果蝇保幼激素受体Met入核及降解调控机制的研究

Methoprene-tolerant (Met), the receptor of juvenile hormone (JH), plays a crucial role in JH signaling pathway. Our previous studies in Drosophila showed that Hsp83 facilitated JH-induced nuclear translocation of Met to modulate JH signaling. Meanwhile, Hsp83 was indispensable for maintaining the stability of Met. However, the molecular mechanism underlying Met nuclear import and stability remains unclear. The nucleoporins and importin β have been reported to play important roles in regulating proteins nuclear import，and the stability of proteins are mainly mediated by the ubiquitin-proteasome system. In this study, we aim to continuously unravel the detail molecular mechanism of Met nuclear translocation and degradation. Firstly, based on the previous data of HPLC-MS/MS analysis on JHRR-binding proteins, and using the technique of molecular biology, biochemistry and genetics, we will investigate whether Nup358 mediate Met nuclear localization through interacting with Hsp83 and importin β. Secondly, the E3 ubiquitin ligase(s) which is (are) involved in the Hsp83 regulated Met degradation will be screened and identified by RNAi mediated knockdown experiments and the detail mechanism will be elucidated. Finally, the relationship between Met nuclear localization and degradation will be clarified based on the above research. Our studies will provide a new insight into JH signalling and pest control.

Met 作为保幼激素（juvenile hormone, JH）受体在JH信号传递中行使重要的功能。我们前期研究发现：果蝇中JH和Hsp83通过促进Met入核而使其发挥活性，并且Hsp83对维持Met蛋白稳定性也是必需的。但Met入核及稳定性调控的分子机制尚不明晰。有报道称核孔蛋白和转运载体β在蛋白入核过程发挥重要的作用，而蛋白的稳定性主要受泛素-蛋白酶体系统的调控。本课题拟深入阐释果蝇Met入核及降解的调控机制。一方面，根据已获得的JHRR亲和蛋白质谱鉴定结果，运用分子生物学、生物化学以及果蝇遗传学等方法和手段确认Nup358与Hsp83、转运载体β相互作用参与Met入核调控；另一方面，利用RNAi技术筛选参与Hsp83调控Met降解的 E3泛素连接酶，并阐明具体分子机制。综合以上结果进一步解析Met入核与降解的关系。本项目将为调控JH信号通路、寻找新的害虫分子靶标提供思路及理论依据。

Met作为保幼激素（juvenile hormone, JH）的受体在JH信号传递中发挥重要的作用。本项目的研究发现核孔蛋白Nup358参与JH对Met的入核调控，并且阐明了Nup358通过影响转运载体importin ꞵ的表达量以及通过其N端的TPR结构域与Hsp83结合从而调控Met入核的机制。同时，我们还对Hsp83抑制剂（geldanamycin，GA）降解Met的分子机制进行了研究，证明GA是通过泛素蛋白酶体系统诱导Met降解。对Met泛素化位点进行了预测及功能验证，发现在线预测获得的位点单突变不足以介导GA诱导的Met降解。此外，阐明了JH初级反应基因Kr-h1启动子区域中的JHRR与核蛋白的结合依赖于JH、Met以及Hsp83。发现并证明了miR-927可通过靶向结合于Kr-h1而调控果蝇幼虫的发育。我们的研究结果进一步深化了JH信号调控网络，为农林业上害虫防治提供了新思路及理论依据。