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. 2018 Oct 31;9:2366.
doi: 10.3389/fimmu.2018.02366. eCollection 2018.

A CARD9 Founder Mutation Disrupts NF-κB Signaling by Inhibiting BCL10 and MALT1 Recruitment and Signalosome Formation

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Free PMC article

A CARD9 Founder Mutation Disrupts NF-κB Signaling by Inhibiting BCL10 and MALT1 Recruitment and Signalosome Formation

Marieke De Bruyne et al. Front Immunol. .
Free PMC article

Abstract

Background: Inherited CARD9 deficiency constitutes a primary immunodeficiency predisposing uniquely to chronic and invasive fungal infections. Certain mutations are shown to negatively impact CARD9 protein expression and/or NF-κB activation, but the underlying biochemical mechanism remains to be fully understood. Objectives: To investigate a possible founder origin of a known CARD9 R70W mutation in five families of Turkish origin. To explore the biochemical mechanism of immunodeficiency by R70W CARD9. Methods: We performed haplotype analysis using microsatellite markers and SNPs. We designed a model system exploiting a gain-of-function (GOF) CARD9 L213LI mutant that triggers constitutive NF-κB activation, analogous to an oncogenic CARD11 mutant, to study NF-κB signaling and signalosome formation. We performed reporter assays, immunoprecipitation and confocal imaging on HEK cells overexpressing different CARD9 variants. Results: We identified a common haplotype, thus providing evidence for a common Turkish founder. CARD9 R70W failed to activate NF-κB and abrogated NF-κB activation by WT CARD9 and by GOF CARD9. Notably, R70W CARD9 also exerted negative effects on NF-κB activation by CARD10, CARD11, and CARD14. Consistent with the NF-κB results, the R70W mutation prevented GOF CARD9 to pull down the signalosome partner proteins BCL10 and MALT1. This reflected into drastic reduction of BCL10 filamentous assemblies in a cellular context. Indeed, structural analysis revealed that position R70 in CARD9 maps at the putative interface between successive CARD domains in CARD9 filaments. Conclusions: The R70W mutation in CARD9 prevents NF-κB activation by inhibiting productive interactions with downstream BCL10 and MALT1, necessary for assembly of the filamentous CARD9-BCL10-MALT1 signalosome.

Keywords: BCL10; CARD9 deficiency; CBM complex; MALT1; NF-κB; filament; founder mutation; signalosome.

