Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome

Abstract

Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis, and nephritis. We described 3 heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C, and p.R1142X) in 6 children from 3 unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts, we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison with mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport, and increased proinflammatory signaling.

Keywords: Cell stress; Immunology; Innate immunity; Monogenic diseases.

Figure 1. Three heterozygous mutations in the CTD of COPA in 6 patients with an autoinflammatory and autoimmune phenotype.

(A) Pedigrees of families A, B, and C with indication of the genotype, assigned with the amino acid changes, below each individual. Affected individuals are indicated by black filled symbols and an arrow, gray half-filled symbols indicate unaffected heterozygous carriers, and open shapes indicate unaffected family members. Squares designate males and circles females. Patient 2 (B.II.1) died at the age of 7 due to hemophagocytic lymphohistiocytosis and multiple organ failure. Patients 5 (C.II.3) and 6 (C.II.4) are dizygotic twins. Sanger sequencing chromatograms for COPA, performed on genomic DNA, are shown, covering a 15 bp snapshot around the mutation. Red arrows indicate the position of the mutation. (B) Medical imaging for patient 1 (A.II.3). Brain MRI (T2-weighted images) shows a hyperintense optic chiasm (red arrow), indicative of neuromyelitis optica with involvement of the optic chiasm (top left panel). Spinal cord MRI (T2 weighted images) illustrates a swollen spinal cord with central hyperintensity (red bracket), ranging from the level of cervical vertebra 4 (C4) to thoracic vertebra 8 (T8), revealing transverse myelitis (bottom left panel). Chest computed tomography (CT) demonstrates bronchiectasis (top right panel), and MRI of the abdomen depicts hepatosplenomegaly (bottom right panel, red arrows). (C) Chest CT of patient 2 (B.II.1) shows signs of an alveolar hemorrhage and centrilobular nodules.

Figure 2. Genetic aspects and in silico pathogenicity prediction of mutations in the CTD of COPA.

(A) Schematic illustration of the COPA protein and its domains. Previously reported mutations in the WD40 domain are depicted in black (filled circle, functionally validated; open circle, functional validation unavailable), mutations in the CTD of COPA in color (color code correlates with Figure 1A). Numbers in parentheses refer to the number of families identified, number of mutation carriers, and number of diseased, respectively. Coatomer WDAD, coatomer WD-associated region. (B) Population genetics of the previously described COPA mutations affecting the WD40 domain, previously published not functionally validated CTD mutations, and CTD COPA mutations. MAF, minor allele frequency; MSC, mutation significance cutoff; CADD, combined annotation-dependent depletion score. (C) Conserved sequence homology at the site of the identified mutations in distantly related eukaryotes. (D) Biomodeling of the mutations affecting the CTD of COPA. The central figure depicts the main proteins of COPI, COPA (orange), COPB (teal), COPB2 (blue), and COPE (purple). Left: The physical interaction between COPA^R1142X and COPA^WT is depicted. In the top representation, COPA^WT is shown as surface and COPA^R1142X as a cartoon inside the surface, illustrating complete removal of the dimerization interface of COPA^R1142X with the neighboring COPA^WT (oval). This exposes the hydrophobic interface of the COPE binding helices, thus disrupting the COPA-COPE dimer. In the bottom representation, the absent residues are colored in gray. Right: The interaction between COPA^R1058C, COPA^C1013S, and COPE is shown. COPA^R1058C and COPA^C1013S likely disturb the conformation of the α-helices on which they are located and subsequently disrupt COPA’s overall structure. (E) Magnification of biomodeling of the α-helices, which compose the main body of the CTD and comprise residues 1013 and 1058, and form a binding site for singleton tryptophan motif (STM). STMs are known to be crucial for COPA homo-oligomerization and ER tethering of COPI vesicles.

Figure 3. Analysis of COPA, COPB2, and COPE mRNA and protein expression in patients affected by mutations in the CTD of COPA.

