Introduction

Inflammatory caspases such as caspase-1 and caspase-5 are activated by the formation of large molecular complexes known as inflammasomes. Activation of inflammatory caspases leads to processing of IL-1b and IL-18, which can evoke numerous cytokine cascades [1–3]. Inflammasomes are formed upon recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and participate in the key stage of inflammation and innate immunity [4, 5]. Inflammasomes are one of the targets for the development of new anti-inflammatory drugs for the treatment of chronic inflammatory disease [6, 7]. Inflammasomes are composed of an NOD-like receptor (NLR), an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 [8]. NALP3 inflammasome, which is composed of NALP3, ASC, and caspase-1, is the most studied and well-known inflammasome [9]. Pyrin domain (PYD)-containing NALPs are members of the NLR family that sense multiple danger signals, including bacteria, microbial toxins, PAMPs, and DAMPs, directly or indirectly [10, 11]. ASC is an adaptor molecule containing two protein interaction modules: PYD at the N-terminus and caspase recruitment domain (CARD) at the C-terminus [12]. Caspase-1 has a CARD domain at the N-terminus. During inflammasome formation, the ASC adaptor acts as a bridge between the caspase and NLR by offering the CARD for CARD–CARD interactions and PYD for PYD–PYD interactions [6, 8]. Therefore, PYDs play a critical role in the activation of those caspases by mediating the formation of such complexes. Many intracellular signaling proteins, especially those involved in inflammation, and the innate immune signaling pathway, such as AIM2, POP1, POP2, and pyrin, also contain PYD for homotypic interactions [13]. To date, 22 PYD-encoding genes have been identified in humans, including 14 members of the NALP family [14]. Given the fact that PYD-mediated inflammation and innate immunity are associated with many human diseases, including cancer, immune disorders, and aging, studies in these areas are of great biological importance [15]. Despite the importance of PYD-mediated NALP3 inflammasome formation, structural and biochemical studies have been limited due to insolubility of the PYD domain and protein components of the NALP3 inflammasome [16, 17]. In this study, we successfully purified and characterized NALP3 PYD and ASC PYD. Our current study showed that ASC PYD easily self-oligomerized under physiological conditions, and this self-oligomerization of the ASC PYD prevents complex formation in the NALP3 inflammasome in vitro.

Materials and Methods

Materials

DNA restriction enzymes (NdeI and XhoI), T4 DNA ligase, and Tag DNA polymerase were obtained from New England Biolabs (NEB). Isopropyl-β-d-thiogalactopyranoside (IPTG), urea, imidazole, and DTT were purchased from Sigma (USA). Escherichia coli BL21 (DE3) cells and plasmid pET vectors were obtained from Novagen. A Superdex 200 gel-filtration column was purchased from GE Healthcare. Inhibitor cocktail was acquired from Roche. All concentrators were obtained from Millipore.

Expression of NALP3 PYD and ASC PYD in E. coli

The cDNA of full-length human NALP3 and human ASC were used as templates for PCR, while plasmid vectors pOKD (a homemade vector) [18] and pET28a were used to add a hexa-hisditine tag to the C-terminus and the N- and C-terminus, respectively, for affinity purification. The PYD domain of NALP3 coding for amino acid residues 1–110 was cloned into pOKD, resulting in pET (NALP3 PYD-6His). Additionally, the PYD domain of ASC PYD coding for amino acid residues 1–96 was cloned into pET 28a, resulting in pET (6His-ASC PYD-6His). The expression methods for NALP3 PYD were the same as previously described [9], while those for ASC PYD were similar to those for NALP3 PYD purification. A brief summary of these methods is presented below. Each clone was streaked onto Luria Broth (LB) agar plates containing appropriate antibiotics (100 μl/ml ampicillin for the pOKD clone and 50 μl/ml kanamycin for the pET 28a clone), after which the plates were incubated at 37 °C overnight. A single colony was then used to inoculate 5 ml of LB medium, which was subsequently incubated at 37 °C in a shaking incubator. Next, 5 ml of the overnight small-scale culture was used to inoculate 1 l of LB in a 2-l culture flask, and the culture growth was continued in a shaking incubator for approximately 4 h. Expression was induced by overnight treatment with 0.5 mM IPTG at 20 °C when the optical density (OD) at 600 nm reached 0.6–0.7.

Purification of Target Proteins

Bacteria expressing each protein were pelleted by centrifugation, resuspended, and lysed by sonication in 80 ml of lysis buffer (20 mM Tris–HCl buffer (pH 8.0), 500 mM NaCl, and 5 mM imidazole) supplied with a protease inhibitor cocktail (Roche, USA). The lysate was then centrifuged at 16,000 rpm for 60 min at 4 °C, after which the supernatant fractions were applied to a gravity column (Bio-Rad) packed with Ni-NTA affinity resin (Qiagen). Next, the unbound bacterial proteins were removed by washing with 120 ml of washing buffer (20 mM Tris buffer at pH 8.0, 500 mM NaCl, and 30 mM imidazole), after which the target proteins were eluted from the column using elution buffer (20 mM Tris–HCl buffer (pH 8.0), 500 mM NaCl, and 250 mM imidazole), with 1-ml elution fractions being collected over a total of 10 ml. Fractions containing greater than 80 % homogeneous target protein (as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)) were then selected and combined. In the final purification step, the sample was loaded onto a Superdex 200 gel-filtration column (GE Healthcare, Sweden) equilibrated with various buffers. Fractions containing the target protein were then pooled and stored at 4 °C for further experiments. The homogeneity of the protein was assessed by SDS-PAGE.

Circular Dichroism Spectroscopy

The secondary structures were measured by circular dichroism (CD) spectroscopy using a J-715 spectropolarimeter at Korea Basic Science Institute in South Korea. The spectra were obtained from 200 to 250 nm at 25 °C in a 0.1-cm path-length quartz cuvette using a bandwidth of 1.0 nm, a speed of 50 mm/min, and a 5-s response time. The protein samples in the buffer containing 20 mM Tris–HCl at pH 8.0 and 150 mM NaCl were diluted to 0.1 mg/ml prior to use. Four scans were accumulated and averaged, and the α-helical content was calculated from the molar ellipticity at 222 nm [19].

Complex Assay by Gel-Filtration Chromatography

Purified NALP3 PYD was incubated with ASC PYD for 1 h at 25 °C, after which the mixture was concentrated to 10–15 mg/ml using a concentration kit (Millipore). The concentrated protein mixture was subsequently applied to a Superdex 200 gel-filtration column 10/30 (GE Healthcare) that had been pre-equilibrated with a solution of 20 mM Tris–HCl buffer at pH 8.0 and 150 mM NaCl or 20 mM sodium citrate buffer at pH 5.0 with 150 mM NaCl. Complex formation was evaluated based on the positions of the eluted peaks monitored at 280 nm followed by SDS-PAGE.

Native PAGE Assay

Formation of the complex between NALP3 PYD and ASC PYD was assayed by native (non-denaturing) PAGE conducted on a PhastSystem (GE Healthcare) with pre-made 8–25 % acrylamide gradient gels (GE Healthcare). Coomassie Brilliant Blue was used for staining and detection of the band patterns. Separately purified NALP3 PYD and ASC PYD were pre-incubated for 1 h at room temperature, after which the mixture was applied to the gel. Complex formation was analyzed based on the appearance of newly formed bands and the disappearance of existing bands.

Molecular Surface Generation

Electrostatic surfaces and ribbon diagrams were generated using the PyMOL program (DeDeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos).

Results

Purification and Characterization of NALP3 PYD and ASC PYD

Inflammasomes, which are formed by pathogens, are large molecular complexes that lead to the activation of caspase-1 and participate in the inflammatory response and adaptive immune response. NOD-like receptors (NLRs) including NALP3, ASC, and caspase-1 are the main components of the NALP3 inflammasome. Each protein contains one member of the death domain (DD) superfamily, CARD for ASC and caspase-1, and PYD for ASC and NALP3 to enable homotypic protein–protein interaction (Fig. 1a). ASC functions as an adaptor for inflammasomes containing both CARD at the C-terminal and PYD at the N-terminal. Interaction of ASC PYD and NALP3 PYD is critical to the formation of inflammasome for recruitment of caspase-1 and its activation. The structures of NALP3 PYD and ASC PYD have been reported and found to be composed of a six-helix bundle, which is a common structural fold for the DD superfamily (Fig. 1b). Although information pertaining to the individual structures is available, there is no structural or biochemical information describing the complex formed by NALP3 PYD and ASC PYD. Based on the structure of NALP3 PYD and ASC PYD, the surface characteristics were deduced and both proteins shown to contain several charged pockets that might be important for the interaction between NALP3 PYD and ASC PYD (Fig. 1c).

