以MyD88TIR二聚化为靶点的抗炎抑制剂筛选模型的建立

Establishment of a Screening Model for The Anti-inflammation Inhibitors Acting on The Dimerization of MyD88 TIR

MyD88是IL-1R/TLR受体超家族向细胞内转导胞外信号时募集到受体胞浆尾部的重要接头蛋白.由TIR结构域介导的MyD88分子同源二聚化是它招募到受体胞浆尾部的前提,然后二聚化的MyD88再募集下游信号分子,传递信号,引发促炎基因的表达.本研究旨在建立一种模型,以实现活细胞原位的、基于荧光信号变化的MyD88二聚化抑制物的高通量筛选.我们分别构建了MyD88 TIR与GFP和RFP的融合蛋白表达质粒,瞬时转染HeLa细胞,在488 nm激发光下,转染GFP-MyD88 TIR和RFP-MyD88 TIR细胞,检测到绿色荧光与红色荧光间的共振能量转移(FRET).而当细胞转染GFP-MyD88 TIR和RFP或RFP-MyD88 TIR和GFP,因TIR二聚化不能实现,FRET效率受到严重影响.实验结果提示,依赖双阳性表达GFP-MyD88 TIR和RFP-MyD88 TIR的细胞株,检测不同化合物对于荧光FRET效率的影响,可以建立MyD88 TIR二聚化抑制药物的筛选模型.此外,我们构建了原核表达质粒,利用纯化的His-MyD88 TIR分别与GST或GST-MyD88 TIR蛋白进行体外结合实验,发现GST-MyD88 TIR(而非GST)可以与His-MyD88 TIR相互结合.结果的差异性提示,利用His-MyD88 TIR和GST-MyD88 TIR体外结合实验分析,可以进一步确定抑制物是否直接阻断了TIR的相互作用.结合真核细胞的荧光FRET阻断结果和原核表达的重组蛋白相互作用分析,可确定MyD88 TIR二聚化的抑制物.利用这一模型可以对商品化的小分子库、自行制备的天然产物组分进行广泛的筛选,从中获得有效抑制MyD88二聚化的化合物,参与对MyD88信号通路依赖的慢性炎症、自身免疫性疾病的药物治疗.

MyD88 is an essential adaptor protein that mediates IL-1R/TLR signals.The homogeneous dimerization of MyD88 is required for its recruitment to the membrane receptors and is achieved by the interaction of its C-terminate TIR domain.After binding with the receptor,MyD88 dimer recruits the downstream molecules transmitting the inflammation signals and inducing gene expression.The present study aimed to establish a living-cell-fluorescence-based and high-through-put model for screening the inhibitors against the dimerization of MyD88 TIR.We constructed GFP-MyD88 TIR and RFP-MyD88 TIR plasmids,and transiently transfected them along with GFP or RFP into HeLa cells with different combinations.Under the excitation at 488 nm,the cells transfected with GFP-MyD88 TIR and RFP-MyD88 TIR plasmids showed the energy transfer from GFP to RFP,which is supposed to be mediated by the interaction of MyD88 TIR.Nevertheless,in the cells transfected with GFP-MyD88 TIR and RFP or RFP-MyD88 TIR and GFP plasmids,FRET was obviously impaired due to the absence of MyD88 TIR dimerization.The results suggest a possibility to establish a model for screening the inhibitors against the dimerization of MyD88 TIR： select the cell line that dually expresses GFP-MyD88 TIR and RFP-MyD88 TIR,and monitor the alteration of FRET upon the adding of different compounds in the medium.Additionally,we isolated and purified recombinant proteins His-MyD88 TIR and GST-MyD88 TIR,and performed in vitro protein binding assay.This recombinant protein binding assay can be used to verify the inhibitor candidates selected from the fluorescence screening model directly blocking the interaction of MyD88 TIR or not.The fluorescence screening model plus the in vitro verifying assay can be broadly used with commercially available compounds banks or lab-made natural products to find effective anti-inflammation inhibitors for therapeutic treatment of MyD88-pathway related chronic inflammation or autoimmune disorders.