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目的 :发现调控Sonic Hedgehog(Shh)信号转导的泛素连接酶。方法 :第一轮筛选,利用荧光素酶报告基因(8×GliBS-Luc)检测系统,检测相应HECT家族E3泛素连接酶的siRNA对Shh信号通路活性的影响;第二轮筛选,利用细胞免疫荧光激光共聚焦检测系统,检测上述siRNA对Shh的受体Ptch1蛋白原纤毛定位的影响。综合2轮筛选结果,初步发现影响Shh信号通路活性的HECT家族E3泛素连接酶。结果:通过2轮筛选,发现Smurf1、Smurf2、Ube3c、Wwp1、Wwp2共5个泛素连接酶,不仅能调节Ptch1蛋白的原纤毛定位,也可以增加通路下游转录因子Gli的活性。结论:建立了筛选调控Shh信号通路的泛素连接酶的平台。在初步筛选的27个HECT E3泛素连接酶中,有5个成员参与调控Shh信号通路。
OBJECTIVE: To discover ubiquitin ligases that regulate Sonic Hedgehog (Shh) signaling. Methods: In the first round of screening, the effect of siRNA of corresponding HECT family E3 ubiquitin ligase on Shh signaling pathway was examined by a luciferase reporter assay (8 × GliBS-Luc). In the second round of screening, Fluorescence confocal laser scanning system was used to detect the influence of the siRNA on the localization of Shch receptor Ptch1 protein. Based on the results of two rounds of screening, the HECT family E3 ubiquitin ligase, which affects the activity of Shh signaling pathway, was initially found. Results: Two ubiquitin ligases of Smurf1, Smurf2, Ube3c, Wwp1 and Wwp2 were found by two rounds of screening, which not only regulated the fibril localization of Ptch1 protein but also increased the activity of the downstream transcription factor Gli. Conclusion: A platform for screening ubiquitin ligases for regulating Shh signaling pathway was established. Of the 27 HECT E3 ubiquitin ligases initially screened, 5 are involved in the regulation of the Shh signaling pathway.