Fig. 1
figure 1

Schematic representation of inflammasome and sequence analysis of PYDs. a Domain boundary of protein components for NALP3 inflammasome, NALP3, ASC, and caspase-1. Each domain is indicated by different colors. CARD caspase recruitment domain, PYD pyrin domain, Casp caspase domain. PYD–PYD and CARD–CARD interactions in the inflammasome are shown by dotted circles. b Sequence alignment of NALP3 PYD and ASC PYD with other PYD domains. c Molecular surface of NALP3 PYD and ASC PYD. The surface was calculated by PYMOL

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To reconstitute the inflammasome in vitro with purified protein components, we initially attempted to purify NALP3 PYD in E. coli. Although it is well known that obtaining functional DD superfamily members is difficult due to their insolubility, following IPTG induction, bacterial cells (BL21-DE3 strain) containing NALP3 PYD in pOKD and ASC PYD in pET28a were found to express recombinant proteins with molecular weights around 16 and 12 kDa, respectively, that were detected in the soluble fraction after sonication. Both proteins were purified by two-quick-step chromatography using Ni affinity chromatography followed by gel-filtration chromatography. Based on previous studies showing that the solubility of many DD superfamily members is sensitive to pH and salt concentration [20], we analyzed the characteristics of each PYD domain of NALP3 and ASC. Our gel-filtration chromatography study showed that NALP3 PYD was soluble and came out in correct position via gel-filtration column at both pH 8.0 and pH 5.0 (Fig. 2a). Interestingly, the oligomeric states of NALP3 PYD varied with pH. At pH 8.0, NALP3 PYD was eluted at around 18 ml, which indicates that it exists as a monomer. Conversely, NALP3 PYD became a dimer at pH 5.0 (Fig. 2a). Upon analysis of the effect of salt on the solubility of NALP3 PYD, salt concentration did not have a great impact on the behavior of NALP3 PYD in solution; however, it did not behave well when no salt was present (Fig. 2b). In the case of ASC PYD, the solubility of ASC PYD was relatively sensitive to pH. Gel-filtration chromatography showed that ASC PYD was well behaved at low pH (pH 5.0) (Fig. 3a); however, at pH 8.0, ASC PYD was not eluted to the correct position on the gel, indicating that ASC PYD is less soluble at pH 8.0 (Fig. 3a). The effect of salt on the solubility of ASC PYD was similar to the effect on NALP3 PYD (Fig. 3b).

Fig. 2
figure 2

Purification and characterization of NALP3 PYD. a pH effect on the solubility of NALP3 PYD shown by gel-filtration chromatograms and fractions. The black and gray lines reflect the gel-filtration profile obtained at pH 5.0 and pH 8.0, respectively. SDS-PAGEs stained by Coomassie blue from the peak fractions of gel-filtration chromatography are shown on the upper left side. b Effect of salt on the solubility of NALP3 PYD shown by gel-filtration chromatograms and fractions. The black line shows the profile obtained under 0 mM NaCl using Tris–HCl buffer at pH 8.0. The gray line shows the profile obtained under 150 mM NaCl using Tris–HCl buffer at pH 8.0. The black dotted line shows the profile obtained under 500 mM NaCl. SDS-PAGEs of the fractions of gel-filtration chromatography stained with Coomassie blue are shown on the upper left side

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Fig. 3
figure 3

Purification and characterization of ASC PYD. a Effect of pH on the solubility of ASC PYD shown by gel-filtration chromatograms and fractions. The black and gray lines reflect the gel-filtration profile obtained at pH 5.0 and pH 8.0, respectively. SDS-PAGEs of the peak fractions of gel-filtration chromatography stained by Coomassie blue are shown on the upper left side. b Effect of salt on the solubility of ASC PYD shown by gel-filtration chromatograms and fractions. The black line shows the profile obtained under 0 mM NaCl using Tris–HCl buffer at pH 8.0. The gray line shows the profile obtained under 150 mM NaCl using Tris–HCl buffer at pH 8.0. The black dotted line shows the profile obtained under 500 mM NaCl. SDS-PAGEs of the fractions of gel-filtration chromatography stained by Coomassie blue are shown on the upper left side

