SUMO/DeSUMO化修饰在抑制性受体膜转运中的作用

Surface expression of inhibitory receptors is crucial for controlling neuronal excitability. Post-translational modifications are essential parts of all signaling pathways and regulatory mechanisms which enable cells to adapt to the rapidly changing environmental conditions. Their tight control in the central nervous system is of critical importance for maintaining neuronal cell viability, function and connectivity. The aim of this project is to eludicate the molecular and cellular mechanisms of the SUMOylation/DeSUMOylation on membrane trafficking of inhibitory receptors, which is dynamically regulated by the relative rates of receptor endocytosis, exocytosis, recycling, and degradation as well as by lateral mobility from and to the postsynaptic loci and receptor stabilization at postsynaptic specializations under normal and activation of ionotropic glutamate receptors using multidisciplinary approaches such as molecular biology, cell biology, electrophysiology, imaging and biochemistry. The expected result of this project will provide the key domain, regulatory site and their interacting proteins of inhibitory receptor on membrane trafficking and the role and regulation of SUMOylation/DeSUMOylation on it, which may understand and disclose an innovative idea or novel mechanism for maintaining homestasis of neuronal excitability. This will lead to illustration of the regulation machanism of SUMO/DeSUMO on the membrane trafficking of inhibitory receptor and indicates a role of sumoylation in modulating neuronal excitability and synaptic transmission and plasticity.

抑制性受体在神经元的正确分布定位是兴奋性的基础。翻译后修饰是信号通路和细胞迅速适应的环境变化调节机制的必要组成部分。中枢神经系统翻译后修饰调控对保持神经细胞的活力，功能和连接至关重要。本课题结合分子生物学、细胞生物学、电生理学、荧光成像和生物化学等研究方法，在培养的正常神经元、SENP1敲除和CNS组织特异SENP1敲除神经元中，研究正常及离子型谷氨酸受体激动剂存在的条件下，神经元表面抑制性受体的数量改变、内吞或侧移、内吞后进入内吞途径、再循环-上膜或降解等膜转运过程的变化，探讨SUMO/DeSUMO化修饰对其膜转运的调控。预期结果可解析调节膜转运过程的受体结构域、调控位点及相互作用蛋白及SUMO/DeSUMO化修饰对其相关环节的调控，从而为揭示和理解神经兴奋性的稳态平衡提供新的观点或新机制。阐明SUMO/DeSUMO化修饰在抑制性受体膜转运的调控机制及其在神经兴奋性和突触功能中的作用。

受体/离子通道在神经元膜表面的数量是由依赖于离子通道/受体的合成，正向转运上膜与内吞、再循环和降解的精细平衡决定的。翻译后修饰包括SUMOyaltion、磷酸化和棕榈化等在是细胞信号转导、蛋白转运和稳定性中起重要作用，因此调控了许多生物化学过程，如细胞存活和神经退行性疾病等中起重要调节作用。本课题结合分子生物学、细胞生物学、电生理学、荧光成像和生物化学等研究方法，在培养的正常神经元、SENP1敲除和CNS组织特异SENP1敲除神经元中，研究正常及离子型谷氨酸受体激动剂存在的条件下，神经元表面抑制性受体的数量改变、内吞或侧移、内吞后进入内吞途径、再循环-上膜或降解等膜转运过程的变化，探讨SUMO/DeSUMO化修饰对其膜转运的调控。我们的结果表明：1）PKC能够被SUMO化并改变其活性，PKC的SUMO化介导了Kainate受体激活引起的甘氨酸受体内吞，PKC的SUMO化状态可以调控这一活动依赖性的Kainate受体引起的甘氨酸受体的内吞过程。因为Kainate受体激活导致甘氨酸受体的下调，这种对抗机体稳态平衡新的调节途径通过影响兴奋-抑制平衡维持神经元合适兴奋性中起重要作用；2)PKC的磷酸化水平能够促进其SUMO化修饰，同时抑制泛素化修饰，进而增强其稳定性。这三种翻译后修饰之间的相互作用，揭示了PMA诱导的PKC降解这一过程的分子机制；3)PKC SUMO修饰抑制了14-3-3蛋白与GluK2的相互作用，且这种抑制作用是通过降低GluK2a磷酸化实现的；4)研究发现SENP1在脑缺血再灌注损伤中发挥神经保护作用，主要作用是减少神经细胞凋亡实现的。