- Open Access
Applications of reconstituted inflammasomes in a cell-free system to drug discovery and elucidation of the pathogenesis of autoinflammatory diseases
© The Author(s) 2017
- Received: 31 January 2017
- Accepted: 6 April 2017
- Published: 3 May 2017
The inflammasome, typically consisting of a Nod-like receptor, apoptosis-associated speck-like protein, and pro-caspase-1, has recently been identified as a huge intracellular complex, which plays a crucial role in interleukin-1 maturation or specific physiological functions. Two Nod-like receptors, such as nucleotide-binding oligomerization domains-containing protein (Nod)1 and Nod2, interact with the receptor-interacting protein serine-threonine kinase (RIPK)2 accompanied by Iκ-B kinase (IKK) complexes to construct the nodosome, leading to nuclear factor (NF)-κB activation. The aberrant activation of inflammasomes or nodosomes causes autoinflammatory diseases. Therefore, inflammasomes may be attractive targets to treat autoinflammatory diseases. Our aim is to develop reconstituted inflammasomes in a cell-free system to discover specific molecular-target drugs and elucidate the molecular pathogenesis of autoinflammatory diseases. In this review, we describe reconstituted inflammasomes in a cell-free system.
Inflammasomes have recently been identified as expanding intracellular complexes that play important roles not only in innate immunity but also in maintaining specific physiological functions [1–3]. The aberrant activation of inflammasomes is thought to be linked to various diseases, including inflammatory diseases, degenerative diseases, and tumors [4, 5]. Therefore, inflammasomes may be attractive targets to treat these diseases. Autoinflammatory diseases are known to be caused by genetic mutations of inflammasome components [6–9]. Thus, we aim to develop reconstituted inflammasomes in a cell-free system in order to identify specific molecular-target drugs and elucidate the molecular pathogenesis of autoinflammatory diseases. In this review, we briefly describe the functions of several inflammasomes and related diseases, and reconstituted inflammasomes in a cell-free system.
To construct reconstituted inflammasomes in a cell-free system, we employed the wheat germ cell-free protein synthesis system rather than Escherichia coli expression system . When we identified ASC a central adaptor protein of inflammasomes, ASC was discovered in the Triton X-100-insoluble fraction of promyelocytic leukemia cell line HL-60 cells , and it was difficult to synthesize recombinant NLRP3 protein using E. coli expression due to its solubility. On the other hand, the wheat germ cell-free protein synthesis has numerous advantages, such as low cost, ease of availability in large amounts, low endogenous incorporation, and the capacity to synthesize high-molecular-weight proteins . In addition, it is suitable for the expression of eukaryotic proteins because it is eukaryotic system .
First, we describe an AIM2 inflammasome in a cell-free system as a prototype  because the AIM2 inflammasome has been well-characterized, and its ligand was reported to be present in poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT). The direct interaction between AIM2 and poly(dA:dT) was elucidated using the amplified luminescent proximity homogeneous assay (Alpha) . In addition, activation of the AIM2 inflammasome has been reported to be related to various diseases [57–62], and it is thought to be an attractive drug target for diseases.
Our reconstituted AIM2 inflammasome basically consists of AIM2 and ASC, and it is considered sufficient for drug and ligand discovery as it assembles without pro-caspase-1 or any other components .
To synthesize the AIM2 inflammasome, PCR products for AIM2 and ASC were inserted into a Gateway™ pDONRTM221 Vector (pDONR221) (Life Technologies, Carlsbad, CA, USA) using Gateway™ BP Clonase™ II Enzyme mix (Life Technologies, Carlsbad, CA, USA) to generate entry clones. The AIM2 entry clone pDONR221-AIM2 was inserted into pEU-E01-GW-bls-STOP for cell-free protein expression. The ASC entry clones pDONR221-ASC and pDONR221-ASC-PYD were inserted into pEU-E01-FLAG-GW-STOP using the Gateway™ LR Clonase™ II Enzyme mix (Life Technologies, Carlsbad, CA, USA). The constructed plasmids were used to synthesize specific proteins with the WEPRO1240 Expression Kit (Cell-Free, Inc., Matsuyama, Japan) .
The AIM2 inflammasome in a cell-free system assembles with its previously reported ligand poly(dA:dT), and the interaction between AIM2 and ASC was disrupted by anti-human ASC mAb, and previously reported inhibitors CRID3 and glycyrrhizin. Thus, our reconstituted AIM2 inflammasome in a cell-free system is useful for investigating novel ligands and drug discovery .
When AIM2 is replaced by NLRP3, we can easily develop the NLRP3 inflammasome in a cell-free system. There are so many mutations in NLRP3 that causes of autoinflammatory diseases including CAPS, and NLRP3 involve various inflammasomopathies. Thus, the reconstituted NLRP3 inflammasome in a cell-free system will be a useful tool for investigating inflammasomopathies and drug discovery. In this context, we are going to develop reconstituted NLRP3 inflammasome in a cell-free system.
The autoinflammatory disease Blau syndrome (BS)/early-onset sarcoidosis (EOS) is caused by a point mutation of Nod2 . Therefore, the Nod2 nodosome may be an attractive drug target for the treatment of BS/EOS. We aimed to develop a reconstituted protein–protein interaction assay system between wild-type Nod2 and the BS/EOS-associated mutants of Nod2 and RIPK2 in a cell-free system, called the reconstituted Nod2 nodosome in a cell-free system .
The plasmids vector pDONR221-Nod2 and BS/EOS-associated mutants, pDONR221-Nod2-R334W and pDONR221-Nod2-N670K, were constructed. pDONR221-RIPK2 and pDONR221-RIPK2-CARD were also constructed. Then, the proteins Nod2-WT-Btn, Nod2-R334W-Btn, Nod2-N670K-Btn, FLAG-RIPK2 and FLAG-RIPK2-CARD were synthesized using the wheat germ cell-free system in the same way as AIM2.
In our Nod2 nodosome, proximity between Nod2 and RIPK2 is basically detected by Alpha using the combination of protein-A-conjugated Alpha acceptor beads for FLAG-tagged proteins and streptavidin-conjugated Alpha donor beads for biotinylated proteins. The Nod2 nodosome in a cell-free system assembles with its previously reported ligand MDP. The Nod2 nodosomes with BS/EOS-associated mutations Nod2-R334W and Nod2-N670K were more sensitive to MDP than Nod2-WT. Therefore, we think that our Nod2 nodosome in a cell-free system can be a useful tool for investigating the pathogenesis of BS/EOS and drug discovery .
Various inflammasomes are thought to play important roles in the maintenance of the homeostasis of cells, tissues, and organs. Excess inflammasome signaling caused by genetic mutations or pathogens may contribute to known or unknown autoinflammatory diseases. Thus, inflammasomes are expected to become attractive targets to treat autoinflammatory diseases. Although our cell-free system is limited in that only an initial event of assembly between ASC or RIPK2 and an upstream protein is detected, reconstituted inflammasomes in a cell-free system will be useful tools for investigating the pathogenesis of autoinflammatory diseases and discovery of their therapeutics.
This work was supported by the Platform for Drug Discovery, Informatics and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan (H.T., T.S., and J.M.), the Center for Clinical and Translational Research of Kyushu University (J.M.), and a Grant-in-Aid for translational research toward the clarification of autoinflammatory mechanisms by familial Mediterranean fever (FMF). Inflammasome based on the Mediterranean fever (MEFV) gene analysis 26310301 from The Ministry of Health, Labour and Welfare, Japan (J.M.), and Grants-in-Aid for Scientific Research (JSPS KAKENHI grant numbers 26293232 26305024 and 17H04656) from The Ministry of Education, Culture, Sports, Science and Technology, Japan (J.M.).
Availability of data and materials
The manuscript was written by all authors. Briefly, NK described about AIM2, TI described about Nod2, YI described about NLRP3. HT and TS synthesized proteins and described about wheat germ cell-free protein synthesis. SM, NN, MK drew figures. NK and JM described about inflammasome-related diseases and organized them. All authors read and approved the final manuscript.
All authors declare that they have no competing interests.
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