N-乙酰葡萄糖胺修饰调节蜕皮激素信号转导途径的分子机制

Ecdysone (20E) plays the main function in the development of insect. There is a non-genomic pathway in addition to the classical nuclear receptor pathway in 20E signaling, which induces rapid post-translational modification to regulate gene transcription. Most of the studies focus on phosphorylation, while the roles of glycosylation have not been reported. O-GlcNAc (O-linkedN-acetylglucosamine) modification is a special glycosylation, which is dynamic and reversible. Many transcription factors and kinases have been shown to be modified by O-GlcNAc, which can influence their modulation on transcription. Here, we intend to explore the molecular mechanism of the regulation of 20E signaling pathway by O-GlcNAc. Firstly, RNA interference and inhibitor of enzyme respond to the cycling of O-GlcNAc will be used to verify whether O-GlcNAc was involved in the 20E signaling pathway. Immunoblotting with antibody anti-O-GlcNAc will be used to screen the target protein. Then, the regulation of O-GlcNAc on the transcription modulation activity of target protein will be analyzed via co-immunoprecipitation , luciferase reporter system and gel mobility shift assay. Finally, mutant the O-GlcNAc modification sites to verify the interaction among the post-translational modification. These studies reveal the mechanism by which O-GlcNAc modification regulates 20E signaling, and then provide new target for pest control.

蜕皮激素20E是调节昆虫发育的主要激素，其作用机制除经典的核受体介导的基因组途径外，还包括膜介导的非基因组途径，主要是通过快速诱导转录激活相关因子的翻译后修饰来调节基因转录。但研究主要集中在磷酸化修饰，糖基化修饰的作用未见报道。N-乙酰葡萄糖胺修饰(O-GlcNAc)是一种可逆的快速的单糖基化修饰，通过调节包括转录因子、激酶在内的多种核质蛋白的功能来参与细胞活动。本项目拟以鳞翅目昆虫棉铃虫为模型，探索O-GlcNAc在20E信号转导途径中的作用机制。首先，确定O-GlcNAc修饰是否参与20E信号途径，并用抗体筛选靶标分子；然后分析O-GlcNAc对靶标分子转录调节能力的影响；最后分析O-GlcNAc与磷酸化、乙酰化的相互作用，以阐明O-GlcNAc调节20E信号转导途径的分子机制。本研究不仅可进一步阐明20E信号转导途径，还可为害虫生物防治提供靶标。

蜕皮激素20E是调节昆虫发育的主要激素，其作用机制除经典的核受体介导的基因组途径外，还包括膜介导的非基因组途径，主要是通过快速诱导转录激活相关因子的翻译后修饰来调节基因转录。但研究主要集中在磷酸化修饰，糖基化修饰的作用未见报道。本研究以鳞翅目昆虫棉铃虫为模型，用蛋白组学和免疫印迹的方法确定了N-乙酰葡萄糖胺 (O-GlcNAc)修饰参与昆虫的变态发育，并初步鉴定了O-GlcNAc修饰的靶标分子如EcR、USP、Br-Z4、catalase等。探索了蜕皮激素20E对糖苷酶OGA的调节机制：20E可在转录水平上调OGA，从而降低取食时期维持的较高O-GlcNAc修饰水平，解除对变态的抑制，使变态正常启动。这些结果完善了O-GlcNAc修饰调节昆虫变态发育的细节，为棉铃虫的生物防治奠定了理论基础。