MEKK1-MKK4-JNK1信号模块与HO-1的结合位点在神经炎症中的作用和机制

Neuroinflammation is considered as an importmant contributor to the occurrence and development of neurodegenerative diseases. Some studies have revealed that the assembly of MEKK1-MKK4-JNK1 signal module can enhance the phosphorylation of JNK1 during nerve inflammation period, ultimately cause neuronal injury and disorder of learning and memory. The preliminary experimental results indicated that inflammation could facilitate heme oxygenase-1 (HO-1) to combine with MEKK1-MKK4-JNK1 signal module, and promote the assembling of the signal module. Activation of HO-1 could induce the disassembling of the module and alleviate the neuronal damage. However, it is still unclear that which target protein(s) of MEKK1, MKK4, JNK1 or JSAP1, can directly combine with HO-1, and where are specific binding sites between them. In this project, a variety of techniques such as mammalian two-hybrid system, GST pull-down will be used to screen and validate the target protein(s) which can directly combine with HO-1. The sites mutagenesis of HO-1 and target protein will be prepared to screen and validate the specific binding sites between them using the above technologies. Finally, the specific binding sites will be mutated to explore the effects and relative mechanisms of neuronal injury and learning and memory disorder induced by neuroinflammation. This project may provide new drug targets for the therapy of neuroinflammation and new ideas for the treatment of neurodegenerative diseases.

神经炎症是促进神经退行性疾病发生发展的关键因素。研究显示：MEKK1-MKK4-JNK1信号模块的组装可增强JNK1的磷酸化，导致神经细胞的损伤和学习记忆障碍。预实验表明：神经炎症中，血红素加氧酶-1（HO-1）可与MEKK1-MKK4-JNK1信号模块结合，并促进其组装；当HO-1激活后，促使该模块解体，减轻神经细胞损伤。但是，HO-1与MEKK1、MKK4、JNK1还是与其支架蛋白JSAP1直接结合，及具体结合位点，尚不明确。本项目拟采用哺乳动物双杂交、GST pull-down等多种技术，从MEKK1、MKK4、JNK1和JSAP1中筛选并验证与HO-1直接结合的靶蛋白；再分别突变HO-1和靶蛋白，用上述技术筛选并验证突变位点；研究该结合位点在神经细胞损伤和学习记忆障碍中的作用及机制。本项目旨在为神经炎症的治疗提供新的药物靶点，为神经退行性疾病的治疗提供新思路。

神经炎症作为激发因素或辅助因素，可以诱发或加重神经系统相关疾病。有研究显示MEKK1-MKK4-JNK信号模块的组装，能促进JNK磷酸化，导致神经细胞的损伤。神经炎症中，血红素加氧酶-1与MEKK1-MKK4-JNK信号模块结合，促进其组装；当HO-1激活后，促进该模块解体，减轻神经细胞损伤，。但是HO-1与MEKK1、MKK4、JNK还是与支架蛋白JSAP1直接结合，及其具体结合位点，尚不明确。本研究从JSAP1、MEKK1、MKK4、JNK中筛选并验证与HO-1直接结合的靶蛋白及其结合位点，并研究其结合位点在神经细胞损伤中的作用及机制。重要结果：①哺乳动物双杂交筛选，GST pull-down和IP验证，HO-1均能够与该模块中MEKK1、 MKK4、 JNK和JSAP1能直接结合。②成功构建重组真核表达质粒pM-HIS-RFP、pM-HIS-RFP-HO-1、pM-HIS-RFP-H25A、pM-HIS-RFP-G143H、pM-HIS-RFP-8M 。③过表达HO-1可增加糖氧剥夺/复氧后细胞存活，降低p-JNK与cleaved Caspase-3蛋白表达，而其突变体无此作用。HO-1的25号的组氨酸、143号的甘氨酸及heme binding pocket结构域可能是HO-1发挥细胞保护作用的关键位点之一，对于调节JNK及其下游信号分子有重要作用。意义：从MEKK1、 MKK4、JNK1 、JSAP1中筛选并验证与HO-1直接结合的蛋白及具体结合位点，为开发治疗神经炎症相关疾病的理想药物提供靶点，为治疗该类疾病提供新思路。