Volume 25, Issue 5, Pages 713-724 (March 2007) Reconstituted NALP1 Inflammasome Reveals Two-Step Mechanism of Caspase-1 Activation  Benjamin Faustin, Lydia Lartigue, Jean-Marie Bruey, Frederic Luciano, Eduard Sergienko, Beatrice Bailly-Maitre, Niels Volkmann, Dorit Hanein, Isabelle Rouiller, John C. Reed  Molecular Cell  Volume 25, Issue 5, Pages 713-724 (March 2007) DOI: 10.1016/j.molcel.2007.01.032 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Expression and Purification of Inflammasome Components by Using Recombinant Baculoviruses (A) Domain structures of NALP1, ASC, and procaspase-1 are depicted, showing Pyrin (PYD), NACHT (with Walker-A and -B motifs forming the nucleotide-binding site), NACHT-associated domain (NAD), leucine-rich repeats (LRR), domain with unknown function (FIIND), caspase-recruitment domain (CARD), and large and small catalytic subunits of procaspase-1. (B) Proteins were expressed in insect cells and affinity purified using either GST or His tags, followed by additional chromatographic separations. Proteins (2–10 ug) were analyzed by SDS-PAGE. For GST-NALP1, gels shown were stained with Coomassie blue (left) and Sypro ruby (right). GST-ASC and His6-procaspase-1 were visualized by staining with Sypro ruby. (C) CARD domain of NALP1 binds procaspase-1. HEK293T cells were transfected with plasmids encoding Myc-tagged NALP1, NALP1ΔCARD, Apaf-1, or NALP3, and Flag-tagged caspase-1, caspase-8, caspase-9, or caspase-12. Cell lysates normalized for total protein content were analyzed directly (bottom) or subjected to immunoprecipitation (IP) (top) using anti-Myc antibody and analyzed by SDS-PAGE/immunoblotting (WB) using anti-Flag and anti-Myc antibodies. Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 MDP Directly Activates NALP1 (A) NALP1 (8.5 nM) and procaspase-1 (8.5 nM) were incubated with either MDP-LD or MDP-DD (0.1 μg/ml), or with γ-tri-DAP (0.1 μg/ml), in the presence or absence of Mg2+ (0.5 mM) and ATP (0.25 mM), with or without caspase-1 inhibitor YVAD-fmk (2 μM). Caspase-1 activity was measured using Ac-WEHD-AMC substrate (20 μM) and expressed as RFU/min (mean ± SD; n = 3). (B) NALP1 induces procaspase-1 processing in an MDP/ATP-dependent manner. NALP1 and/or procaspase-1 was incubated with biotinyl-VAD-fmk (30 μM) and subsequently stimulated with or without MDP-LD, MDP-DD, or γ-tri-DAP, in the presence or absence of ATP/Mg2+ for 30 min at 37°C. NT, not treated. Active caspase-1 was recovered from samples using streptavidin-Sepharose (SA) and was analyzed by SDS-PAGE immunoblotting using anti-p10 caspase-1 antibody. (C) Various protein amounts of procaspase-1 were incubated with NALP1 (8.5 nM), with or without active MDP (MDP-LD, 0.1 μg/ml), Mg2+ (2.5 mM), and ATP (0.25 mM) for 30 min at 37°C. Caspase-1 activity was then measured using cleavage of Ac-WEHD-AMC (20 μM) as specific substrate. Data are expressed as RFU per min (mean ± SD; n = 3). (D) Comparison of MDP and LPS. NALP1 (8.5 nM) and procaspase-1 (8.5 nM) were incubated with various amounts of either MDP-LD or LPS, in the presence of ATP/Mg2+. Caspase-1 activity was measured using Ac-WEHD-AMC (20 μM) (mean ± SD; n = 3). (E) NALP1 (8.5 nM) and/or procaspase-1 (8.5 nM) was incubated for 30 min at 37°C with increasing amounts of MDP-LD in the presence of Mg2+ (0.5 mM) and ATP (0.25 mM). Experimental data points (n = 3) were analyzed by a nonlinear regression method to fit the Michaelis-Menten equation. Bar graphs represent apparent maximal velocity values (Vmax app) determined from the fitting procedure for MDP-LD (mean ± SD; n = 3). Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Characterization of NTP Dependence of NALP1-Induced Caspase-1 Activation (A) Ribonucleoside triphosphates (NTPs) are required for NALP1-mediated caspase-1 activation. NALP1 (8.5 nM) and procaspase-1 (8.5 nM) were incubated with or without active MDP-LD (0.1 μg/ml), and various NTPs or analogs (0.25 mM) in the presence of Mg2+ (0.5 mM) for 30 min at 37°C. Caspase-1 activity was then measured by hydrolysis of Ac-WEHD-AMC (20 μM). Comparison of activity of NTPs is shown. NALP1 (8.5 nM) and/or procaspase-1 (8.5 nM) was incubated with MDP-LD (0.1 μg/ml) and various NTPs (60 nM) in the presence of Mg2+ (0.5 mM) for 30 min at 37°C. Caspase-1 activity was then measured by hydrolysis of Ac-WEHD-AMC (20 μM) (mean ± SD; n = 3). (B–D) Kinetic features of NALP1-mediated caspase-1 activation. NALP1 (8.5 nM) and/or procaspase-1 (8.5 nM) was incubated for 30 min at 37°C with (B) increasing concentrations of ATP in the presence of MDP-LD (0.1 μg/ml), Mg2+ (0.5 mM), and Ac-WEHD-AMC (20 μM); or (C) increasing concentrations of Mg2+ in the presence of MDP-LD (0.1 μg/ml), ATP (0.25 mM), and Ac-WEHD-AMC (20 μM); or (D) increasing concentrations of substrate Ac-WEHD-AMC (0–20 μM) with MDP-LD (0.1 μg/ml), ATP (0.25 mM), and Mg2+ (0.5 mM). Caspase-1 activity was measured by hydrolysis of the fluorogenic peptide substrate, expressing data as RFU/min (mean ± SD; n = 3). Experimental data points (n = 3) were analyzed by a nonlinear regression method to fit the Michaelis-Menten equation. Bar graphs represent Vmax app determined from the fitting procedure for each component: ATP (B), Mg2+ (C), and Ac-WEHD-AMC (D). Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 ASC Is an Enhancer of NALP1-Mediated Caspase-1 Activation (A) Concentration dependence of ASC influence on NALP1-mediated caspase-1 activation. NALP1 (4.25 nM) and procaspase-1 (8.5 nM) were incubated with increasing concentrations of ASC in the presence of MDP-LD (0.1 μg/ml), ATP (0.25 mM), and Mg2+ (0.5 mM) for 30 min at 37°C. Caspase-1 activity was measured by hydrolysis of Ac-WEHD-AMC (20 μM) (RFU/min; mean ± SD; n = 3). (B) ASC does not activate procaspase-1 without NALP1. Procaspase-1 (8.5 nM) was combined with various concentrations of ASC (x axis) in the presence or absence of 4.25 nM NALP1. All reactions were supplied with 0.1 ug/ml MDP, 0.25 mM ATP, 0.5 mM Mg2+, and Ac-WEHD-AMC (20 uM) for 30 min at 37°C. The fold increase in protease activity (y axis), relative to procaspase-1 alone, was determined (mean ± SD; n = 3). (C) Kinetic features of NALP1 inflammasome in the presence of ASC. NALP1 and/or procaspase-1 was incubated with or without ASC in the presence of MDP-LD (0.1 μg/ml), Mg2+ (0.5 mM), and ATP (0.25 mM) for 30 min at 37°C, measuring caspase-1 as above (left). Data (n = 3) were analyzed as in Figure 3. Bar graphs (right) represent Vmax app (mean ± SD; n = 3). Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 MDP and ATP/Mg2+ Induce NALP1 Oligomerization via a Two-Step Process (A) Purified NALP1 monomers obtained from gel filtration (∼150 kDa fraction) were incubated with or without Mg2+ (1 mM); ATP, ADP, or AMP-PNP (1 mM); or MDP-LD or MDP-DD (20 μg/ml) for 30 min at 37°C. Proteins were separated by a first native-PAGE dimension, then by a second denaturating SDS-PAGE dimension, and analyzed by immunoblotting using anti-GST antibody. Molecular weight markers are indicated. (B) MDP is required for ATP binding to NALP1. (Left) NALP1 was incubated with various FL-conjugated ATP analogs (10 nM) and Mg2+ (0.5 mM) for 5 min in ice in the presence of MDP-LD (20 μg/ml). ATP binding was analyzed by FPA (mean ± SD; n = 3 millipolars [mP]). (Middle) GST-NALP1, GST, or procaspase-1 was incubated with FL-ATP (10 nM) and Mg2+ (0.5 mM) with or without MDP-LD or MDP-DD (20 μg/ml). (Right) LRRs repress ATP binding to NALP1. NALP1ΔLRR or GST was incubated with FL-ATP (10 nM) and Mg2+ (0.5 mM) with or without MDP-LD (20 μg/ml). Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 EM Analysis of NALP1 (A) Field of view of negatively stained NALP1 after incubation with ATP (1 mM), MDP (20 μg/ml), Mg2+ (1 mM), and DTT (1 mM). Protein densities are white on a dark background. For display purposes, the oligomers in this image have been boxed and a few small particles (out of the ∼70 in this image) have been circled. (B) Images of two oligomers. (C) Average of 424 oligomers showing five discernible density units. (D) Average of 266 oligomers showing seven discernible density units. (E) Image of eight small particles. (F) Eight representative reference-free averages of the small particles (see Experimental Procedures). Scale bars are 5 nm. (G) Quantification of the number of oligomers present per micrograph before and after incubation with MDP and ATP (mean ± SD). Molecular Cell 2007 25, 713-724DOI: (10.1016/j.molcel.2007.01.032) Copyright © 2007 Elsevier Inc. Terms and Conditions