(A and B) RT-qPCR analysis of transcript levels of COPA, COPB2, and COPE analyzed in cDNA extracted from whole blood (for patients and carriers of families A and C) or PBMCs (for B.I.2 and B.II.1). (A) COPA mRNA expression, evaluated using 4 probes covering exons 2–3, 4–5, 11, and 32–33. (B) Transcript levels of COPA (calculated as mean of transcript levels of the 4 COPA probes, shown in A), COPB2, and COPE. Relative mRNA level depicts fold increase of gene expression normalized to GAPDH (ΔCt) and to mean ΔCt of control samples. Three to four samples from separate time points were analyzed for A.II.3 and A.I.1. (C) Western blot analysis of COPA, COPB2, and COPE in whole-cell lysates of PBMCs of A.I.1, A.II.3, and 3 healthy controls. Immunoblotting of COPA with an antibody specific for the N-terminal region of COPA (N-COPA) and an antibody specific for the C-terminal region of COPA (C-COPA). (D) Quantification of protein level of COPA, detected by N-COPA and C-COPA antibody (left), and COPB2 and COPE (right), as observed in C. Band intensity was determined relative to GAPDH and normalized to mean of healthy controls (n = 8, 5 adults, 3 children). (E) Western blot analysis of COPA, COPB2, and COPE in EBV LCLs of A.I.1, A.II.3, C.I.1, and C.II.1–4 compared with 4 adult healthy controls. (F) Western blot analysis of COPA, COPB2, and COPE in fibroblasts of A.I.1, A.II.3, and B.II.1 compared with 2 healthy controls. Results in C–F are representative of 2–3 independent experiments. In A, B, and D, columns and bars represent mean ± SEM values, statistically analyzed using 2-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 4. COPA^R1142X and COPA^R1058C disrupt COPI integrity and impair anterograde ER-to-Golgi and retrograde Golgi-to-ER trafficking.

(A) Western blot analysis of FLAG, N-terminal COPA, COPB2, COPE, and β-actin antibody in whole-cell extract (input) or eluate of IP of HEK293T cells cotransfected with both WT or mutant COPA and WT or EV of COPB2 and COPE. IP was performed with an antibody against FLAG (IP COPA). (B) Quantification of COPB2 and COPE protein levels coimmunoprecipitated with COPA-FLAG (COPA IP) compared with the input signal, as observed in A. (C) Immunofluorescence analysis of PCI transport assay in 4 different control fibroblasts (1 representative control is shown), COPA^R1142X fibroblasts, derived from A.I.1 and A.II.3, and COPA^R1058C fibroblasts, derived from B.II.1, at 0 and 60 minutes. Scale bars: 10 μm. (D) Graphs represent quantification of the ER exit (top) and Golgi entry (bottom) of PCI, as shown in C. (E) Immunofluorescence analysis of CtxB transport assay in 4 different control fibroblasts, COPA^R1142X fibroblasts, derived from A.I.1 and A.II.3, and COPA^R1058C fibroblasts, derived from B.II.1. Analysis was performed 2 hours and 10 hours after exposure to CtxB. Scale bars: 10 μm. (F) Graphs represent quantification of the Golgi release (top) and ER entry (bottom) of CtxB, as observed in E. In B, D, and F, results are shown as mean ± SEM, and significance levels were calculated using 1-way (D and F) or 2-way (B) ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Data are representative of 2–3 independent experiments. (G) Electron microscopy of COPA^R1142X fibroblasts, derived from A.II.3, compared with fibroblasts from a healthy control. Original magnification, ×15,000; ×40,000 (bottom right); scale bars: 200 nm. The images demonstrate a fragmented and disorganized Golgi apparatus (blue box) and an accumulation of vesicles (red box) in the cytosol of COPA^R1142X fibroblasts.

Figure 5. Mutations in the CTD of COPA induce type I IFN pathway activation in 3 of 6 patients.

(A and B) IFN scores (A) and ISG expression (B) in peripheral whole blood of patients and their families. (B) mRNA expression of the individual ISGs for 3 healthy controls, 1 IFN-α– and IFN-β–stimulated PBMC sample of a healthy control, 1 SAVI patient (carrying a p.V155M mutation in STING), 1 AGS patient (heterozygous for a p.S962A fs*92 and a p.P193A mutation in ADAR), and the patients, carriers, and healthy family members included in A. The first number in parentheses is the decimalized age at the time of sampling, the second the IFN score. Mean ± SEM values of different time points, if available, are shown (n = 7 for A.II.3 [prior to transplantation]; n = 3 for A.I.1, A.I.2, A.II.4; n = 2 for A.II.2; and n = 1 for other individuals). (C) Evolution of type I IFN score (left) and CRP value (right) through the disease course of patient A.II.3. Timing is indicated in days prior to hematopoietic stem cell transplantation. Relevant clinical manifestations and treatments are indicated. (D) Flow cytometry analysis of p-STAT1 in monocytes of A.II.3 compared with a control and a STAT1 gain-of-function (GOF) patient. The ratio of the number of p-STAT1–positive cells in comparison with the unstimulated condition is indicated. Data are representative of 2 independent experiments. (E) Immunofluorescent analysis of STING localization in fibroblasts (top) and EBV LCLs (bottom) of healthy controls, A.I.1, A.II.3, B.II.1, C.I.1, and C.II.1–4. Cells were stained for STING-TMEM173, cis-Golgi (giantin), and nucleus (Hoechst). The merge column represents an overlay between the stains. Scale bars: 10 μm. (F) Quantification of colocalization of STING and cis-Golgi, expressed as the Pearson coefficient. Results are representative of at least 3 independent experiments. Results are shown as means ± SEM, and significance levels were calculated using 1-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Figure 6. Overexpression of the CTD COPA mutants does not induce STING-dependent type I IFN signaling in HEK293T cells.

