Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants Reiko Kagawa,

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Presentation transcript:

Alanine-scanning mutagenesis of human signal transducer and activator of transcription 1 to estimate loss- or gain-of-function variants Reiko Kagawa, MD, Ryoji Fujiki, MS, Miyuki Tsumura, PhD, Sonoko Sakata, MD, Shiho Nishimura, MD, Yuval Itan, PhD, Xiao-Fei Kong, MD, PhD, Zenichiro Kato, MD, PhD, Hidenori Ohnishi, MD, PhD, Osamu Hirata, MD, PhD, Satoshi Saito, MD, Maiko Ikeda, MD, Jamila El Baghdadi, PhD, Aziz Bousfiha, MD, Kaori Fujiwara, MD, Matias Oleastro, MD, Judith Yancoski, PhD, Laura Perez, BSc, Silvia Danielian, PhD, Fatima Ailal, MD, Hidetoshi Takada, MD, PhD, Toshiro Hara, MD, PhD, Anne Puel, PhD, Stéphanie Boisson-Dupuis, PhD, Jacinta Bustamante, MD, PhD, Jean-Laurent Casanova, MD, PhD, Osamu Ohara, PhD, Satoshi Okada, MD, PhD, Masao Kobayashi, MD, PhD Journal of Allergy and Clinical Immunology Volume 140, Issue 1, Pages 232-241 (July 2017) DOI: 10.1016/j.jaci.2016.09.035 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Family trees and known pathogenic STAT1 mutations. A, Human STAT1α isoform with its known pathogenic mutations. Mutations identified in patients with an AR form of complete (orange) or partial (green) STAT1 deficiency are shown above. Mutations identified in patients with AD MSMD are shown in red (current study) and blue (previously reported). The reported GOF mutations are shown in magenta below the protein. TA, Transcriptional activation domain; TS, tail segment. B-D, Family trees. Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 GAS transcription assay based on systematic alanine-scanning mutagenesis. GAS transcriptional activity in response to IFN-γ was measured in 176 STAT1 proteins mutated in the CCD (A) and 166 STAT1 proteins mutated in the DBD (B). Of alanine mutants, 34.7% (61/176) and 30.1% (50/166) were deemed to be GOF mutations, whereas 2.2% (4/176) and 15.7% (26/166) were deemed to be LOF mutations in the CCD and DBD, respectively. Experiments were performed in triplicate, and data are expressed in relative luciferase units (RLU). Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Distributions of LOF and GOF mutations in antiparallel and parallel STAT1 dimers. GOF (red) and LOF (blue) alanine substituents were mapped to the antiparallel (A) and parallel (B) dimeric structures of STAT1. Fig 3, A, GOF alanine mutants predominantly localized at the interface of the antiparallel dimer, whereas LOF alanine mutants localized inside the protein. Fig 3, B, Many LOF alanine mutants localized close to the DNA-binding site. Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Functional assays of E157K and G250E STAT1 identified in patients with MSMD. A, Levels of pSTAT1 were reduced in the E157K and G250E mutants in response to IFN-γ to levels similar to those in the K673R mutant. B and C, E157K and G250E STAT1, as well as the K673R hypomorphic mutant, severely impaired the ability of GAF to bind GAS (Fig 4, B) and induce IRF1 reporter activity after IFN-γ stimulation (Fig 4, C). In contrast, Q463H and Y701C STAT1 completely abolished DNA binding to GAS (Fig 4, B) and IRF1 activation (Fig 4, C). D, All the mutants exerted a dose-dependent negative effect on WT STAT1–mediated GAS activation after IFN-γ stimulation. The amounts of plasmids used are shown below the x-axis. Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Subcellular localization of WT and mutant STAT1. U2OS cells stably expressing V5-tagged WT or mutant STAT1 were stimulated with IFN-γ for 20 minutes and subjected to immunostaining. A, Before IFN-γ stimulation, the WT and all mutant STAT1 proteins were localized to the cytoplasm. B, Like the K673R mutant, the nuclear translocation of E157K and G250E STAT1 was partially impaired in cells expressing either protein. In contrast, Y701C completely abolished the nuclear translocation of STAT1 after IFN-γ stimulation. Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Interaction between E157 and E394 residues affects GAS transcriptional activity. A-C, E157K STAT1 was analyzed in silico with MOE. Unlike WT STAT1 (Fig 6, A), the E157K (Fig 6, B) and E394K (Fig 6, C) mutants were predicted to form hydrogen bonds with E394 and E157 in their dimeric partners, respectively. D, The E157K+E394K double mutant was predicted to allow access to their side chains, leading to electrostatic repulsion between the two. E, Both E157K and E394K were LOF mutations, whereas the E157K+E394K double mutant was predicted to be a GOF mutant by using the reporter assay. Similar results were observed with arginine substitutions. Journal of Allergy and Clinical Immunology 2017 140, 232-241DOI: (10.1016/j.jaci.2016.09.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions