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J Immunol Methods. 2015 Nov;426:76-81. doi: 10.1016/j.jim.2015.08.004. Epub 2015 Aug 8.

Reconstituted AIM2 inflammasome in cell-free system.

Author information

1
Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
2
Divison of Cell-free Sciences, Ehime University Proteo-Science Center, Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan.
3
Clinical Research Center, Nagasaki Medical Center, Kubara 2-1001-1, Omura, Nagasaki 856-8562, Japan.
4
Department of Infectious Immunology, Shinshu University Graduate School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan.
5
Unit of Translational Medicine, Department of Immunology and Rheumatology, Nagasaki University Graduate School of Biomedical Sciences, Medicine, Sakamoto 1-7-1, Nagasaki 852-8501, Japan.
6
Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan. Electronic address: masumoto@m.ehime-u.ac.jp.

Abstract

Absent in melanoma 2 (AIM2) is an intracellular pattern-recognition receptor, which is a member of the PYHIN protein family, consisting of a PYD domain and an IFN-inducible nuclear localization (HIN) domain. AIM2 is reported to oligomerize with adaptor protein ASC upon sensing bacterial and viral cytosolic DNA in order to form the AIM2 inflammasome, which activates caspase-1 leading to IL-1β secretion. Dysregulation of AIM2 inflammasome is supposed to result in autoinflammatory and autoimmune diseases. Thus, the development of new targeted drugs against AIM2 inflammasome would be important for the treatment of these diseases. However, since AIM2 inflammasome is an intracellular receptor, enforced internalization of both ligands and candidate molecules is necessary for the screening of AIM2-inflammasome-targeted molecules. We developed a reconstituted AIM2 inflammasome in a cell-free system with amplified luminescent proximity homogeneous assay (Alpha). Strong Alpha signal was detected upon incubation with poly-deoxyadenylic-deoxythymidylic acid, poly(dA:dT), whereas no Alpha signal was detected upon incubation with muramyl dipeptide, one of the NLR ligands of Nod2 ligand. The interaction between AIM2 and ASC was disrupted by an anti-human ASC monoclonal antibody, CRID3, a class of diarylsulfonylurea-containing compounds, and glycyrrhizin, a substance found in liquorice root. Thus, the reconstituted AIM2 inflammasome in a cell-free system is useful for screening AIM2-inflammasome-targeted therapeutic molecules.

KEYWORDS:

AIM2; ASC; Cell-free; Inflammasome; Reconstitution

PMID:
26259507
DOI:
10.1016/j.jim.2015.08.004
[Indexed for MEDLINE]

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