Heterozygous mutations in the C-terminal domain of COPA underlie a complex autoinflammatory syndrome
Selket Delafontaine, … , Jérôme Delon, Isabelle Meyts
Selket Delafontaine, … , Jérôme Delon, Isabelle Meyts
Published January 4, 2024
Citation Information: J Clin Invest. 2024;134(4):e163604. https://doi.org/10.1172/JCI163604.
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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.

Authors

Selket Delafontaine, Alberto Iannuzzo, Tarin M. Bigley, Bram Mylemans, Ruchit Rana, Pieter Baatsen, Maria Cecilia Poli, Daisy Rymen, Katrien Jansen, Djalila Mekahli, Ingele Casteels, Catherine Cassiman, Philippe Demaerel, Alice Lepelley, Marie-Louise Frémond, Rik Schrijvers, Xavier Bossuyt, Katlijn Vints, Wim Huybrechts, Rachida Tacine, Karen Willekens, Anniek Corveleyn, Bram Boeckx, Marco Baggio, Lisa Ehlers, Sebastian Munck, Diether Lambrechts, Arnout Voet, Leen Moens, Giorgia Bucciol, Megan A. Cooper, Carla M. Davis, Jérôme Delon, Isabelle Meyts

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Figure 6

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

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Overexpression of the CTD COPA mutants does not induce STING-dependent t...
(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 COPAWT 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.