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Haploinsufficiency of A20 Impairs Protein–Protein Interactome and Leads Into Caspase-8-Dependent Enhancement of NLRP3 Inflammasome Activation

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Haploinsufficiency of A20 Impairs Protein–Protein Interactome and Leads Into Caspase-8-Dependent Enhancement of NLRP3 Inflammasome Activation Autoinflammatory disorders RMD Open: first published as 10.1136/rmdopen-2018-000740 on 17 October 2018. Downloaded from ORIGINAL ARTICLE Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation Kristiina Rajamäki,1 Salla Keskitalo,2 Mikko Seppänen,3 Outi Kuismin,4 Paula Vähäsalo,5 Luca Trotta,6 Antti Väänänen,7 Virpi Glumoff,8 Paula Keskitalo,5 Riitta Kaarteenaho,9,10 Airi Jartti,11 Nina Hautala,12 Päivi Jackson,12 Dan C Nordström,13 Janna Saarela,6 Timo Hautala,14 Kari K Eklund,15,16,17 Markku Varjosalo2,18 To cite: Rajamäki K, ABSTRACT Key messages Keskitalo S, Seppänen M, Objectives TNFAIP3 encodes A20 that negatively et al. Haploinsufficiency regulates nuclear factor kappa light chain enhancer of of A20 impairs protein– What is already known about this subject? activated B cells (NF-κB), the major transcription factor protein interactome and A20 is a negative regulator of nuclear factor kappa B coordinating inflammatory gene expression.TNFAIP3 ► leads into caspase-8- (NF- B) having a key role in immune activation and polymorphisms have been linked with a spectrum of κ dependent enhancement autoimmunity. inflammatory and autoimmune diseases and, recently, of NLRP3 inflammasome Loss-of-function mutations in A20 are reported to loss-of-function mutations in A20 were found to cause ► activation. RMD Open lead to an inflammatory disease termed haploinsuf- 2018;4:e000740. doi:10.1136/ a novel inflammatory disease ‘haploinsufficiency ofA 20’ ficiency ofA 20 (HA20). rmdopen-2018-000740 (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate What does this study add? ► Prepublication history and the upstream molecular mechanisms linking HA20 to ► We report a novel p.(Lys91*) A20 mutation and use it additional material for this dysregulation of NF-κB and the related inflammasome as a model to uncover molecular mechanisms relat- paper are available online. To pathway. ed to the role of A20 in HA20 development. http://rmdopen.bmj.com/ view these files, please visit Methods NF-κB activation was studied in a mutation- ► The p.(Lys91*) fails to suppress NF-κB activity and the journal online (http:// dx. doi. expressing cell line using luciferase reporter assay. org/ 10. 1136/ rmdopen- 2018- has severely impaired protein interactome com- Physical and close-proximity protein–protein interactions 000740). pared with the wild-type A20 in HEK cells, and caus- of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were es aberrant NLRP3 inflammasome responsiveness analysed using mass spectrometry. NF-κB -dependent in patient peripheral blood mononuclear cells. KR and SK contributed equally. transcription, cytokine secretion and inflammasome TH, KKE and MV contributed activation were compared in immune cells of the HA20 How might this impact on clinical practice? equally. ► This study significantly deepens our understanding patients and control subjects. on September 26, 2021 by guest. Protected copyright. on the role of A20 in rheumatic diseases and may Results The protein–protein interactome of Received 11 June 2018 provide novel approaches for the development of p.(Lys91*) mutant A20 was severely impaired, Revised 3 August 2018 therapeutic approaches. Accepted 7 September 2018 including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB INTRODUCTION signalling, which led to increased NF-κB -dependent Tumour necrosis factor alpha-induced proinflammatory cytokine transcription. Functional protein 3 (TNAP3, also known as A20), © Author(s) (or their experiments in the HA20 patients’ immune cells encoded by the TNFAIP3 gene, is a key employer(s)) 2018. Re-use uncovered a novel caspase-8-dependent mechanism negative regulator of nuclear factor kappa permitted under CC BY-NC. No commercial re-use. See rights of NLRP3 inflammasome hyperresponsiveness that B (NF-κB) activation downstream a large and permissions. Published mediated the excessive secretion of interleukin-1β and number of immune receptors, including the by BMJ. interleukin-18. TNF and Toll/interleukin(IL)-1 receptor For numbered affiliations see Conclusions The current findings significantly deepen families and the T and B cell antigen recep- end of article. our understanding of the molecular mechanisms tors.1–5 A20 achieves this via its dual function underlying HA20 and other diseases associated with as a ubiquitin-editing enzyme: an N-terminal Correspondence to reduced A20 expression or function, paving the way for ovarian tumour (OTU) domain removes Dr Markku Varjosalo; future