Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptosis. The anthrax lethal factor metalloprotease and small-molecule DPP8/9 inhibitors both activate the NLRP1B inflammasome, but the molecular mechanism of NLRP1B activation is unknown. In this study, we used genome-wide CRISPR-Cas9 knockout screens to identify genes required for NLRP1B-mediated pyroptosis. We discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves NLRP1B, inducing the N-end rule–mediated degradation of the NLRP1B N terminus and freeing the NLRP1B C terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the NLRP1B N terminus but not via the N-end rule pathway. Thus, N-terminal degradation is the common activation mechanism of this innate immune sensor.

NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death 1-6 . Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis 7-14 . NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains [15][16][17][18] . Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT) 19,20 . Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms 11,19,21 . Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a "bomb-diffuser" to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover..

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) activate both NLRP1 and CARD8. Interestingly, DPP9 binds directly to NLRP1 and CARD8, and this interaction 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 CARD8 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 that DPP9’s catalytic activity and not its binding to CARD8 restrains the CARD8 inflammasome and thus suggests the binding interaction likely serves some other biological purpose.

Intracellular pathogens and danger signals trigger the formation of inflammasomes, which activate inflammatory caspases and induce pyroptotic cell death. The anthrax lethal factor metalloprotease and small molecule DPP8/9 inhibitors both activate the Nlrp1b inflammasome, but the molecular mechanism of Nlrp1b activation is not known. Here, we used genome-wide 5 CRISPR/Cas9 knockout screens to identify genes required for Nlrp1b-mediated pyroptosis, and discovered that lethal factor induces cell death via the N-end rule proteasomal degradation pathway. Lethal factor directly cleaves Nlrp1b, which induces the N-end rule-mediated degradation of the Nlrp1b N-terminus and thereby frees the Nlrp1b C-terminus to activate caspase-1. DPP8/9 inhibitors also induce proteasomal degradation of the Nlrp1b N-terminus, but, in 10 contrast, not through the N-end rule pathway. Overall, these data reveal that N-terminal degradation is the common mechanism for activation of this innate immune sensor protein. 15 One Sentence Summary: Proteasome-mediated degradation of the Nlrp1b N-terminus releases the Nlrp1b C-terminus to activate caspase-1 and induce pyroptotic cell death. 20 3 Main Text: Mammals express a diverse array of intracellular pattern-recognition receptors (PRRs) that detect cytoplasmic microbial structures and activities, including bacterial flagellin, double stranded DNA, and pathogen modification of host Rho GTPases (1, 2). Upon recognition of their cognate danger signals, several PRRs form large, multiprotein complexes called inflammasomes, which recruit and activate the inflammatory protease caspase-1. Active caspase-5 1, in turn, cleaves and activates inflammatory cytokines and Gsdmd, which mediates a form of lytic cell death called pyroptosis and stimulates a powerful immune response (1,3,4).Mouse Nlrp1b is a member of the nucleotide-binding domain and leucine rich repeat containing (NLR) family of intracellular PRRs that can form an inflammasome (1, 2,5). Anthrax lethal toxin (LT) is the best-characterized activator of the Nlrp1b inflammasome (5,6). LT is a 10 bipartite toxin consisting of lethal factor (LF), a zinc metalloprotease, and protective antigen (PA), a cell binding-protein that transports LF into the host cell cytosol. LF directly cleaves Nlrp1b after lysine 44, and this cleavage is required for inflammasome formation (7-9). However, it remains unknown how proteolytic cleavage results in the activation of Nlrp1b. We recently discovered that small molecule inhibitors of the host serine dipeptidases DPP8 and DPP9 (DPP8/9) also 15 activate Nlrp1b (10-12). It is not known how DPP8/9 inhibition stimulates formation of the Nlrp1b inflammasome, but, unlike LT-induced activation, it does not involve the direct cleavage of Nlrp1b (11). Intriguingly, proteasome inhibitors block both LT-and DPP8/9 inhibitor-induced pyroptosis (11, 13,14), but do not block pyroptosis mediated by other inflammasomes (14, 15). Thus, although LT and DPP8/9 inhibitors activate Nlrp1b in different ways, a key component of the 20 Nlr...

Inflammasomes are multiprotein complexes that activate inflammatory cytokines and induce pyroptosis in response to intracellular danger‐associated signals. NLRP1 and CARD8 are related germline‐encoded pattern recognition receptors that form inflammasomes, but their activation mechanisms and biological purposes have not yet been fully established. Both NLRP1 and CARD8 undergo post‐translational autoproteolysis to generate two non‐covalently associated polypeptide chains. NLRP1 and CARD8 activators induce the proteasome‐mediated destruction of the N‐terminal fragment, liberating the C‐terminal fragment to form an inflammasome. Here, we review the danger‐associated stimuli that have been reported to activate NLRP1 and/or CARD8, including anthrax lethal toxin, Toxoplasma gondii, Shigella flexneri and the small molecule DPP8/9 inhibitor Val‐boroPro, focusing on recent mechanistic insights and highlighting unresolved questions. In addition, we discuss the recently identified disease‐associated mutations in NLRP1 and CARD8, the potential role that DPP9’s protein structure plays in inflammasome regulation, and the emerging link between NLRP1 and metabolism. Finally, we summarize all of this latest research and consider the possible biological purposes of these enigmatic inflammasomes.

Pathogen-related signals induce a number of cytosolic pattern-recognition receptors (PRRs) to form canonical inflammasomes, which activate pro-caspase-1 and trigger pyroptotic cell death. All well-studied inflammasome-forming PRRs oligomerize with the adapter protein ASC (apoptosis-associated speck-like protein containing a CARD) to generate a large structure in the cytosol, which induces the dimerization, autoproteolysis, and activation of the pro-caspase-1 zymogen. However, several PRRs can also directly interact with pro-caspase-1 without ASC, forming smaller “ASC-independent” inflammasomes. It is currently thought that little, if any, pro-caspase-1 autoproteolysis occurs during, and is not required for, ASC-independent inflammasome signaling. Here, we show that the related human PRRs NLRP1 and CARD8 exclusively form ASC-dependent and ASC-independent inflammasomes, respectively, identifying CARD8 as the first canonical inflammasome-forming PRR that does not form an ASC-containing signaling platform. Despite their different structures, we discovered that both the NLRP1 and CARD8 inflammasomes require pro-caspase-1 autoproteolysis between the small and large catalytic subunits to induce pyroptosis. Thus, pro-caspase-1 self-cleavage is a required regulatory step for pyroptosis induced by human canonical inflammasomes.

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Highlights d A 2PCA-biotin probe enables chemical enrichment of protease substrates (CHOPS) d CHOPS rapidly identifies DPP substrates with a gel-based readout d DPP9 preferentially cleaves short peptides, not whole proteins (e.g., Nlrp1b) d CHOPS enables unbiased protease substrate profiling with quantitative proteomics