Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: Structural comparison of unliganded and liganded states of IL-17RA. (A) Structure of unliganded human IL-17RA, represented as a blue cartoon diagram with the three disordered loops A’A (15 missing residues), C’E (7 missing residues), and FG (9 missing residues) shown as dotted lines and the BC loop highlighted by a dotted ellipsoid. (B) Structural overlay of the three known liganded states of IL-17RA with IL-17A (black, from PBD: 4hsa), IL-17F (yellow, from PDB: 3jvf), and IL-17A/F (red, from PDB: 5nan) in ribbon representation, with the cytokine ligands omitted for clarity. The overlay is based on the D1 domain only. The orientation is identical to (A). Note the shift of the D2 domain induced by the different IL-17 cytokines, and the structural transition of the BC loop on top (dotted ellipsoid). (C) Close-up view of the D1 domain, in a different orientation that better shows the conformational switch of the BC loop. Note the key role of the A’A loop in cytokine binding. (D) Close-up view of the D2 domain. Note the change in the domain orientation in the IL-17A (black), IL-17F (yellow), and IL-17A/F (red) complexes compared with the unliganded state (blue).

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Comparison, Binding Assay

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: Conserved crystal contact between two IL-17RA receptor chains (blue ribbon) in the crystal structures of the IL-17A (left view; IL-17A subunits as carmine and gray ribbon, respectively; figure based on PDB: 4hsa), IL-17A/F (center; carmine ribbon: A-subunit; orange ribbon: F-subunit; figure based on PDB: 5nan), and IL-17F complexes (right view; IL-17F subunits as orange and gray ribbon, respectively; figure based on PDB: 3jvf). (A) The top panel shows a side view of two copies of the complex related by exact, crystallographic 2-fold symmetry (IL-17A and IL-17F complexes) or non-crystallographic 2-fold symmetry (IL-17A/F complex). The black arrow points to the BC loop that adopts a helical conformation upon binding of the cytokine and plays a critical role in forming these crystal contacts. The lower panel shows a top view (90° rotation) with the dimerization interface involving the BC loop indicated by a black square. Note that the three X-ray structures were determined from crystals with different unit cells and space group symmetries. (B) Close-up view of the BC loop at the IL-17RA dimer interface, down the 2-fold symmetry axis. The core interface residues Thr102 to Ser105 (PDB: 5nan numbering scheme) are shown in thick stick representation. The green mesh shows the electron-density map contoured at 1.5σ. (C) Stereo views showing the full IL-17RA dimer interface from the top (left panel) or from the side (right view). All residues within 4.0 Å from the other subunit are included. The side-chain of Ala104, buried near the center of the interface, is shown in bold stick.

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Binding Assay

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: Structure of the heteromeric IL-17A (carmine ribbon) complex with IL-17RA (blue ribbon) and IL-17RC (gray ribbon). (A) The asymmetric unit contains one-half of the full 2:2:2 complex shown on (B). (B) Full extracellular, hexameric complex (two views related by 90° rotation). The blue dots represent the flexible linker region of IL-17RA that connects the cytokine-binding domains to the transmembrane region. Note the presence of the same dimerization interface as found in all known IL-17RA binary complexes and highlighted with a black square. The bottom panel is a close-up view of the IL-17RA dimerization interface. The alanine 104 to glutamate mutation, used in this study to disrupt IL-17RA dimerization, is shown in stick representation. (C) Schematic model of the IL-17 signalosome; the intracellular SEFIR domains are depicted in arbitrary positions and orientations, with an arbitrary number of Act1 and Traf6 molecules. For more details about the composition of the IL-17 signalosome, please refer to .

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Binding Assay, Mutagenesis

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: SPR analysis (A) and isothermal calorimetry (B) of IL-17A binding to WT and A104E IL-17RA. (A) Representative sensorgrams are plotted as response in resonance units (RUs) versus time and shown with colored lines. The concentrations of the injected analytes are indicated in the top left corner of the sensorgrams. The kinetic parameters are calculated using a Langmuir 1:1 binding model with the fitted curves depicted as black lines. The indicated Kd represents the mean from four independent experiments ± the standard error of the mean (SEM) and are also summarized in . (B) ITC titration curves for the binding of IL-17A to WT and A104E IL-17RA. For each experiment, the top panel shows the raw data with the differential electrical power (DP) plotted against time. The bottom panel represents the binding isotherm (heat change versus cytokine/receptor molar ratio) obtained from the integration of the raw data and fitted to a “one-site” model. Note that the 2:2 stoichiometry cannot be evidenced from the ITC data; the 2:2 model highlighted with a star sign was inferred from the crystallographic (PDB: 4hsa) and SEC-MALS data.

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Binding Assay, Injection, Titration

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: SEC-MALS analysis of IL-17A binary (IL-17RA) and ternary (IL-17RA/IL-17RC) complexes. SEC chromatograms for isolated proteins (A) and their complexes (B) are color-coded as indicated in the insert table and overlaid with the molecular mass distribution determined by MALS (shown as horizontal lines). Experiments using the A104E IL-17RA point mutant are shown as dotted lines. Peaks are labeled with the corresponding protein or protein complex, depicted schematically, with the star indicating the A104E mutation. The calculated molecular weights in (A) do not consider the glycosylation. Therefore, the observed values are larger, except for IL-17A, which was expressed in E. coli and was thus not glycosylated. For an improved comparison between calculated and observed molecular weights in the case of the receptor complexes, the calculated values in (B) were derived from the observed molecular weights in (A) for the isolated proteins.

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Isolation, Mutagenesis, Labeling, Glycoproteomics, Comparison, Derivative Assay

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: Binding of IL-17A and IL-17F to WT and A104E IL-17RA cells. Biotinylated human IL-17A (left) or biotinylated human IL-17F (right) was incubated with the cells for 1 h at 4°C, and bound IL-17 was determined by flow cytometry using Alexa Fluor 647 conjugated streptavidin. IL-17A: mean ± standard error of the mean (SEM) (n = 4). IL-17F: mean ± SEM (n = 2); highest possible concentration for IL-17F (1 mg/mL). Color code: blue = IL-17RA WT cells; red = A104E IL-17RA cells.

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Binding Assay, Incubation, Flow Cytometry, Concentration Assay

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet: Functional response of WT and A104E IL-17RA N/TERT cells stimulated for 6 h with (A) IL-17A ± 1 ng/mL TNFα or (B) IL-17F ± TNFα after which IL36G mRNA induction was quantified by qRT-PCR. Left side: Concentration-response curves of IL36G mRNA induction (n = 4). Right side: EC 50 values and statistical significance of response between WT and A104E IL-17RA cells. (C) EC 50 values for CXCL1 mRNA induction by IL-17A or IL-17F ± 1 ng/mL TNFα in WT and A104E IL-17RA cells. Shown is mean ± SEM (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001. Color code: blue, WT IL-17RA cells; red, A104E IL-17RA cells.

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Functional Assay, Quantitative RT-PCR, Concentration Assay

Journal: Cell reports

Article Title: IL-17-induced dimerization of IL-17RA drives the formation of the IL-17 signalosome to potentiate signaling

doi: 10.1016/j.celrep.2022.111489

Figure Lengend Snippet:

Article Snippet: For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice.

Techniques: Control, Recombinant, Transfection, Plasmid Preparation, Software