A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly

Abstract

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci.

Figure 1.

VHH_ASC specifically binds human ASC CARD. (A) Resins with covalently coupled VHH_ASC and VHH_Enhancer were used to immunoprecipitate the respective VHH targets from THP-1 cells expressing EGFP. Proteins bound to the resin were eluted with acidic buffers, separated by SDS-PAGE, and stained with colloidal Coomassie. The highlighted protein bands were cut out and subjected identification by mass spectrometry. (B) Differentiated THP-1 cells were left untreated or treated with LPS and nigericin to trigger NLRP3 inflammasome activation. Cells were subjected to immunofluorescence staining with rabbit anti-ASC and Alexa Fluor 647–coupled VHH_ASC or VHH NP1. Maximum intensity projections of representative images of at least three experiments are presented. Bars, 10 µm. (C) A549 cells transiently expressing HA-tagged VHHs and the designated bait proteins fused to Renilla luciferase were lysed to immunoprecipitate VHHs with immobilized anti-HA. Renilla luciferase activity of the coimmunoprecipitated proteins was measured and normalized by the activity in the input. Mean values ± SEM from three independent experiments are displayed. (D) MBP-ASC^CARD was incubated with an excess of VHH_ASC and complexes separated from monomeric VHH_ASC by size-exclusion chromatography. MBP was cleaved off purified complexes with TEV protease, and the yielded protein samples were subjected to size-exclusion chromatography as well. Samples of the fractions were analyzed by SDS-PAGE and Coomassie staining. (E) A549 cells were transiently transfected with expression vectors for EGFP, EGFP-ASC, EGFP-ASC^PYD, or EGFP-ASC^CARD. 24 h after transfection, cells were lysed and subjected to SDS-PAGE and immunoblot with anti-EGFP, anti-ASC (raised against the PYD of ASC), and VHH_ASC combined with the respective HRP-coupled secondary antibodies. Representative immunoblots of at least three experiments are shown.

Figure 2.

VHH_ASC binds to an interface of ASC^CARD that is required for CARD–CARD interactions. (A) Structure of VHH_ASC at 1.9 Å resolution. (B) Structure of VHH_ASC in complex with the N128A/E130R monomeric mutant of ASC^CARD at 4.2 Å resolution. Residues at the interaction interface are indicated. Because the resolution of the complex was not sufficient to resolve the displayed amino acid side chains, we used the side chain conformations from the VHH crystal structure at 1.9 Å resolution and the full-length ASC NMR structure. (C) Comparisons of the structures of the complex of VHH_ASC/ASC^CARD N128A/E130R with VHH_ASC and ASC^CARD alone.

Figure 3.

Binding of VHH_ASC to ASC^CARD sterically occludes ASC^CARD type II interface. (A) Electrostatic surface representation of the VHH_ASC–ASC^CARD complex, highlighting tyrosine 59 in VHH_ASC and tyrosine 187 in ASC^CARD. (B) The MBP-ASC^CARD-SUMO fusion protein was incubated with TEV protease to cleave off the MBP protein, which led to polymerization of ASC^CARD-SUMO. A representative electron micrograph of the resulting fibers subjected to negative staining is shown. Bar, 100 nm. (C) Representation of the ASC^CARD filament modeled using the known MAVS^CARD filament structure (left), a zoomed view of the type II interface of two highlighted ASC^CARD subunits (middle), and illustration of the complex of VHH_ASC with ASC^CARD subunit 1 (right). The VHH sterically clashes with ASC^CARD subunit 2 bound via the type II interface (shown in transparent cyan). (D) Interaction of ASC WT and type II mutant Y187A with His-tagged caspase-1. The indicated combinations of His₆-GFP-caspase-1^CARD and WT or mutant His₆-MBP-ASC were incubated. Where indicated, His₆-MBP was cleaved off ASC using TEV protease, and His-tagged proteins were purified using Ni-NTA resin. Samples of the protein mixtures were analyzed by SDS-PAGE and Coomassie staining. Positions of the indicated proteins are marked on the right. The intensity of the ASC^FL bands was determined from the scanned gel and normalized by the band intensity of GFP-caspase-1^CARD.

Figure 4.

