NLRP1 and CARD8 Inflammasomes: Assembly, Regulation and Stress Sensing

Abstract Nucleotide-binding domain (NBD)-like and leucine-rich repeat (LRR)-containing proteins (NLRs) perform diverse functions in cellular biology, mediating a broad set of fundamental biological pathways from immune signaling to embryonic development. In immunity, several NLRs form supramolecular protein signaling complexes called inflammasomes. Inflammasomes activate caspase-1, an inflammatory protease that processes the cytokine interleukin-1β (IL-1β) and the pore-forming protein gasdermin D to potentiate pyroptotic cell death. One such inflammasome- forming protein, NLRP1, is directly activated in response to intracellular pathogens and the inhibition of DPP9, an endogenous peptidase, serving as both a pattern recognition receptor and a signaling complex. While most NLRs share a common domain architecture, the multifunctionality and regulation of NLRP1 requires additional structural components. In addition to the characteristic NBD and LRR domains, human NLRP1 contains an N- terminal pyrin domain (PYD) and a rare function-to-find domain (FIIND) followed by a caspase activation and recruitment domain (CARD) on its C-terminus. The only other protein with a FIIND is CARD8, which has also been shown to form inflammasomes, leading to cytokine secretion and cell death. Mechanistically, the NLRP1 and CARD8 FIIND domains constitutively catalyze autoproteolytic cleavage, leading to the formation of two noncovalently associated peptides: an autoinhibitory N-terminal fragment and an inflammatory C-terminal fragment. Additionally, Dipeptidyl peptidase 8 or 9 (DPP8/9) binds and inhibits NLRP1 and CARD8, and small molecule inhibitors of DPP8/9 induces NLRP1 and CARD8 activation through some poorly understood pathway. In this application, we propose to elucidate the activation and regulation of NLRP1 and CARD8 using a structure-guided approach. We will determine structures of NLRP1 or CARD8 in complex with DPP8 or DPP9, both WT and mutants. We will analyze the structures and perform additional biochemical and cellular biological experiments to test our hypotheses. In one central aim, we propose that NLRP1 and CARD8 are stress sensors for endogenous cellular dysregulation and play important roles in unwanted inflammation and diseases.