. 614
615
FIG 2. Constitutively activated mutations of TMEM173. A. Schematic 616
representation of the TMEM173 protein highlighting the four transmembrane domains 617
(TM1, 2, 3 and 4), the cGAMP binding domain (CBD) and the C terminal tail domain 618
(CTD), as well as four phosphorylation sites (P) at p.S345, p.S358, p.S366 and 619
p.S379. In black, previously described mutations in exon 5 (p.V147L; p.V147M; 620
p.N154S; p.V155M). In red, 3 novel mutations in exons 6 and 7 (p.C206Y: P3; 621
p.R281Q: P1; p.R284G: P2). B. Ribbon representation of the 3D structure of the 622
STING CTD dimer in complex with cyclic-di-GMP (PDB reference 4ef4), indicating 623
the position of the three novel amino acids found mutated in this study, as well as 624
amino acids previously reported with mutations and involved in the CTD dimer 625
interface, at the bottom of the ligand binding site. C. Focus on the 3D aligned 626
positions of p.C206, p.R281 and p.R284. D. Surface representation of the 3D 627
structure of the STING CTD dimer (orthogonal view relative to panel B), showing the 628
accessibility of the three amino acids with patient-associated substitutions (p.C206, 629
p.R281 and p.R284). E. IFNß-Luc reporter assay. HEK293T cells were transfected 630
with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING plasmids 631
(EV: Empty vector; WT: Wild type STING; p.N154S p.V155M; p.C206Y; p.R281Q; 632
p.R284G) and stimulated with increasing doses of cGAMP (none; 4 µg/µl; 12 µg/µl). 633
Luciferase activity was determined by Dual-luc assay 24 hours post-stimulation. 634
Mean and SDs shown, representative of 5 different experiments in triplicate. 635
-
636
FIG 3. Analysis of substitutions conferring constitutive activation of STING. A. 637
and B. Localization of amino acids on surface and ribbon representations of the 638
STING dimer 3D structure (PDB reference 4ef4)31. Colored amino acids in which 639
mutations confer constitutive activation: p.D205 in green, p.C206 in yellow, p.R281 640
in red and p.R284 in blue. Amino acids where substitutions did not confer constitutive 641
activation are shown in grey. C. IFNß-Luc reporter assay. HEK293T cells were 642
transfected with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING 643
plasmid (EV: Empty vector; WT: Wild type STING; p.N154A; p.V155M; p.D205Y 644
or H; p.C206Y, F, L or S; p.R281Q, K or V; p.R284G, H or K) and stimulated with 645
increasing doses of cGAMP (none; 4 µg/µl; 12 µg/µl). Luciferase activity was 646
determined by Dual-luc assay 24 hour post-stimulation. Mean and SDs shown, 647
representative of 3 different experiments in triplicate. 648
649
FIG 4. Constitutive activation conferred by p.C206Y, p.R281Q and p.R284G is 650
independent of cGAMP binding. A. IFNß-Luc reporter assay. HEK293T cells were 651
transfected with IFNß-Firefly Luc, Renilla Luciferase and indicated human STING 652
plasmid (EV: Empty vector; WT: Wild type STING; p.V155M; p.C206Y; p.R281Q; 653
p.R284G with or without a second mutation p.R232A) and stimulated with increasing 654
doses of cGAMP (none; 4 µg/µl; 12 µg/µl). Luciferase activity was determined by 655
Dual-luc assay 24 hours post-stimulation. Mean and SDs shown, representative of 4 656
different experiments in triplicate. B. Immunoblot (IB) analysis of STING, 657
phosphorylated IRF3 (p.S396), total IRF3 and cofilin in whole cell lysates of 658
HEK293T cells transfected with empty vector (EV), wild type (WT) human STING 659
with or without cGAMP stimulation, p.V155M, p.C206Y, p.R281Q and p.R284G 660
(unstimulated), either alone or in combination with a second mutation at p.R232A. Of 661
note, in HEK293T cells transfected with WT plasmids, the amount of STING was 662
greater at basal state than upon stimulation with cGAMP, which is consistent with a 663
model of STING degradation, possibly through an autophagic-mediated mechanism24. 664
665
FIG 5. Constitutive activation conferred by p.C206Y, p.R281Q and p.R284G 666