VHH_ASC prevents ASC^CARD-nucleated polymerization of caspase-1^CARD and ASC^CARD. (A) ASC-nucleated caspase-1^CARD polymerization in the presence of VHH_ASC and VHH_Enhancer. Fluorescently labeled monomeric MBP-caspase-1^CARD-SUMO (4 µM) was mixed with oligomeric GFP-ASC^CARD (1.1 µM) preincubated with VHH_ASC or VHH_Enhancer (1.1 µM) where indicated. MBP was cleaved from fluorescent caspase-1^CARD-SUMO to allow ASC^CARD-nucleated polymerization of caspase-1^CARD. Polymerization was followed by measuring fluorescence polarization over time. (B) ASC-nucleated caspase-1^CARD polymerization in presence of different VHH_ASC concentration. Polymerization of caspase-1^CARD-SUMO by oligomeric GFP-ASC^CARD was measured by fluorescence polymerization assays as described in A, except that GFP-ASC^CARD was preincubated with the indicated concentrations of VHH_ASC. (C) Apparent inhibition constant of VHH_ASC. The initial slope of the fluorescence polarization processes in B was plotted against the logarithm of the VHH_ASC concentration. A single-site antagonist model was fitted to this plot to extract the apparent inhibition constant K_app. (D) ASC^CARD polymerization. Fluorescently labeled monomeric MBP-ASC^CARD-SUMO (5 µM) was preincubated with VHH_ASC at the indicated concentrations. MBP was cleaved off fluorescent ASC^CARD-SUMO to self-nucleated polymerization of ASC^CARD. Polymerization was followed by measuring fluorescence polarization over time. (E) Stability of ASC^CARD oligomers in presence of VHH_ASC. Oligomeric GFP-ASC^CARD was incubated with excess VHH_ASC for 30 min at room temperature or left untreated. Protein samples were subsequently subjected to size-exclusion chromatography. OD₂₈₀ profiles are displayed.

Figure 5.

VHH_ASC prevents activation of NLRP3 and AIM2 inflammasomes in THP-1 cells. (A) THP-1 cell lines inducibly expressing VHH_ASC-HA or VHH NP1-HA were cultivated for 24 h in the absence or presence of doxycycline, fixed, stained for HA, and analyzed by flow cytometry. (B–E) Wild-type THP-1 cells and the cell lines described in A were differentiated and VHH expression induced for 24 h. (B) Cells were treated with 200 ng/ml LPS for 3 h, harvested, and cell lysates analyzed by immunoblot with anti-ASC, anti-HA, and anti-GAPDH. (C) Cells were treated with 200 ng/ml LPS for 4 h or left untreated. Supernatants were analyzed by TNF ELISA. (D and E) Cells were treated with LPS for 3 h and nigericin for 45 min, or left untreated. Supernatants were analyzed by IL-1β ELISA (D) and by immunoblots with anti–IL-1β and anti-caspase-1 p10 (E). Immunoblots representative of three experiments are shown. The asterisk indicates a statistically significant difference (Student‘s t test; P < 0.001). (F and G) Cells treated as in D were fixed and stained for DNA and ASC; images were recorded by wide field fluorescence microscopy (F) and the fraction of cells containing ASC foci was quantified (G). Data from three independent experiments with at least 500 cells per condition ± SEM is shown. Bars, 20 µm. (H) THP-1 cell lines cultivated as described in B were treated with LPS for 3 h and transfected with poly (dA:dT) or transfection agent only. Supernatants were harvested after 4 h and IL-1β levels were quantified by ELISA. The asterisk indicates a statistically significant difference (Student‘s t test; P = 0.002). Data from three independent experiments was quantified for all ELISA results and mean values ± SEM are shown.

Figure 6.

VHH_ASC prevents IL-1β secretion, but not cell death, by NAIP/NLRC4 inflammasomes in THP-1 cells. (A and B) WT THP-1 cells and cell lines inducibly expressing VHH_ASC-HA or VHH NP1-HA were differentiated and VHH expression induced for 24 h. Cells were treated with PA and LFn-MxiH WT or 2A for 3 h or left untreated. IL-1β in the supernatants was quantified by ELISA (A), LDH activity was quantified and normalized to cells lysed in 1% Triton X-100 (B). Data from three independent experiments was quantified and mean values ± SEM are shown. The asterisk indicates a statistical significant difference (Student‘s t test; P = 0.039). (C and D) THP-1 cell lines were differentiated and induced as described in A, and subsequently treated with PA and MxiH WT or 2A for 2 h in the presence of 50 µM z-YVAD-cmk. Cells were fixed and stained for DNA and ASC; images were recorded by wide field fluorescence microscopy (C) and the fraction of cells containing ASC foci was quantified (D). Bars, 20 µm. Data from three independent experiments with at least 500 cells per condition ± SEM is shown.

