Autolytic proteolysis within the function to find domain (FIIND) is required for NLRP1 inflammasome activity

Abstract

Nucleotide-binding domain leucine-rich repeat proteins (NLRs) play a key role in immunity and disease through their ability to modulate inflammation in response to pathogen-derived and endogenous danger signals. Here, we identify the requirements for activation of NLRP1, an NLR protein associated with a number of human pathologies, including vitiligo, rheumatoid arthritis, and Crohn disease. We demonstrate that NLRP1 activity is dependent upon ASC, which associates with the C-terminal CARD domain of NLRP1. In addition, we show that NLRP1 activity is dependent upon autolytic cleavage at Ser(1213) within the FIIND. Importantly, this post translational event is dependent upon the highly conserved distal residue His(1186). A disease-associated single nucleotide polymorphism near His(1186) and a naturally occurring mRNA splice variant lacking exon 14 differentially affect this autolytic processing and subsequent NLRP1 activity. These results describe key molecular pathways that regulate NLRP1 activity and offer insight on how small sequence variations in NLR genes may influence human disease pathogenesis.

FIGURE 1.

NLRP1 inflammasome activity requires the adaptor protein, ASC, elements encoded with exon 14 and the C-terminal CARD. A, IL-1β ELISA of supernatants from U2OS cells transduced with increasing MOI of BacMam virus expressing NLRP3 or increasing amounts of NLRP1, procaspase-1 (MOI of 1.25), and pro-IL-1β (MOI of 25) in the presence or absence of ASC (MOI of 1.25). B, domain architecture of NLRP1. PYD (pryin domain), nucleotide binding domain (NBD), LRR, FIIND (function to find), CARD (caspase-recruitment domain). Exon 14 is located within FIIND domain (amino acids 1262–1305). C, IL-1β ELISA from supernatants of HEK293T cells transfected with NLRP1 truncation mutants, ASC, procaspase-1, and pro-IL-1β. D, immunoprecipitation of NLRP1 truncation mutants from transfected HEK293T cells. Immunoblots (IB) were probed for ASC.

FIGURE 2.

NLRP1 is cleaved within the FIIND. A, immunoblot (IB) analysis of HEK293T cells transfected with NLRP1 truncation mutants. B, immunoblot analysis of HEK293T cells transfected with full-length NLRP1 expressing an N-terminal HA tag and a C-terminal FLAG tag. C, immunoblot analysis of endogenous NLRP1 in different cell lines with antibodies directed to the C terminus. D, upper panel, IL-1β ELISA of supernatants from HEK293T cells expressing wild type NLRP1 or the indicated SNP. The lower panel shows the NLRP1 immunoblot probed with anti-HA antibodies.

FIGURE 3.

The C-terminal cleavage fragment of NLRP1 is necessary and sufficient for NLRP1 inflammasome activity. A, multiple species sequence alignment of the NLRP1 cleavage site. Asterisks denote completely conserved residues; colon denotes highly conserved residues; arrow denotes site of peptide cleavage. B, diagram depicting the N- and C-terminal cleavage fragments. C, Western blot showing expression of N- and C- terminal NLRP1 cleavage fragments. D, IL-1β ELISA of supernatants from HEK293 cells expressing full-length NLRP1 or the indicated cleavage fragment. Hs, Homo sapiens; Mam, Macaca mulatta; Bt, Bos taurus; Ec, Equus caballus; Mum, Mus musculus; Rn, Rattus norvegicus.

FIGURE 4.

Cleavage within the FIIND is critical for NLRP1 inflammasome activity. A, immunoblot (IB) analysis of HEK293T cells transfected with NLRP1 containing point mutations within the cleavage site. NLRP1 was identified with antibodies directed to HA or FLAG tags located at the N or C termini, respectively. B, IL-1β ELISA of supernatants from HEK293 cells expressing the indicated point mutation. C, NLRP1 was immunoprecipitated with N-terminal HA antibodies, and immunoblots were probed with antibodies specific for the C terminus of NLRP1. D and F, wild type NLRP1 or the indicated point mutant was immunoprecipitated from cell extracts via an N-terminal HA tag. Captured proteins were treated with NH₂OH and analyzed by immunoblot using antibodies specific for the C terminus of NLRP1. Some samples were treated with SDS to disrupt tertiary structure. E, predicted ribbon structure of the NLRP1 FIIND domain showing the position of the three conserved His residues and the cleavage site.