title: Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.
abstract: 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-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory 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 amplified 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. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly 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. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Iwasaki
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Naoe
authors.initials: N
authors.lastname: Kaneko
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Division of Cell-Free Sciences, Ehime University Proteo-Science Center, Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.affiliation: Division of Cell-Free Sciences, Ehime University Proteo-Science Center, Bunkyocho 3, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.affiliation: Department of Pediatrics, Kyoto University Graduate School of Medicine, Shogoin Kawaramachi 54, Kyoto 606-8507, Japan.
authors.forename: Toshio
authors.initials: T
authors.lastname: Heike
authors.affiliation: Clinical Research Center, Nagasaki Medical Center, Kubara 2-1001-1, Omura, Nagasaki 856-8562, Japan.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Migita
authors.affiliation: Department of Infection and Host Defense, Shinshu University Graduate School of Medicine, Asahi 3-1-1, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Kazunaga
authors.initials: K
authors.lastname: Agematsu
authors.affiliation: 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.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kawakami
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Shinnosuke
authors.initials: S
authors.lastname: Morikawa
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Mokuda
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Kurata
authors.affiliation: Department of Pathology, Ehime University Proteo-Science Center and Graduate School of Medicine, Shitsukawa 454, Toon, Ehime 791-0295, Japan.
authors.forename: Junya
authors.identifier: 0000-0003-0847-0456
authors.initials: J
authors.lastname: Masumoto
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000378:metabolism; D001168:Arthritis / Q000378:metabolism* / Q000473:pathology; D002474:Cell-Free System; D055808:Drug Discovery; D006801:Humans; D053473:Nod2 Signaling Adaptor Protein / Q000378:metabolism*; D012507:Sarcoidosis / Q000378:metabolism* / Q000473:pathology; D013585:Synovitis / Q000378:metabolism* / Q000473:pathology; D014605:Uveitis / Q000378:metabolism* / Q000473:pathology; D056660:Hereditary Autoinflammatory Diseases
chemical_list: C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D000119:Acetylmuramyl-Alanyl-Isoglutamine