mutants is independent of non-canonical pathway STING engagement. A. IFNß-667
Luc reporter assay. HEK293T cells were transfected with IFNß-Firefly Luc, Renilla 668
Luciferase, and indicated human STING plasmid (EV: Empty vector; WT: Wild type 669
-
STING; p.N154S; p.V155M; p.C206Y; p.R281Q; p.R284G with or without a second 670
mutation p.R169A) and stimulated with increasing doses of cGAMP (none; 4 µg/µl; 671
12 µg/µl). Luciferase activity was determined by Dual-luc assay 24 hours post-672
stimulation. Mean and SDs shown, representative of 4 different experiments in 673
triplicate. B. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at p.S396, 674
total IRF3 and cofilin in whole cell lysate of HEK293T cells transfected with empty 675
vector (EV), wild type (WT) human STING with or without cGAMP stimulation, 676
p.N154S, p.V155M, p.C206Y, p.R281Q and p.R284G unstimulated, with or without 677
double mutant p.R169A. 678
679
FIG 6. Activation of newly identified disease-causing STING mutants relies on 680
IRF3 phopshorylation. A. IFNß-Luc reporter assay. HEK293T cells were 681
transfected with IFNß-Firefly Luc, Renilla Luciferase, and indicated human STING 682
plasmid (EV: Empty vector; WT: Wild type STING; p.V155M; p.C206Y; p.R281Q; 683
p.R284G with or without a second mutation p.S366A) and stimulated with cGAMP 684
(12 µg/µl) or not. Luciferase activity was determined by Dual-luc assay 24 hours 685
post-stimulation. Mean and SDs shown, representative of 3 different experiments in 686
triplicate. B. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at p.S396, 687
total IRF3 and cofilin in whole cell lysate of HEK293T cells transfected with empty 688
vector (EV), wild type (WT) human STING with or without cGAMP stimulation, 689
p.V155M, p.C206Y, p.R281Q and p.R284G unstimulated, with or without a second 690
mutation p.S366A. 691
692
FIG 7. STAT1 phosphorylation status in CD4, CD8 and CD19 cells before and 693
after treatment with ruxolitinib in vitro. Increased basal STAT1 phosphorylation 694
was observed in CD4, CD8 and CD19 cells from P3, which normalized after in vitro 695
treatment with the JAK1/2 blocker ruxolitinib. 696
697
698
TABLE EI. Primer sequences used for site directed mutagenesis. 699
700
TABLE EII. TMEM173 primer sequences. 701
702
TABLE EIII. Summary data of substitutions of STING tested in vitro. WT: Wild 703
-
type. 704
705
FIG E1. Multiple sequence alignment of STING proteins. Sequences are 706
designated using UniProt identifiers. The regular secondary structures, as observed 707
from experimental 3D structure (pdb 4ef4) are reported at the top. MAFFT and 708
ESPript were used for building and rendering of the multiple alignment32,33. BOVIN: 709
Bovine; XENTR: Xenopus tropicalis; DANRE: Danio rerio, NEMVE: Nematostella 710
vectensis. 711
712
FIG E2. Double mutant p.D205H with p.R232A, p.R169A or p.S366A. A, B and 713
C. IFNß-Luc reporter assay. HEK293T cells were transfected with IFNß-Firefly Luc, 714
Renilla Luciferase, and indicated human STING plasmid (EV: Empty vector; WT: 715
Wild type STING; p.D205H with or without a second mutation p.R232A, p.R169A or 716
p.S366A respectively) and stimulated with increasing doses of cGAMP (none; 4 717
µg/µl; 12 µg/µl). Luciferase activity was determined by Dual-luc assay 24 hours post-718
stimulation. Mean and SDs shown, representative of 2 different experiments in 719
triplicate. D and E. Immunoblot (IB) analysis of STING, IRF3 phosphorylated at 720
p.S396, total IRF3 and cofilin in whole cell lysate of transfected HEK293T 721
transfected with wild type (WT) human STING with or without cGAMP stimulation 722
and p.D205H unstimulated, with or without double mutant p.R232A, p.S366A or 723
p.R169A. 724
* Uncharacterized band. Of note, in HEK293T cells transfected with WT plasmids, 725
the amount of STING was greater at basal state than upon stimulation with cGAMP, 726
which is consistent with a model of STING degradation, possibly through an 727
autophagic-mediated mechanism24. 728
top related