Hedgehog信号通路在胚胎发育、组织再生中发挥重要的作用,且与癌症发生发展密切相关.其胞内调控组分SuppressorofFused(SuFu)蛋白通过结合转录因子Gli(S),负调控该信号通路,但其作用的分子机制仍不甚清楚.在本项研究中,以人SuFu作为诱饵蛋白,利用酵母双杂交技术成功地筛选到1个新的相互作用因子一蛋白酶体成熟蛋白(POMP).通过免疫共沉淀、体外GSTpull-down和免疫细胞化学实验验证其相互作用.为了探究POMP与SuFu的相互作用对Hedgehog信号通路的影响,构建了POMP的过表达质粒和干扰质粒(miR.RNAi)以及转录因子Gli活性检测系统,即荧光素酶报告基因法,结果显示,过表达SuFu蛋白时POMP正调控Hedgehog信号通路,而下调POMP的表达则抑制Gli的活性.该研究结果揭示了POMP新的生物学功能,为阐明Hedgehog信号通路的具体分子机制提供了新的线索.
Hedgehog signaling pathway plays an important role in embryogenesis and tissue regeneration, which is also related closely to carcinogenesis and tumor formation. Suppressor of Fused (SuFu) , the intracellular component of Hedgehog signaling, negatively regulates the pathway via binding to transcription factors Gli (s), but the specific molecular mechanism still remains unclear. Herein, proteasome maturation protein (POMP) human SuFu using yeast two hybrid was screened out successfully as a new interacting protein of system. Their interaction was further confirmed by co- immunoprecipitation and GST pull-down, and the co-localization was also observed under confocal microscope. To explore the influence of their interaction on Hedgehog signaling, POMP overexpression plasmid, POMP RNAi plasmids and Gli transcription activity detection system (dual luciferase reporter assay) were constructed. Co-transfected with SuFu, exogenous POMP can positively regulate Hedgehog signaling, while knockdown of POMP restrained Gli activity. Our studies will provide new clues for elucidating the specific molecular mechanisms of Hedgehog signaling and reveal a new biology function of POMP.