Research Article Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis Tomoyuki Iwasaki, 1 Naoe Kaneko, 1 Yuki Ito, 1 Hiroyuki Takeda, 2 Tatsuya Sawasaki, 2 Toshio Heike, 3 Kiyoshi Migita, 4 Kazunaga Agematsu, 5 Atsushi Kawakami, 6 Shinnosuke Morikawa, 1 Sho Mokuda, 1 Mie Kurata, 1 and Junya Masumoto 1 1 Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan 2 Division of Cell-Free Sciences, Ehime University Proteo-Science Center, Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan 3 Department of Pediatrics, Kyoto University Graduate School of Medicine, Shogoin Kawaramachi 54, Kyoto 606-8507, Japan 4 Clinical Research Center, Nagasaki Medical Center, Kubara 2-1001-1, Omura, Nagasaki 856-8562, Japan 5 Department of Infection and Host Defense, Shinshu University Graduate School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan 6 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 Correspondence should be addressed to Junya Masumoto; masumoto@m.ehime-u.ac.jp Received 11 March 2016; Accepted 23 May 2016 Academic Editor: Rolando Cimaz Copyright © 2016 Tomoyuki Iwasaki et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-B-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinfammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplifed luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. Te ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more signifcantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Tus, we successfully developed Nod2-nodosome in a cell-free system refecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous infammatory diseases. 1. Introduction Nucleotide-binding oligomerization domain-containing pro- tein (Nod) 2 is a nuclear factor- (NF-) B-activating intracel- lular pattern recognition receptor, which was identifed as a susceptibility gene product of Crohn’s disease, an infamma- tory bowel disease [1–3]. Nod2 was reported to be oligomerized with adaptor protein RICK (RIP2/RIPK2) and IKK complexes, which can activate NF-B by muramyl dipeptide (N-Acetylmuramyl- L-Alanyl-D-Isoglutamine: MDP), one of the components of bacterial cell-wall peptidoglycan, and is utilized as an immune-stimulatory adjuvant for vaccination and for devel- oping antibodies [4–8]. Hindawi Publishing Corporation e Scientific World Journal Volume 2016, Article ID 2597376, 7 pages http://dx.doi.org/10.1155/2016/2597376