调控带绦虫成虫早期发育关键SUMO化修饰靶蛋白的鉴定与功能研究

Taeniidae species seriously threaten animal husbandry and public health. The host transition is a key step in the normal growth and development, survival and pathogenesis of tapeworms. It is crucial to excavate the specific drug targets and analyse the key factors in regulating growth and development of tapeworms for effectively controlling taeniasis/cysticercosis. In previous studies, it was investigated that the core components of SUMOylation pathway are integrated in tapeworm. And the interaction was verified in tapeworm between SUMO conjugating enzyme UBC9 and the key metabolic enzymes. It is indicated that protein SUMOylation in the tapeworm is vital for the worm development and survival adaptability in host. However, SUMOylation is not yet explored in Taeniidae and the SUMOylation substrates are scarce. Therefore, using Taenia pisiformis, the objectives of the present project are to detect the large-scale SUMOylation targets, by an integrated research approach using SUMO-modified proteome, LC-MS/MS, bioinformatics analysis, Co-immunoprecipitation，real-time PCR, RNA-seq, RNAi technology and so on, to screen differentially and specifically expressed SUMOylation substrates, to validate the key SUMOylation substrates related with regulating worm development in early stage of adult tapeworm and study its function. The expected results will reveal the melocular basis of some important targets in terms of the SUMOylation pathway in Taeniidae and provide the novel targets to develop new drugs and vaccines for the prevention and surveillance of animal taeniasis/cysticercosis.

带绦虫严重威胁畜牧业发展及公共卫生安全。宿主转换是带绦虫实现正常生长发育、生存及致病的关键环节。解析调控虫体生长发育的关键分子进而挖掘高效药靶对于从源头防控带绦/蚴病至关重要。前期研究发现，带绦虫表达完整SUMO化修饰途径的核心组件，且SUMO结合酶UBC9与虫体代谢酶具有相互作用；提示SUMO化修饰在带绦虫的生长发育和适应宿主生存中发挥重要作用。但目前对带绦虫SUMO化修饰缺乏相关研究。本项目拟选择豆状带绦虫为材料，通过定量SUMO化蛋白质组分析，结合LC-MS/MS、生物信息学、免疫共沉淀、荧光定量PCR、RNA干扰和RNA-seq等多种技术和手段，筛选并验证豆状带绦虫阶段性差异表达的SUMO化靶蛋白，鉴定调控成虫早期发育的关键SUMO化靶蛋白分子及其功能。预期结果将为阐明带绦虫细胞内蛋白质SUMO化修饰的分子机制提供重要理论依据，为防控动物带绦/蚴病的药物和疫苗研制提供新的重要靶标。

SUMO化修饰对寄生虫生长发育及生存具有重要的调控作用，但目前对带绦虫SUMO化修饰缺乏相关研究。本项目系统研究了带绦虫SUMO化修饰系统核心组件分子特征及其重要底物分子功能，主要结果为：（1）带绦虫具有完整的SUMO化修饰系统，且存在单个的SUMO基因和唯一的E2结合酶UBC9；其中TpSUMO序列N端11位赖氨酸（FK11MG）为多聚链形成的主要位点，其C端GG缺失对多聚链形成无明显影响；早期成虫组织中TpSUMO表达丰度高，提示SUMO修饰在成虫早期生长发育过程中具有重要作用。（2）应用RNA-Seq和iTRAQ技术分析了豆状带绦虫幼虫期和成虫期差异基因/蛋白表达趋势，结果获得4093个差异表达基因（DEGs），其中成虫期有3186个DEGs上调表达，907个DEGs下调表达；而差异表达蛋白（DEPs）有1665个，其中成虫期上调表达672个DEPs，下调表达993个DEPs。关联分析二者相关性较低，r=0.44，共有525个DEGs/DEPs表达趋势一致；GO和KEGG功能分析显示共有蛋白主要参与细胞过程和代谢过程。（3）利用蛋白质谱技术进行了SUMO修饰组和外泌体组分析，结果筛选出 22个带绦虫TpSUMO底物分子和59个修饰位点；丰度较高且在外泌体组和SUMO蛋白质组中均鉴定出的分子主要包括actin、major vault protein、calpain-A、14-3-3和热休克蛋白等。（4）大肠杆菌SUMO化重构系统证实Tp14-3-3可被TpSUMO化修饰，且依赖于TpSUMO分子C末端GG残基；但虫体内Tp14-3-3互作分子主要为其同型或异型分子。外泌体与宿主细胞相互作用表明，LoVo细胞可主动摄入绦虫外泌体；Western blot显示LC3B-II、Beclin I 和 P62 的表达水平及pAMPK/AMPK显著降低（P<0.01）；免疫电镜结果发现绦虫外泌体中携带TpSUMO和TpUBC9，与Tp14-3-3共转染HEK293T细胞检测Bax表达明显降低，Bcl2/Bax比值升高；提示外泌体介导了SUMO化底物等虫源因子对宿主靶细胞增殖、自噬等生物学功能的调控。以上研究结果为揭示带绦虫SUMO化底物与宿主细胞相互作用机制提供了重要理论依据，也为动物带绦/蚴病的源头防控和治疗提供了新通路和新靶标。