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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 important adapter protein recruited to the tail of the receptor cytoplasm when the IL-1R / TLR receptor superfamily transduces an extracellular signal into the cell.HerD88 molecule homodimerization mediated by the TIR domain was recruited to Receptor preconditions in the tail of the cytoplasm, and dimerization of MyD88 then recruits downstream signaling molecules to transmit signals that trigger the expression of proinflammatory genes.This study aimed to establish a model that enables living cells to be in situ based on fluorescence signals High-throughput screening of the modified MyD88 dimerization inhibitor.We constructed the fusion protein expression plasmid of MyD88 TIR, GFP and RFP, transiently transfected into HeLa cells, transfected GFP-MyD88 TIR under 488 nm excitation and RFP-MyD88 TIR cells detected a resonance energy transfer (FRET) between green and red fluorescence, whereas FRET was not achieved due to TIR dimerization when cells were transfected with GFP-MyD88 TIR and RFP or RFP-MyD88 TIR and GFP The efficiency is seriously affected.Experimental results suggest that, depending on the double-positive expression of GFP-MyD88 TIR and RFP-MyD88 TIR cell lines to detect the effect of different compounds on the fluorescence FRET efficiency, we can establish MyD88 TIR dimerization inhibitor drug screening model.In addition , We constructed a prokaryotic expression Plasmid, GST-MyD88 TIR (but not GST) was found to bind to His-MyD88 TIR using an in vitro binding assay of purified His-MyD88 TIR with GST or GST-MyD88 TIR protein respectively.Results of the variability suggest that using His -MyD88 TIR and GST-MyD88 TIR in vitro, it can be further determined whether the inhibitor directly blocks the interaction of TIR.According to the analysis of the FRET blocking result of eukaryotic cells and the prokaryotic recombinant protein interaction, we can confirm MyD88 TIR dimerization inhibitors.Using this model can be commercial small molecule libraries, self-prepared natural product components for a wide range of screening, obtained from the effective inhibition of dimerization of MyD88 compounds, involved in the MyD88 signaling pathway Dependent on chronic inflammation, autoimmune disease medication.