DPP9 activity and not protein binding inhibits the CARD8 inflammasome

Inflammasomes are multiprotein complexes formed in response to pathogens. NLRP1 and CARD8 are related proteins that form inflammasomes, but the pathogen-associated signal(s) and the molecular mechanisms controlling their activation have not been established. Inhibitors of the serine dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) were recently discovered to activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction, in addition to DPP9’s catalytic activity, may contribute to the inhibition of NLRP1. Here, we use activity-based probes, reconstituted inflammasome assays, and mass spectrometry-based proteomics to further investigate the DPP9-CARD8 interaction. We show that the DPP9-CARD8 interaction, unlike the DPP9-NLRP1 interaction, is not disrupted by DPP9 inhibitors or mutations that block autoproteolysis. Moreover, wild-type, but not catalytically-inactive mutant, DPP9 rescues CARD8-mediated cell death in DPP9 knockout cells. Together, this work reveals DPP9 activity and not direct protein binding restrains the CARD8 inflammasome, and suggests the binding interaction likely serves some other biological purpose.