Fig 2: Human caspase-1 autoproteolysis is critical for pyroptosis.(A, B) HEK 293T cells stably expressing the indicated pro-caspase-1 constructs were transiently transfected with plasmids encoding CARD8-Flag (0.05 µg) or NLRP1-Flag (0.1 µg) and ASC (0.01 µg) without GSDMD (A) or with GSDMD (0.01 µg) (B). After 24 h, the cells were treated with VbP (10 µM, 6 h) before lysates were analyzed by immunoblotting. (C) HEK 293T cells stably expressing the indicated pro-caspase-1 were transiently transfected with residues 1–328 of NLRC4 for 24 h before lysates were evaluated by immunoblotting. (D) Schematic of the DmrB-caspase-1 constructs. Predicted cleavage sites, sizes of potential cleavage products, and the catalytic cysteine are indicated. (E) HEK 293T cells stably expressing GSDMD were transiently transfected with the indicated DmrB-caspase-1 constructs for 24 h before addition of AP-20187 (500 nM, 1 h). GSDMD and CASP1 cleavage were evaluated by immunoblotting. All data, including immunoblots, are representative of three or more independent experiments. CL, cleaved; FL, full-length.

Fig 3: CARD8’s disordered region is removed in cells. A, domain organization of CARD8. CARD8 undergoes autoproteolysis between the ZU5 and UPA subdomains. The size in kilodaltons (kDa) of each fragment is indicated. B, lysates of the indicated cell lines were analyzed by immunoblotting. FL, CARD8 full-length; CT, CARD8 C-terminal fragment; p44, CARD8 44 kDa species; asterisks (*) denote nonspecific bands. C, HEK 293T cells were transfected with plasmids encoding the indicated FLAG-tagged constructs (left). Lysates were analyzed by immunoblotting (right). D, the specific residues of CARD8 that the antibodies target are depicted on the cartoon (top). HEK 293T cells were transiently transfected with a plasmid encoding C-terminally HA-tagged CARD8FL WT construct and lysates were analyzed by immunoblotting (bottom). E, C-terminally FLAG-tagged CARD8FL WT expressed from HEK 293T cells were purified with anti-FLAG beads. The 44 kDa ponceau-stained band was analyzed by Edman degradation (right). pm, picomoles. F, Edman degradation analysis in E revealed residues F150 to Y156 (colored red) as the N-terminus of CARD8p44. F150 is shown on the CARD8 structure predicted by Alpha-Fold (26). G–H, CARD8–/– THP-1 cells stably expressing CARD8 WT, S297A, or ?1-149 were treated with compounds for 5 h (G) or 3 h (H) before LDH release and immunoblotting analyses. ****p < 0.0001, by Student’s two-sided t test. NS, not significant. I, HEK 293TCASP1+GSDMD cells were transfected with plasmids encoding the indicated proteins. After 24 h, cells were treated with dTAG-13 for 5 h before LDH release and immunoblot analyses. Data in (G–I) (n = 3) are means ± standard deviation (SD) of replicates. All data, including immunoblots, are representative of three or more independent experiments. HA, hemagglutinin. LDH, lactate dehydrogenase.

Fig 4: VbP activates CARD8FLlacking NT lysines. A, HEK 293T cells were transiently transfected with C-terminally FLAG-tagged CARD8FL S297A and incubated for 24 h. The cells were treated with the indicated compounds in the last 6 h prior to harvest. FLAG-tagged proteins were enriched with anti-FLAG beads, and the eluates were analyzed by immunoblotting. It should be noted that the signals detected on the anti-ubiquitin immunoblots in the BORT-treated samples were likely due to ubiquitinated substrates that bound non-specifically to the anti-FLAG beads, as no laddering pattern of CARD8 (in both the anti-UBQ and FLAG immunoblots) was observed. B, CARD8–/– THP-1 stably expressing the indicated constructs were treated with the compounds for 8 h before LDH release and immunoblotting analyses. PARP (poly ADP-ribose polymerase 1) cleavage to its p89 fragment was assessed as an indicator of apoptosis. C, CARD8–/– THP-1 cells stably expressing the indicated constructs were treated with the indicated compounds for 24 h before LDH release and immunoblotting analyses. Data in (B) and (C) are means ± standard deviation (SD) of three replicates. ****p < 0.0001 by Student’s two-sided t test. NS, not significant. D, lysates of CARD8–/– THP-1 cells stably expressing the indicated constructs were analyzed by immunoblotting. E, HEK 293TCASP1+GSDMD cells were transiently transfected with plasmids encoding the WT and K26R CT fragments and incubated for 16 h prior to immunoblotting analyses. All data, including immunoblots, are representative of three or more independent experiments. VbP, Val-boroPro.

Fig 5: NLRP1 is ASC-dependent and CARD8 is ASC-independent.(A) Human NLRP1, CARD8, and ASC domain organization. The autoproteolysis sites are indicated. (B, C) HEK 293T cells stably expressing CASP1 and GSDMD (HEK 293TCASP1 + GSDMD) were transiently transfected with constructs encoding the indicated proteins and treated with DMSO or VbP (10 µM, 6 h). (B, C) Supernatants were evaluated for LDH release (B) and lysates were analyzed by immunoblotting (C). Data are means ± SEM of three biological replicates. ***P < 0.001 by two-sided t test. (D, E) HEK 293T cells were transfected with constructs encoding GFP-tagged ASC and NLRP1 or CARD8, treated with DMSO or VbP (10 µM, 6 h), and evaluated for ASC speck formation by fluorescence microscopy. The cells were not fixed before analysis. (D, E) Shown are the mean ± SEM (D) and representative images (E) from 10 replicates from one of two independent experiments. ***P < 0.001 by two-sided t test. (F) HEK 293T cells transiently transfected with constructs encoding the indicated proteins and treated with DMSO or VbP (10 µM, 6 h). Lysates were harvested, subjected to disuccinimidyl suberate cross-linking, and evaluated by immunoblotting. All data, including immunoblots, are representative of three or more independent experiments. FL, full-length; WCL, whole cell lysate.