基于磷酸化蛋白质组学发现的血吸虫重要信号分子调控虫体生殖发育的分子机理研究

Schistosomes are dioecious trematodes responsible for schistosomiasis in humans and other animals. Sexual maturation and subsequent egg production in female schistosomes require a permanent pairing with male schistosomes at the gynecophornal canal. Matured female schistosomes produce a large number of eggs which are primarily responsible for the pathogenesis of schistosomiasis as well as dissemination of the parasite. However, the current understanding of molecular mechanism of female worm maturation induced by the male worm after pairing is very limited. In our previous study, a phosphoproteomic approach was employed to investigate the phosphoproteins in Schistosoma japonicum. We then built a protein-protein interaction map (PPI) based on several hundred identified phosphoproteins in Schistosome japonicum by using comparative genomics combined with bioinformatics. The resulted PPI indicated that there were several potentially important regulatory nodes and crosstalk points mainly including Heat shock protein 90 (HSP90), Glycogen synthase kinase 3 beta (GSK3B), and p38 motigen-activated protein kinase (p38MAPK). Our preliminary study showed that the block of HSP90 function in Schistosoma japonicum by applying drug inhibitor led to the significant reduction of worms in the final host and the eggs in the host's liver, suggesting these molecules in the regulatory nodes and crosstalk points of PPI play an important role in the regulation of schistosome development, sexual maturation and egg production. Additionally, recent study indicated that HSP90 could facilitate/mature a large number of signaling molecules involved in many biological processes such as GSK3B and p38MAPK in both human and yeast. Here, we propose to use co-immunoprecipitation, yeast two hybrid system and RNA interference (RNAi) to experimentally determine the functions of the HSP90, GSK3B and p38MAPK in female schistosomes for sexual maturation and egg production and also investigate their partners and signal pathways involved in schistosome development and sexual maturation. Then, we plan to apply RNAi and chemical inhibitors to block the functions of these molecules and/or their involved signal pathways to evaluate the potential of these molecules as drug targets against schistosomiasis. The expected results of the proposal will not only provide important information for theoretically understanding the mechanism of schistosome development and sexual maturation but also may facilitate to develop effective anti-schistosome vaccines and drugs for schistosomiasis control.

血吸虫雌雄合抱是雌虫性发育成熟的关键，性发育成熟的雌虫所产大量虫卵又是造成宿主病理损害和疾病传播的根本原因。目前，有关血吸虫生殖发育的分子机理认识仍非常有限。基于日本血吸虫磷酸化蛋白质组研究构建的蛋白质相互作用图，发现了数个重要调控节点和对话点(HSP90，GSK3Beta 和p38MAPK)。我们已研究表明阻断血吸虫HSP90 可显著降低动物体内虫体的寄生及肝脏的虫卵量，提示关键信号分子在血吸虫生殖发育和产卵中的重要作用。为此，本项目拟利用免疫共沉淀、酵母双杂交和RNAi 等技术研究重要调控节点HSP90，GSK3Beta 和p38MAPK 的分子功能及其相互作用分子，发现其参与血吸虫发育生殖和产卵的信号通路，探索干扰关键分子以阻断虫体发育成熟和产卵的可行性。研究结果不仅对阐明血吸虫的生殖发育机理具有重要的理论意义，还可为研发抗血吸虫生殖发育疫苗和药物提供新思路，具有重要的现实意义。

血吸虫雌雄异体，雌虫所产大量虫卵是造成宿主病理损害和疾病再传播的根本原因。目前，有关血吸虫生殖发育的分子机理认识非常有限。本项目根据前期研究发现，深入揭示关键分子参与血吸虫生殖发育和产卵的调控功能。首先，课题完成Hsp90，Gsk3b和p38MAPK分子的重组蛋白表达、抗体制备及其组织表达研究，发现了Hsp90，Gsk3b和p38MAPK在血吸虫多个发育时期均表达，且主要定位于虫体肌肉和体被。然后，完成了Hsp90，Gsk3b和p38MAPK互相作用分子的分离和鉴定和验证工作，利用免疫共沉淀获得40多个潜在的互作分子，通过筛选酵母双杂交文库及验证，获得15个与Gsk3b的互作蛋白分子。还有，课题完成了Hsp90，Gsk3b和p38MAPK三个基因的RNA干扰研究和化学抑制剂抑制研究，并评估对血吸虫存活和产卵的影响，结果表明干扰或抑制Hsp90或p38MAPK可影响血吸虫的存活和产卵。另外，课题还开展了日本血吸虫雌雄虫差异蛋白/磷酸化蛋白的定量蛋白质组学研究，在雌雄虫中共鉴定了2058个蛋白和924个磷酸化蛋白，其中雌虫有88个蛋白和166个磷酸化蛋白呈现差异，雄虫有144个蛋白和174个磷酸化蛋白呈现差异。进一步分析表明Hsp90在差异蛋白的互作网络中处于关键节点，可能在雌虫的性成熟和产卵中发育重要的调控作用。总之，课题深入研究了Hsp90，Gsk3b和p38MAPK在血吸虫的存活和产卵中功能，发现了Hsp90-Gsk3b/Cdc37-p38MAPK调控通路在血吸虫的寄生和产卵中可能发挥重要作用，该结果不仅对丰富了血吸虫生殖发育的基础理论，还对开发高效的抗血吸虫疫苗和药靶具有一定指导意义。