Molecular mechanisms for the regulation and assembly of the NLRP1 and CARD8 inflammasomes

Abstract

Inflammasomes are germline-encoded cytosolic surveillance complexes that respond to a diverse array of intracellular threats including pathogens and sterile damage. Signaling through the inflammasome pathway usually induces pyroptosis, an inflammatory form of cell death, recruiting immune cells to the site of perceived danger. While essential to combating pathogens, dysregulation of the inflammasome pathway leads to a number of autoinflammatory disorders such as cryopyrin-associated periodic syndromes and keratoacanthomas.

There are several different inflammasome sensors in humans that each respond to specific cognate ligand(s), cellular perturbations, and/or pathogens. In this dissertation, we investigated regulation of the NLRP1 and CARD8 inflammasomes. Unique among inflammasome sensors, these related proteins contain a function-to-find domain (FIIND) that autoproteolyzes into two noncovalently associated fragments: a repressive N-terminal fragment (NT) and an inflammatory C-terminal fragment (CT). Processive NT degradation by the proteasome releases the CT, resulting in its oligomerization, inflammasome assembly, and pyroptosis. The cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with and repress NLRP1 and CARD8, and small-molecule DPP8/9 inhibitors activate them by unclear mechanisms. Here, our structure-guided studies reveal that these inflammasome sensors assemble a repressive ternary complex comprising DPP9, full-length NLRP1 or CARD8, and the released bioactive CT. The CT only binds DPP9 in the presence of its full-length counterpart, suggesting that the DPP9 ternary complex acts as a biological rheostat to threshold inflammasome activation. Upon overcoming DPP9 blockade, CTs oligomerize through multivalent interactions that drive its filament formation, creating a signalling hub that recruits downstream adaptors and/or effectors to the inflammasome.

In summary, our structural and cellular data demonstrate that DPP9 acts as a checkpoint for inflammasome activation by directly binding to and sequestering the NLRP1 and CARD8 inflammatory CTs. DPP9 binding sterically occludes a key interface for CT filament assembly, repressing pyroptotic signalling. Since these CTs are generated by homeostatic protein turnover, we propose that disease-associated polymorphisms accelerate this steady-state turnover rate to overcome DPP9 blockade. This mechanism suggests versatile regulation of inflammasome activity, and in the final chapter of this dissertation, I discuss these implications on future inflammasome research and therapeutic discovery.