In Vitro Reconstitution of the NALP1 Inflammasome Reveals Requirements for Caspase Activation.
Authors: Faustin Benjamin , Lartigue Lydia , Bruey Jean-Marie , Luciano Frederic , Hanein Dorit , Rouiller Isabelle , Reed John C. 
Published: October 2019

Abstract
NLRs constitute a large family of host defense proteins that minimally contain a nucleotide-binding domain responsible for oligomerization and Leucine Rich Repeat (LRR) domains that bind microbial ligands, typically together with additional domains such as CARD or PYRIN that link NLRs to downstream components of either the NF-kB activation machinery or pro-inflammatory caspases involved in proteolytic processing of cytokines such as Interleukin-1b (IL-1b). Hereditary mutations in gene encoding some NLRs are associated with autoimmune and chronic inflammatory diseases. The NLR-family protein NALP1 (NAC; CARD7; CLR17.1; DEFCAP) contains a nucleotide-binding NACHT domain, LRRs, CARD, PYRIN, and other domains. NALP1 has been reported to bind the bipartite adapter protein, ASC, and pro-inflammatory caspases, forming a large multimer protein complex termed the “inflammasome.” The molecular details of inflammasome assembly and function however are unclear. Here, we have expressed the components of inflammasome (NALP1, ASC, pro-caspase-1) using recombinant baculoviruses and have purified the proteins to accomplish an in vitro reconstitution of this caspase-activating machine. Purified NALP1 monomers were induced to oligomerize in a two-step mechanism requiring ATP and the microbial ligand, muramyl-dipeptide (MDP), as determined by non-denaturing gel-electrophoresis experiments. MDP induced a conformational change in NALP1 detectable by non-denaturing gel-electrophoresis, followed by ATP-induced oligomerization. Non-hydrolyzable analogs of ATP blocked oligomerization. Inactive stereoisomers of MDP also failed to induce NALP1 oligomerization, further demonstrating specificity. Oligomerization of NALP1 was independently confirmed by cryo-electron microscopy, showing a striking increase in particle size when ATP and MDP were supplied. These changes in oligomerization correlated with NALP1-mediated activation of pro-caspase-1. NALP1 was found to bind pro-caspase-1 directly via its CARD domain. In reactions containing only NALP1 and pro-caspase-1, the combination of ATP and MDP induced caspase-1 activation, as determined by hydrolysis of fluorigenic substrate Ac-WEHD-AMC. The Kd values for ATP and for MDP were 13.3 ± 9.06 and 2.89 ± 1.9 nM, respectively. Several nucleotide triphosphates, including GTP, CTP and TTP, effectively substituted for ATP, but not deoxy- or dideoxy-ATP, not ADP or AMP, and not non-hydrolyzable ATP analogs. Adapter protein ASC was not required for NALP1-induced caspase-1 activation, but it enhanced caspase-1 activation, with optimal activity occurring at a ratio of 1:1:2 (ASC: NALP1; pro-caspase-1). The catalytic efficiency (kcat/Km) of NALP1 increased from 2715 ± 564 to 4623 ± 770 M−1.s−1 when ASC was supplied. Taken together, studies of the reconstituted NALP1 inflammasome reveal for the first time:ribonucleotide triphosphates are required for oligomerization and caspase-1 activation;MDP directly stimulates NALP1 oligomerization, without requiring a protein co-factor;ASC is non-essential but enhances inflammasome activity when present at optimal ratios relative to NALP1 and procaspase-1.The in vitro reconstituted inflammasome will be useful for further structural and biophysical characterization of this caspase-activating machine and for analysis of chemical inhibitors.