Figure 7.

VHH_ASC-EGFP assembles along ASC filaments in response to inflammasome triggers. (A–D) THP-1 cell lines inducibly expressing VHH_ASC-EGFP or VHH NP1-EGFP were differentiated and VHH expression induced for 24 h. Cells were left untreated (A), treated with LPS for 3 h and nigericin for 45 min to trigger NLRP3 inflammasomes (B), treated with LPS for 3 h and transfected with DNA for 4 h to trigger AIM2 inflammasomes (C), or treated with PA and LFn-MxiH WT in the presence of 30 µM z-YVAD-cmk for 2 h to trigger NAIP/NLRC4 inflammasomes (D). Cells were fixed and stained for DNA and ASC; images of single planes were recorded by spinning disc confocal microscopy. Bars, 10 µm. Representative images from at least three experiments are shown. (E–G) THP-1 cell lines were differentiated, VHH expression induced, and NLRP3, AIM2, and NLRC4 inflammasomes activated or cells left untreated as in A–D, but in the presence of 50 µM z-YVAD-cmk. Cells were fixed and mounted with mounting medium containing DAPI; Z stacks were recorded by spinning disc confocal microscopy. At least 300 cells per condition were analyzed and cells containing green-fluorescent filaments or foci were manually counted. One typical experiment representative of at least three independent experiments was quantified.

Figure 8.

VHH_ASC stabilizes ASC filaments formed through ASC^PYD polymerization in vitro and in cells. (A) MBP-ASC or MBP-ASC preincubated with VHH_ASC was incubated with TEV protease to remove the MBP fusion and allow polymerization. At the indicated times, the reaction was stopped and the resulting macromolecular structures were visualized by negative stain electron microscopy. Bars, 500 nm. (B) A549 cells were transiently transfected with expression vectors for EGFP-ASC^PYD or EGFP-ASC in the absence or presence of expression vectors for VHH_ASC or VHH NP1. 24 h after transfection, cells were fixed and stained for DNA and filamentous actin. Z-stacks of representative cells were recorded by spinning disc confocal microscopy and maximum intensity projections are displayed. Bars, 10 µm. (C) Human monocyte-derived macrophages were differentiated in the presence of 10% FBS and 100 ng/ml M-CSF for 5 d and subsequently infected with mature virions of vaccinia virus WR E VHH_ASC-EGFP L mCherry or WR E VHH NP1-EGFP L mCherry at an MOI of 20. Cells were fixed 18 h after infection and stained for DNA. Z stacks of representative cells were recorded by spinning disc confocal microscopy and images of maximum intensity projections are shown. Bars, 10 µm.

Figure 9.

Molecular model of inflammasome activation. (A) Mechanism of ASC filament formation nucleated by NLRP3 (left) and NAIP/NLRC4 (right). (B) Model of ASC filament cross-linking mediated by ASC^CARD. (C) Modeled structure of ASC filament covered with VHH_ASC. Relative arrangement of ASC^PYD, ASC^CARD, and VHH_ASC is shown. ASC^CARD–VHH_ASC unlikely follows the exact helical symmetry of the PYD filament core as a result of the flexible linker between PYD and CARD. (D) Schematic representation of ASC focus formation in unperturbed conditions, and formation of ASC filaments coated with VHH_ASC-EGFP.

Figure 10.

Model of small oligomer of ASC^CARD complexed by VHH_ASC. (A) An ASC^CARD oligomer (orange) with bound VHH_ASC (gray) was modeled based on the MAVS^CARD filament symmetry and the ASC^CARD/VHH_ASC structure. VHH_ASC occludes type IIb interface of ASC^CARD, but does not interfere with small oligomer assembly. (B) A few ASC molecules bound to activated NLRC4 are sufficient to nucleate the formation of ASC^PYD filaments even in the presence of VHH_ASC.