Figures

Figure 1
Figure 1
Pedigrees. CARD9 R70W mutations were identified in five families. Each kindred is identified by a number (F1–F5), each generation by a Roman numeral (I–V), and each subject by an Arabic numeral (1–12). Square, circle and diamond shapes indicate male, female, and sex unknown, respectively. A double line represents reported consanguinity. Diagonal lines indicate deceased individuals. Filled symbols represent affected individuals, clear symbols represent unaffected individuals. The index patients are indicated with arrows. Where available, the genotype is mentioned. WT, wild-type allele.
Figure 2
Figure 2
CARD9 structure and haplotype analysis of the R70W (c.208C>T) mutation. (A) Schematic CARD9 gene structure and reported disease-associated mutations. Roman numbered boxes represent exons. Gray boxes situate the CARD and Coiled Coil domains at protein level. The R70W mutation is shown in bold, the gain-of-function L213LI mutation in red. (B) Segregation analysis of flanking microsatellites and single nucleotide polymorphisms revealed a common haplotype of 1.03 Mb. Patients codes refer to the pedigrees in Figure 1. Segregation of the haplotype in all available family members is shown in Supplemental Figure 1. M, mutant allele; F, family.
Figure 3
Figure 3
R70W CARD9 inhibits NF-κB transcriptional activity. (A, C) Schematic summaries of the experimental conditions in (B,D) respectively, adjusted from (13). (B, D) MALT1-deficient HEK293T cells were transfected with WT, R70W, L213LI and R70W/L213LI CARD9 as indicated, with fixed total DNA quantities, with or without MALT1 as indicated, together with an NF-κB-dependent luciferase reporter expression plasmid and a constitutively expressed β-galactosidase reporter gene. Luciferase values were normalized against β-galactosidase and expressed as fold induction compared to WT CARD9 without MALT1. Expression of MALT1 and CARD9 protein measured by western blot are shown below the graphs. (E) WT HEK293T cells were co-transfected with decreasing doses of L213LI DNA and increasing doses of R70W/L213LI CARD9 DNA (ng/well, doses specified under graph). The total DNA amount was kept constant at 400ng/well. Luciferase values were expressed as fold induction compared to L213LI CARD9 alone. All values under the dotted line are significant.Results shown in (B,D,E) are mean +/- standard deviation of 4 replicates. One out of 2–3 representative experiments is shown. Statistical analysis was performed on reporter assay data with one-way ANOVA and Tukey's multiple comparison's (B, D, E) post-testing. The most relevant statistical differences are shown, a list of all p-values is provided in Supplemental Tables 2–4. p < 0.001 (***) in all panels, in (B,D) only for reporter assays with MALT1 reconstitution.
Figure 4
Figure 4
CARD9 premature stop mutants reveal the presence of an N-terminal auto-inhibition domain (A). NF-κB luciferase reporter assay of R70W CARD9 co-transfected with auto-active mutants of CARD9, CARD10, CARD11, and CARD14, all lacking the C-terminal inhibitory domain. Luciferase values were expressed as fold induction compared to WT CARD9 alone. (B) NF-κB luciferase reporter assay of CARD9 constructs, harboring a premature stop after the first CC (Q295*) and the second CC (E419*) domain. Results shown in both panels are mean +/- standard deviation of 4 replicates. Statistical analysis was performed on reporter assay data with one-way ANOVA and Tukey's multiple comparison's post-testing. The most relevant statistical differences are shown: each CARD protein compared with and without CARD9 R70W in (A) and each mutant compared to WT CARD9 in (B). A list of all p-values is provided in Supplemental Tables 5, 6. ***p < 0.001.
Figure 5
Figure 5
R70W CARD9 fails to pull down BCL10 and MALT1 and inhibits BCL10 filamentous network formation. (A) WT HEK293T cells were co-transfected with MALT1 and E-tagged BCL10 with indicated Flag-tagged CARD9 constructs. The CARD9 variants were immunoprecipitated with anti-Flag antibody and co-immunoprecipitation of BCL10 and MALT1 was detected (“IP”, upper panel) by anti-E-tag (BCL10) and anti-MALT1 antibody. CARD9 was detected with anti-Flag-HRP antibody. Input controls as immunoblotted on total lysate are shown in the lower panel. Density quantification of CARD9 on the total lysates is shown in Supplemental Figure 4. (B) A structural model for human CARD9 was created from structure-based sequence alignments of the sequence encoding the CARD domain of human CARD9 (uniprot Q9H257) and further improved computed via I-TASSER and QUARK. The R70 residue is depicted in red. (C) The homology model of the CARD domain of CARD9 was docked into a three-dimensional model representing the human BCL10 filament using the segment fitting algorithms implemented in Chimera. (D) E-tagged BCL10 was co-transfected with indicated Flag-tagged CARD9 constructs in un-stimulated HEK293T cells. Confocal imaging was performed with anti-E (BCL10, Green) and anti-Flag (CARD9, Red). 3D reconstructions of 50 Z-stacks are shown. Scale bar = 10μm.

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References

    1. Puel A, Cypowyj S, Bustamante J, Wright JF, Liu L, Lim HK, et al. . Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science (2011) 332:65–8. 10.1126/science.1200439 - DOI - PMC - PubMed
    1. Okada S, Puel A, Casanova J-L, Kobayashi M. Chronic mucocutaneous candidiasis disease associated with inborn errors of IL-17 immunity. Clin Trans Immunol. (2016) 5:e114–9. 10.1038/cti.2016.71 - DOI - PMC - PubMed
    1. Li J, Vinh DC, Casanova J-L, Puel A. Inborn errors of immunity underlying fungal diseases in otherwise healthy individuals. Curr Opin Microbiol. (2017) 40:46–57. 10.1016/j.mib.2017.10.016 - DOI - PMC - PubMed
    1. Gross O, Gewies A, Finger K, Schäfer M, Sparwasser T, Peschel C, et al. . Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature (2006) 442:651–6. 10.1038/nature04926 - DOI - PubMed
    1. Glocker EO, Hennigs A, Nabavi M, Schäffer AA, Woellner C, Salzer U, et al. . A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N Engl J Med. (2009) 361:1727–35. 10.1056/NEJMoa0810719 - DOI - PMC - PubMed

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