Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.
Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis.
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DOI：
10.1155/2016/2597376
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发表时间：
2016
影响因子：
--
通讯作者：
Masumoto J
中科院分区：
文献类型：
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作者：
Iwasaki T;Kaneko N;Ito Y;Takeda H;Sawasaki T;Heike T;Migita K;Agematsu K;Kawakami A;Morikawa S;Mokuda S;Kurata M;Masumoto J
含有核苷酸结合寡聚化结构域的蛋白 (Nod) 2 是一种细胞内模式识别受体，可识别细菌肽聚糖成分胞壁酰二肽 (N-乙酰胞壁酰-L-丙氨酰-D-异谷氨酰胺：MDP)，并与接头蛋白 RICK (RIP2/RIPK2) 形成称为结节体的 NF-κB 激活复合物。 Nod2 突变体与自身炎症性疾病、布劳综合征 (BS)/早发性结节病 (EOS) 相关。对于 BS/EOS 的药物发现，我们尝试在无细胞系统中开发 Nod2-nodosome。合成了 FLAG 标记的 RICK、生物素化的 Nod2 和 BS/EOS 相关的 Nod2 突变体，并通过放大发光邻近均质测定 (ALPHA) 检测 FLAG 标记的蛋白质和生物素化的蛋白质之间的邻近信号。与 MDP 一起孵育后，Nod2-WT 和 RICK 之间相互作用的 ALPHA 信号以剂量依赖性方式增加。 RICK 和 BS/EOS 相关 Nod2 突变体之间相互作用的 ALPHA 信号比 Nod2-WT 更显着增加。值得注意的是，Nod2-WT 和 RICK 之间的 ALPHA 信号在与 MDP 一起孵育时增加，但与相同浓度的 L-丙氨酸、D-异谷氨酸或 MDP-D-亚型一起孵育时则没有增加。因此，我们在无细胞系统中成功开发了Nod2-nodosome，反映了其体内功能，可用于筛选针对BS/EOS和肉芽肿性炎症性疾病的Nod2-nodosome靶向治疗分子。
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.