(A–F) HEK293T cells were cotransfected with EV or WT STING and WT or mutant COPA as indicated. (A) Immunoblotting of whole-cell lysates for COPA (FLAG), p-IRF3, total IRF3, STING, and β-actin. (B) Quantification of p-IRF3 protein relative to total IRF3, as demonstrated in A. (C) Relative mRNA expression of IFIT1 and ISG15, normalized to GAPDH and to HEK293T cells expressing COPA EV and STING EV. First, expression was compared between cells cotransfected with STING and COPA^WT and cells cotransfected with STING and mutant COPA (lines and asterisks). Second, expression in cells cotransfected with STING EV and WT or mutant COPA was compared with the corresponding condition cotransfected with STING (asterisks above error bars). Cells stimulated with 2′3′-cGAMP served as a positive control. (D) ISRE luciferase reporter assay. Luciferase activity was measured in total cell lysate. (E) Confocal microscopy of COPA and STING colocalization. Cells were stained for COPA (FLAG), STING, Golgi (GM130), and ER (calnexin). The additional squares in the STING column contain an enlargement of the image. Scale bar: 25 μm. (F) Quantification of the ratio of STING localized to the Golgi over total STING, as demonstrated in E. Results in A–F are representative of 2–4 independent experiments. In B–D and F, columns and bars represent mean ± SEM, analyzed using 1-way (F) or 2-way (B–D) ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (G) Relative mRNA expression of IFIT1 in HEK293T cells cotransfected with different ratios of WT and mutant COPA and EV or WT STING. Triangles depict the amount of transfected WT, EV, or mutant COPA cDNA. Dotted lines and the right y axis illustrate fold change of IFIT1 corresponding to HEK293T cells cotransfected with different percentages of WT COPA. Variants are classified based on their effect on IFIT1 expression. The mean of 3 technical replicates is shown.

Figure 7. CTD COPA mutations cause activation of ER stress and proinflammatory signaling pathways such as the NF-κB pathway.

(A) Relative mRNA expression of HSPA5, ATF4, DDIT3, and COPA in EBV LCLs of 4 healthy controls, A.I.1, A.II.3, C.I.1, and C.II.1–4. LCLs were unstimulated (white, –) or treated for 6 hours with thapsigargin (black, +). Results were normalized to GAPDH (ΔCt) and to the control samples (ΔΔCt). (B) Representative images of immunofluorescent analysis of BiP intensity in fibroblasts of healthy controls, A.I.1, A.II.3, and B.II.1. Cells were stained for BiP, F-actin, and nucleus and stimulated with thapsigargin. Graphs represent quantification of MFI of BiP. (C) Representative images of immunofluorescent analysis of p65–NF-κB nuclear translocation in fibroblasts of healthy controls, A.I.1, A.II.3, and B.II.1. Cells were stimulated with LPS and stained for p65–NF-κB and nucleus. Nuclear translocation of p65 appears violet. Graphs represent quantification of nuclear translocation of NF-κB. Scale bars: 10 μm. In A–C, columns and bars represent mean ± SEM, representative of 2 (A) to 3 (B and C) independent experiments. Statistical analysis was performed using 1-way (A) or 2-way ANOVA (B and C) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (D–F) Analysis of bulk RNA sequencing data of whole-blood RNA of 4 controls (black), 2 carriers (A.I.1 and C.I.1), 1 SAVI patient (green), and 5 patients (A.II.3, C.II.1–4). (D) Principal component analysis (PCA) plot of bulk RNA sequencing data, based on the 1,000 genes with the largest intersample variance (after a variance stabilizing transformation removing the variance dependence on the mean). (E) Top 10 differentially expressed pathways determined by IPA analysis of differential gene expression for patients A.II.3 (left) and C.II.4 (right) versus the group consisting of carriers (A.I.1, C.I.1), controls, SAVI patient, and C.II.1–3. (F) Heatmaps represent differential expression analysis for the eIF2 pathway, 24 autophagy genes, and a limited list of ISGs.