Toll样受体7胞内区功能关键位点筛选及其机制研究(英文)

A critical amino acid in Toll-like receptor 7 TIR domain impacting its binding with MyD88

目的筛选出TLR7 TIR区对其信号传递起关键作用的位点,并解释该位点的作用机制。方法针对TLR7野生型序列,设计构建一系列TLR7缺失突变与点突变质粒。将这些质粒转染入HEK293T细胞,利用双荧光素酶报告基因检测这些质粒对NF-κB信号通路的影响。利用免疫共沉淀与蛋白质印迹实验验证TLR7突变体与其下游接头蛋白MyD88结合能力的变化。结果通过对TLR7胞内区截短突变分析,发现在其TIR区存在一个由16个氨基酸残基组成的区域对其信号传递至关重要。进一步的研究发现,该区域存在一个RXR信号基序。该基序位于1004位与1006位的两个保守精氨酸残基,为TLR7正常信号传递所必需。通过免疫共沉淀与蛋白质印迹实验,可以发现TLR7位于1004位的精氨酸对其与下游蛋白MyD88的结合至关重要。结论成功鉴定出TLR7 TIR区1004位的精氨酸对其信号传递起重要作用,该位点是通过影响TLR7与MyD88结合的方式发挥功能。

Objective To study the mechanism of Toll-like receptor 7（TLR7） signal transduction by identifying the critical amino acid in TLR7 Toll/Interleukin-1 receptor（TIR） domain.Methods We carried out a series of truncating mutagenesis and site-direct mutagenesis to obtain TLR7 mutants.These TLR7 mutants were transfected into HEK293T cells.NF-κB signal pathway activation was determined by the reporter gene assay.Immunoprecipitation and immunoblotting were employed to determine the binding between TLR7 and MyD88.Results We identified a RXR signal motif with two arginines at position 1004 and 1006,which plays a significant role in TLR7 signal transduction.We further demonstrated that only the arginine at position 1004 could affect the binding between TLR7 and MyD88.Conclusion We conclude that the arginine at position 1004 of human TLR7 is critical for its binding with MyD88 in signal transduction.