Figure 5. Two Distinct Ways to Activate the CARD8 Inflammasome.

(A) Schematic of a direct displacement assay for DPP9 inhibition. Co-expressed FLAG-DPP9, NLRP1 or CARD8 CT-V5 and full-length NLRP1 or CARD8 SA mutant (FL-SA-MYC) (Input) were captured on FLAG beads. The bound components were displaced with DPP8/9 inhibitors or control compounds (Wash), and then eluted with 3x-FLAG peptide (Eluate).

(B) Displacement of NLRP1-CT from DPP9 by the DPP8/9 inhibitors VbP and 8J in vitro as depicted in (A). The displacement was apparent in the Wash, and also visible in the Eluate. The vehicle control DMSO or the non-selective aminopeptidase inhibitor MeBs did not cause displacement.

(C) Lack of displacement of CARD8-CT from DPP9 in vitro by any of the compounds. Immunoblots are representative of 2 independent experiments.

(D) FP biotin reduces capture of DPP9 by CARD8. Lysates from HEK 293T cells ectopically expressing full-length CARD8 and CARD8 CT-FLAG, were incubated with DMSO, VbP, or FP biotin prior to FLAG co-immunoprecipitation. Immunoblots are representative of 2 independent experiments.

(E) Further enhancement of cell death by VbP in dTAG13-induced CARD8 activation in the presence of FIIND-SA rescue. Reconstituted HEK 293T cells expressing dTAG-CARD8 and increasing amount of FIIND-SA were treated with dTAG-13 (500 nM) alone, or both dTAG-13 and VbP (10 μM) for 3 hr. Cell death was measured by LDH release (top) and GSDMD processing (bottom). The enhancement was most apparent at a lower dose of transfected FIIND-SA. Mean ± SEM are shown from 3 independent biological replicates. *** and ns denote p < 0.001 and not significant, respectively, by 2-way ANOVA with Tukey’s multiple comparison correction.

(F) VbP disruption of FIIND-SA mediated cell death rescue independent of VbP-induced CARD8 degradation. Reconstituted HEK 293T cells were expressed with dTAG-CARD8-ZUC, which does not contain the N-terminal disordered sequence to undergo VbP-induced degradation, and increasing amount of FIIND-SA, and treated with dTAG-13 (500 nM), VbP (10 μM), or dTAG13 plus VbP for 3 hr. Mean ± SEM are shown from 3 independent biological replicates. * and *** denote p < 0.05 and p < 0.001, respectively, by 2-way ANOVA with Tukey’s multiple comparison correction.

(G) Functional disruption of FL-SA rescue of dTAG13-induced CARD8 activation by VbP in monocytes. THP-1 CARD8^−/− cells were stably reconstituted with dTAG-ZUC, with or without the Tet-inducible FL-SA (S297A) construct. While doxycycline (DOX)-dependent FL-SA expression dampened dTAG-13 induced pyroptosis, VbP disrupted this rescue. Mean ± SEM are shown from 3 independent biological replicates. *** and ns denote p < 0.001 and not significant, respectively, by 2-way ANOVA with Tukey’s multiple comparison correction.

(H) Reduction of co-immunoprecipitated DPP9 by FLAG-tagged CARD8-FIIND-S297A (FIIND-SA). HEK 293T cells were co-expressed with FLAG-FIIND-SA and dTAG-CARD8 (FL) or dTAG-CARD8-ZUC (ZUC) constructs and treated with dTAG13 (500 nM) alone or dTAG13 plus VbP (10 μM) for 24 hr. * indicates non-specific band.

(I) Model of CARD8 inflammasome activation upstream of the proteosome (signal 1) and downstream of the proteasome (signal 2).

See also Figure S5.