G蛋白偶联受体M1 mAChR的SUMO修饰新方式及其对受体功能调控的研究

M1 muscarinic acetylcholine receptor(M1 mAChR)is a member of class A of G-protein-coupled receptors (GPCRs).The predominantly post-synaptic M1 mAChR in the central nervous system plays an important role in Alzheimer's disease (AD)-like pathological features and is a pivotal target that links major hallmarks of AD. It is much attention to M1 mAChR agonists as potential disease-modifying therapy for AD and a target of significant interest for the development of drugs to treat AD. To date, M1 mAChR agonists are still used in clinical trials and have not shown much promise. Besides unfavorable side effects , another important limiting factor for theses compound is that a deficit in AD in muscarinic receptor activation is associated with G-protein signaling dysfunction, which may limit the efficacy of cholinomimetic pharmacotherapies. So, it is necessary to investigate the underlying mechanisms on activity and functions of M1 mAChR. .SUMO (small ubiquitin-related modifier), an important post-translational modification, controls diverse cellular processes. SUMO covalently modifies the lysines in target proteins to regulate their localization, stability, interactions and physiological functions. Sumoylation is a highly dynamic process that is reversed by a family of Sentrin/SUMO-specific protease (SENPs). Desumoylation process must be important for regulation of the fate and function of SUMO-conjugated proteins as well as SUMOylation process.. Our previous study has discovered that SUMO can be conjugated to M1 mAChR and M1 mAChR is a physiologically relevant SUMO substrate.The present study，firstly, will further elucidate more interaction between M1 mAChR and SUMO for virtual screening by homology modeling, protein-protein docking, and molecular dynamics simulation.Secondly，it will be to investigate the detailed mechanisms on M1 mAChR by dynamic process of SUMO/SENPs in CHO cells transfected with M1 mAChR, SAM/P8 mice, APPswe/PS-1 mice, and SENP+/- mice, through immunoprecipitation,radioligand binding assay of receptors, surface plasmon resonance technology, and high content screening. .In conclusion, Our study will elucidate the novel mode on dynamic SUMOylatin of M1 mAChR and the regulatory mechansims on receptor functions. It may give an chance to a better understanding of epigenetic modifications and regulation on GPCRs. It also may provide insights into the pathophysiology of AD and bring about the development of novel therapeutic interventions for AD.

研究表明G蛋白偶联受体M1胆碱受体(M1 mAChR)在调控阿尔茨海默病(AD)样病理变化中发挥关键作用，是AD药物研发最重要标靶之一。然而AD患者常存在M1 mAChR活性不足，与G蛋白偶联障碍而制约受体后级联效应，使M1 mAChR激动剂效能降低，因此探索M1 mAChR的调节新方式及机制有极为重要的科学意义。课题组前期研究首次发现M1 mAChR 存在SUMO修饰且影响信号转导。本研究拟进一步通过蛋白-蛋白对接、分子动力学模拟等方法解析M1 mAChR的SUMO修饰作用；通过转基因细胞及动物模型，应用表面等离子共振、高内涵筛选等技术，深入探讨SUMO修饰平衡对受体活性和功能的调节。以期阐明M1 mAChR 的SUMO 修饰新方式及其对受体功能的调节机制，加深对G蛋白偶联受体表观遗传学修饰与调节方式的认识，为理解AD的发病机制提供新线索，也为寻找AD治疗新靶点提供新思路和新方法。

M1 mAChR是G蛋白偶联受体家族(GPCRs )A组重要成员，在认知、记忆等方面发挥重要生理作用，是治疗阿尔茨海默病（AD）等疾病的重要药物靶标。M1 mAChR内在活性的调控直接影响其生理作用和靶标药物的效能。小分子泛素样相关修饰物（SUMO）是调节蛋白质活性及功能的重要修饰因子，已鉴定出的SUMO修饰靶蛋白大多数位于胞核内，近年越来越多研究表明胞核外蛋白SUMO修饰发挥重要作用，M1 mAChR作为重要细胞膜受体，其SUMO修饰及对受体功能调控的研究尚不清楚。.本项目采用同源建模、分子对接等方法构建受体复合物模型，基于分子动力学模拟研究M1 mAChR与SUMO的相互作用。采用免疫共沉淀及免疫荧光方法验证M1 mAChR与SUMO的相互作用，定点突变筛选出对M1 mAChR修饰关键位点。通过SiRNA干扰技术及SUMO1质粒转染，运用放射配基、激光共聚焦等研究SUMO1对M1 mAChR活性及受体后信号转导的影响。通过AD模型鼠研究探讨脑组织SUMO修饰水平的变化，以及与M1 mAChR受体关系。.结果表明：(1)分子动力学模拟出SUMO1稳定受体复合物模型结构，SUMO1与M1 mAChR不仅存在共价结合形式还存在一定的静电吸引作用。(2)M1 mAChR存在SUMO修饰，K327位点为M1 mAChR与SUMO1结合的主要位点。卡巴胆碱激动后引起受体去SUMO化及SUMO1入核，与M1 mAChR及受体后PKC信号有关。(3)干扰SUMO1表达、或M1 mAChR突变K327后，受体亲和力下降，解离加快，内吞减慢，并且受体后信号IP1累积量减少，Ca2+内流减弱，PKC、ERK、以及GSK-3β激酶活性降低；反之，提高SUMO1表达水平，受体活性提高，受体后信号转导能力加强。（4）APPswe/PS-1双转基因小鼠海马M1 mAChR与SUMO1有相互作用，其认知功能障碍与SUMO修饰水平下降有关。.综上，本研究发现M1 mAChR存在一类新的修饰方式—SUMO修饰， M1 mAChR的SUMO1修饰稳定受体活性及调控受体后信号转导。SUMO1可能作为一个潜在的调控因子在M1 mAChR生理学和药理学作用方面发挥重要作用，对深化GPCRs结构及功能的认识，和M1 mAChR生理功能及靶标药物的研发提供新的方向和靶标，具有重要理论意义和潜在应用价值。