Journal Article Nod2-nodosome in a cell-free system: Implications in pathogenesis and drug discovery for blau syndrome and early-onset sarcoidosis

Iwasaki, Tomoyuki  ,  Kaneko, Naoe  ,  Ito, Yuki  ,  Takeda, Hiroyuki  ,  Sawasaki, Tatsuya  ,  Heike, Toshio  ,  Migita, Kiyoshi  ,  Agematsu, Kazunaga  ,  Kawakami, Atsushi  ,  Morikawa, Shinnosuke  ,  Mokuda, Sho  ,  Kurata, Mie  ,  Masumoto, Junya

20162016 , Hindawi Publishing Corporation
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 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.

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