therapeutic targeting of the pathway. markku. varjosalo@ helsinki. fi activating K63-linked polyubiquitin from key Rajamäki K, et al. RMD Open 2018;4:e000740. doi:10.1136/rmdopen-2018-000740 1 RMD Open RMD Open: first published as 10.1136/rmdopen-2018-000740 on 17 October 2018. Downloaded from intermediate NF-κB signalling molecules to destabilise searched for rare heterozygous variants, according to protein–protein interactions, whereas a C-terminal zinc the inferred autosomal-dominant inheritance pattern finger domain catalyses the attachment of K48-linked (figure 1A). The variant identified in the TNFAIP3 polyubiquitin to induce proteasomal degradation.3 6 7 In gene (Ensembl ENSG00000118503:ENST00000433680: addition, A20 inhibits NF-κB activation via a non-cata- exon2:c.A271T: p.(Lys91*) was confirmed using PCR lytic mechanism involving its binding to linear ubiquitin and capillary electrophoresis. on NF-κB essential modulator (NEMO).8 9 The mRNA expression of A20 is NF-κB-inducible,10 thus generating Creating A20 expressing Flp-In 293 T-REx cell lines a negative feedback loop to attenuate NF-κB responses. A20 mutant plasmids were created using QuickChange Recently Zhou et al reported six families with hetero- Site-Directed Mutagenesis kit (Agilent Technologies) to zygous loss-of-function mutations in the TNFAIP3 gene wt TNFAIP3 obtained from the human Orfeome collec- that led to haploinsufficiency of A20 (HA20) and caused tion (Horizon Discovery). TNFAIP3 constructs were 11 further subcloned into N-terminal MAC-tag Gateway an early-onset Behçet-like disease. Studies in mouse 24 models have previously demonstrated a crucial role for destination vector. All generated constructs were A20 in autoinflammation and autoimmunity as mice with confirmed with direct sequencing. The MAC-tagged a germline deletion of Tnfaip3 spontaneously develop expression constructs were transfected into Flp-In severe multiorgan inflammation and tissue damage 293 T-REx cells (Invitrogen, Life Technologies) resulting in perinatal death,12 and cell-specific ablation with FuGENE HD (Promega). The cells were grown of Tnfaip3 results in diverse symptoms of immune dysreg- according to manufacturer’s instructions under selec- ulation.13 14 Similar to Tnfaip3 deficiency in mice,12–15 tion with Hygromycin B (Thermo Fisher Scientific) human HA20 led to exaggerated NF-κB responses to create stable cell lines. Expression of stable trans- and increased secretion of NLR family pyrin domain genes was confirmed by western blotting using anti-he- containing 3 (NLRP3) inflammasome target cytokines.11 magglutin (HA) primary (Biolegend, MMS-101R) and Additional reports from these16 and further identified horseradish peroxidase-conjugated secondary anti- patients17–21 have expanded the clinical spectrum of body. HA20 to comprise diseases such as autoimmune lymph- Luciferase assay oproliferative syndrome, systemic juvenile arthritis, psori- For reporter assays, HEK293 cells were transfected with atic arthritis, Crohn’s disease and Hashimoto’s thyroiditis. in total 50 ng A20 mutant or wt MAC-tagged construct The increasing number of diseases associated with A20 24 and GFP or empty MAC-tag-vector, 40 ng of NF-κB lucif- has emphasised its role in inflammatory diseases and in erase reporter plasmid (Cignal 45), and 1.4 ng Renilla autoimmunity and raised the possibility that mutations in luciferase reporter plasmid (pRL-SV40). Transfected TNFAIP3 may be an unexpectedly common underlying cells were stimulated with indicated amounts of tumour factor in the pathogenesis of rheumatic and other auto- http://rmdopen.bmj.com/ necrosis factor alpha TNF-α (R&D Systems) for 16 hours, immune diseases. lysed with 1× passive lysis buffer (Promega) and luciferase We describe here a family with polyautoimmunity and assays performed with Dual-Glo Luciferase Assay System autoinflammatory symptoms caused by a novel hetero- according to manufacturer’s instructions (Promega). zygous TNFAIP3 p.(Lys91*) loss-of-function mutation resulting in haploinsufficiency. Our aim was to further Affinity purification and mass spectrometry elucidate the upstream molecular mechanisms linking For each pull-down approximately 5×107 cells (5×15 cm HA20 to dysregulated NF-κB activation and NLRP3 dishes) were induced with 2 µg/mL tetracycline (Thermo inflammasome responses. Thus, we analysed protein– Fisher Scientific) for 24 hours (for BioID additional 50 on September 26, 2021 by guest. Protected copyright. protein interactions of wild-type (wt) and p.(Lys91*) A20 µM biotin was added). After induction, cells were washed, in HEK cells and performed functional experiments in harvested, pelleted by centrifugation, snap-frozen and patients’ immune cells to study NLRP3 inflammasome stored at −80°C until analysis. Affinity purification
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