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Structures of Purified NALP3 PYD and ASC PYD Are Typical α-Helix Bundle Folds

To confirm the correct folding and analyze the secondary structures of NALP3 PYD and ASC PYD, the overall secondary structure and α-helicities were analyzed by measuring the far UV circular dichroic spectra. As shown in Fig. 4, NALP3 PYD showed CD spectrum patterns typical of α-helical proteins, exhibiting two pronounced minima at 208 and 222 nm and a maximum at 195 nm, which matched well with the molecular structure of other members of the death domain superfamily (Jang and Park). Although the data for ASC PYD are not clear, ASC PYD also showed CD spectrum patterns typical of α-helical proteins. The percentage of helix contents predicted and measured agreed well, with 58 % predicted vs. 54 % measured for NALP3 PYD and 61 % predicted vs. 52 % measured for ASC PYD.

Fig. 4
figure 4

Circular dichroic spectra of purified NALP3 PYD (a) and ASC PYD (b). The spectra were recorded at 25 °C, and four scans were conducted and averaged using a J-715 spectropolarimeter (Jasco, Japan). The predicted percentage of helix contents was calculated using the GOR IV secondary structure prediction server (http://npsa-pbil.ibcp.fr/cgi-bin/npsaautomat.pl?page=npsagor4.html)

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NALP3 PYD Did Not Interact with ASC PYD In Vitro

After successful characterization and purification of NALP3 PYD and ASC PYD, we tested whether purified PYDs can interact with each other in vitro. Because it is well known that NALP3 PYD interacts with ASC PYD directly for the assembly of the NALP3 inflammasome, we expected a close interaction in vitro. To accomplish this, purified NALP3 PYD was incubated with ASC PYD for 1 h at room temperature, after which the mixture was applied to a Superdex 200 gel-filtration column 10/30 (GE Healthcare) that had been pre-equilibrated with a proper solution. Since the interaction can be affected by various conditions, we tested the interaction studies under various pH values and salt concentrations. Complex formation was evaluated based on the positions of the eluted peak monitored at 280 nm followed by SDS-PAGE. Because the individual components and the protein mixture were both eluted at around 17–18 ml without any possible new complex peak, we concluded that NALP3 PYD did not interact with ASC PYD in vitro, regardless of how much salt was added or what pH the solution was (Fig. 5a, b). SDS-PAGE showed that NALP3 PYD and ASC PYD did not co-migrate, indicating that they had not formed a complex (Fig. 5a, b).

Fig. 5
figure 5

Complex formation assay by gel-filtration chromatography. Separately purified NALP3 PYD (gray line) and ASC PYD (black line) were mixed together, incubated at room temperature for 1 h, and then loaded onto a gel-filtration column that had been pre-equilibrated with buffer containing 20 mM sodium citrate and 150 mM NaCl (a) or 20 mM Tris–HCl (pH 8.0) and 150 mM NaCl (b). SDS-PAGEs of gel-filtration fractions obtained from the mixture (red-dot line) are shown on the left side of the peak

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Self-oligomerization of ASC PYD Domain Prevents Complex Formation in the NALP3 Inflammasome In Vitro

To confirm the complex formation between NALP3 PYD and ASC PYD, we conducted native (non-denaturing) PAGE with purified protein samples. Purified NALP3 PYD and ASC PYD were loaded onto SDS-PAGE before performing native PAGE to be used as a control (Fig. 6a). After mixing and incubating the two PYDs, they were applied to the native gel (8–25 % gradient gel from GE Healthcare). The complex formation was then evaluated based on the appearance of newly formed bands and the disappearance of existing bands. On native PAGE, NALP3 PYD was shown as a single band at the bottom of the gel. However, ASC PYD was shown as a smeared single band at the top of the gel. The locations of each band indicate that NALP3 PYD exists as a monomer or dimer in solution but that ASC PYD forms a higher oligomeric self-complex in solution (Fig. 6b). Based on the fact that no remarkable changes in band pattern were observed in the mixed sample, NALP3 PYD cannot form a stable complex with ASC PYD (Fig. 6b). These two different PAGE experiments showed that NALP3 PYD and ASC PYD did not form a stable complex in vitro and that ASC PYD easily generated a highly oligomeric form. Furthermore, the results indicate that this self-oligomerization prevents complex formation between NALP3 PYD and ASC PYD.

