Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

Autor: Kiyoshi Migita, Toshio Heike, Hiroyuki Takeda, Tatsuya Sawasaki, Tomoyuki Iwasaki, Kazunaga Agematsu, Mie Kurata, Sho Mokuda, Junya Masumoto, Yuki Ito, Atsushi Kawakami, Naoe Kaneko, Shinnosuke Morikawa
Rok vydání: 2016
Předmět:
Zdroj: The Scientific World Journal, Vol 2016 (2016)
The Scientific World Journal
ISSN: 1537-744X
2356-6140
DOI: 10.1155/2016/2597376
Popis: 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.
Databáze: OpenAIRE
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