ABSTRACT
Several cytosolic pattern-recognition receptors (PRRs) form multiprotein complexes called canonical inflammasomes in response to intracellular danger signals. Canonical inflammasomes recruit and activate caspase-1 (CASP1), which in turn cleaves and activates inflammatory cytokines and gasdermin D (GSDMD), inducing pyroptotic cell death. Inhibitors of the dipeptidyl peptidases DPP8 and DPP9 (DPP8/9) activate both the human NLRP1 and CARD8 inflammasomes. NLRP1 and CARD8 have different N-terminal regions but have similar C-terminal regions that undergo autoproteolysis to generate two non-covalently associated fragments. Here, we show that DPP8/9 inhibition activates a proteasomal degradation pathway that targets disordered and misfolded proteins for destruction. CARD8's N terminus contains a disordered region of â¼160 amino acids that is recognized and destroyed by this degradation pathway, thereby freeing its C-terminal fragment to activate CASP1 and induce pyroptosis. Thus, CARD8 serves as an alarm to signal the activation of a degradation pathway for disordered and misfolded proteins.
Subject(s)
CARD Signaling Adaptor Proteins/chemistry , CARD Signaling Adaptor Proteins/metabolism , Inflammasomes/metabolism , Intrinsically Disordered Proteins/metabolism , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Animals , Boronic Acids/pharmacology , Dipeptides/pharmacology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , HEK293 Cells , Humans , Lysine/metabolism , Mice , Proteolysis , Proteostasis , RAW 264.7 Cells , THP-1 CellsABSTRACT
Canonical inflammasomes are innate immune signaling platforms that are formed in response to intracellular pathogen-associated signals and trigger caspase-1-dependent pyroptosis. Inflammasome formation and signaling is thought to mainly occur in myeloid cells, and in particular monocytes and macrophages. Here we show that small molecule inhibitors of dipeptidyl peptidases 8 and 9 (DPP8/9), which activate the related CARD8 and NLRP1 inflammasomes, also activate pyroptosis in human and rodent resting lymphocytes. We found that both CD4+ and CD8+ T cells were particularly sensitive to these inhibitors, although the sensitivity of T cells, like macrophages, varied considerably between species. In human T cells, we show that CARD8 mediates DPP8/9 inhibitor-induced pyroptosis. Intriguingly, although activated human T cells express the key proteins known to be required for CARD8-mediated pyroptosis, these cells were completely resistant to DPP8/9 inhibitors. Overall, these data show that resting lymphoid cells can activate at least one inflammasome, revealing additional cell types and states poised to undergo rapid pyroptotic cell death in response to danger-associated signals.
Subject(s)
CARD Signaling Adaptor Proteins/metabolism , Cell Cycle , Dipeptidases/antagonists & inhibitors , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/antagonists & inhibitors , Inflammasomes/metabolism , Lymphocytes/metabolism , Neoplasm Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Cell Cycle/drug effects , Cells, Cultured , Dipeptidases/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Humans , Lymphocyte Activation/drug effects , Lymphocytes/drug effects , Mice , NLR Proteins , Protease Inhibitors/pharmacology , Pyroptosis/drug effects , RatsABSTRACT
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.
Subject(s)
Caspase 1/metabolism , Pyroptosis/physiology , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis , Apoptosis Regulatory Proteins/metabolism , CARD Signaling Adaptor Proteins/metabolism , Carrier Proteins/metabolism , HEK293 Cells , Humans , Inflammasomes/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Signal Transduction , THP-1 CellsABSTRACT
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.