Fig. 6
figure 6

Self-oligomerization of ASC PYD detected by native PAGE assay. a SDS-PAGE of purified NALP3 PYD and ASC PYD. Lane 1 marker, lane 2 purified ASC PYD, lane 3 purified NALP3 PYD. b Native PAGE analysis of the specific interactions between NALP3 PYD and ASC PYD. Lane 1 NALP3 PYD, lane 2 ASC PYD, lane 3 NALP3 PYD + ASC PYD. Arrows indicate the location of each protein band on native PAGE

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Discussion

NALP3 inflammasome, which is composed of NALP3, ASC, and caspase-1, is a caspase-activating molecular complex that plays a pivotal role in inflammation and innate immunity. NALP3 contains the PYD domain at the N-terminus. PYD of NALP3 is a critical protein–protein interaction module that oligomerizes and recruits PYD-containing adaptor proteins ASC (apoptosis-associated speck-like protein containing CARD), leading to the activation of inflammatory caspases such as caspase-1. During inflammasome formation, the ASC adaptor acts as a bridge between caspase and NALP by offering the CARD for CARD–CARD interactions and PYD for PYD–PYD interactions. Therefore, NALP3 PYD and ASC PYD play a critical role in the formation of inflammasome.

As a first step in elucidation of the molecular basis for the assembly of inflammasome via PYD domains, we attempted to express and purify NALP3 PYD and ASC PYD and to reconstitute the complex in vitro for further structural studies. Because it is well known that obtaining functional PYD domains is difficult due to their insolubility under physiological conditions, we generated various plasmid constructs to obtain plasmids that could be over-expressed in bacterial cells. Based on this approach, we realized that the PYD domain of NALP3 coding for amino acid residues 1–110 and the PYD domain of ASC coding for amino acid residues 1–96 were the only constructs that were over-expressed and solubilized in bacterial cells. Purified NALP3 PYD and ASC PYD, which were used for our current study, contained an α-helix that matched well with the molecular structure of other members of the death domain superfamily.

Since the solubility of many DD superfamily members is sensitive to pH and salt concentration, we analyzed the characteristics of each PYD domain of NALP3 and ASC to identify the optimal conditions for purification and reconstitution in vitro. We found that NALP3 PYD was soluble and came out in correct position via gel-filtration column at both pH 8.0 and pH 5.0. In addition, we showed that the oligomeric states of NALP3 PYD changed with pH. Specifically, at pH 8.0, NALP3 PYD exists as a monomer, while it became a dimer at pH 5.0. Because the dimeric crystal structure of NALP3 PYD has been reported, it is not surprising that changes in stoichiometry were detected. In the case of ASC PYD, the solubility of ASC PYD was relatively sensitive to pH. ASC PYD was soluble and came out in correct position on the profile at pH 5.0, but not pH 8.0. Upon analysis of the effect of salt on the solubility of NALP3 PYD and ASC PYD, salt concentration did not have a great effect on the behavior of either protein in solution. Since it has been reported that NALP3 PYD can interact directly with ASC PYD for the assembly of the NALP3 inflammasome in the cell, we tested whether purified NALP3 PYD could interact with purified ASC PYD in vitro. We found that separately purified PYDs did not form a stable complex. Based on the native PAGE study, we concluded that NALP3 PYD and ASC PYD did not interact with each other because of self-oligomerization of ASC PYD. PYD domain-mediated self-oligomerization of ASC might be regulating process for the assembly of inflammasome.

Although several hetero-oligomeric structures of the DD superfamily have been elucidated, including the RAIDD DD–PIDD DD complex [21], Fas DD–FADD DD complex [22], IRAK2 DD–IRAK4 DD–MyD88 DD complex [23], and procaspase-9 CARD–Apaf-1 CARD complex [24], structural information pertaining to the PYD–PYD complex is still not available [25]. The human PYD-containing proteins consist of approximately 22 members, many of which have been studied intensively due to their important role in the inflammation and innate immunity signaling pathways [14]. Given the paucity of structural information regarding PYD complexes, it is still not known if the three types of interactions detected in other DD superfamily members will be observed in PYD complexes and what stoichiometry will be formed for the assembly of inflammasome. More biochemical and structural studies are required to address these questions and fully understand the molecular basis of PYD-mediated interactions in the assembly of inflammasome.