Lingo-2与Ring-1相互作用及在维持神经元存活中作用的研究

Lingo-2, a member of LRR gene family, has been linked with Essential tremor and Parkinson's disease. The similar protein structure between Lingo-2 and Lingo-1 indicates that Lingo-2 may participate in the activities of neurons through protein-protein interaction. Recently, we identified Ring-1, a functional E3 ubiquitin ligase, as a binding partner of Lingo-2 through yeast two-hybrid screening. Furthermore, we found that the MG132, the proteosome inhibitor, blocked Lingo-2 degradation and overexpression of Lingo-2 could cause neuronal death. Based on our previous findings, we propose that Ring-1 may function as E3 ubiquitin ligase in the degradation of Lingo-2 and the disruption of the interaction between two proteins may impair the degradation of Lingo-2 and the following neuronal death. In this study，we will try to provide the direct evidence that Ring-1 is a molecule involved in the degradation of Lingo-2 which is very important for the neuronal survival. Understanding the biological function of Lingo-2 and its role in the activities of neurons will help us to explore the molecular mechanism of related neurological disorders.

研究发现Lingo-2基因改变会增加原发性震颤及帕金森病的发生风险，目前尚无法解释基因改变导致相关疾病发生的可能机制。Lingo-2的结构与Lingo-1高度相似，提示其可能通过蛋白相互作用参与神经元的生命活动。运用酵母双杂交系统我们筛选出Lingo-2的互作蛋白Ring-1，Ring-1具有E3泛素连接酶活性，我们还发现蛋白酶体抑制剂MG132可抑制Lingo-2的降解，而神经元中过表达Lingo-2后会导致神经元的死亡。据此，推测Ring-1与Lingo-2蛋白的降解有关，影响两者相互作后可能会导致Lingo-2的异常积聚和神经元的死亡。本课题将（1）证实Lingo-2通过泛素蛋白酶系统降解；（2）证实Ring-1和Lingo-2蛋白具有相互作用；（3）了解两者相互作用对Lingo-2蛋白降解及神经元存活的影响。课题的开展将为明确相关神经系统疾病的发病机制提供实验依据。

研究发现 LINGO-2 基因改变会增加原发性震颤及帕金森病的发生风险，目前尚无法解释基因改变导致相关疾病发生的可能机制。LINGO-2的结构与LINGO-1 高度相似，提示其可能通过蛋白相互作用参与神经元的生命活动。运用酵母双杂交系统我们筛选出LINGO-2 的互作蛋白RNF-2应用免疫荧光和免疫共沉淀技术我们在SH-SY5Y 细胞中验证了两者的相互作用。在神经元中过表达LINGO-2 后会导致神经元的死亡，敲除其互作蛋白RNF-2 后，LINGO-2 产生积聚。我们还发现溶酶体抑制剂Leupeptin 可抑制LINGO-2 的降解，而LINGO-2 和早期内体、晚期内体共定位。文献报道RNF-2 具有E3 泛素连接酶活性，因此我们认为RNF-2 可能在LINGO-2 蛋白的降解过程中起到了泛素化修饰的作用，被泛素化修饰的LINGO-2 可以被内体分选蛋白识别进入早期内体和晚期内体并且最终在溶酶体降解，影响两者相互作后可能会导致LINGO-2 的异常积聚和神经元的死亡。本课题的开展为明确相关神经系统疾病的发病机制提供实验依据。在项目资助下，已在Parkinsonism & Related disorders杂志上发表研究论文1篇，在中文核心期刊《内科理论与实践》杂志上发表研究论文1篇；培养博士研究生1名。