【作者】 孙晓琳；

【导师】 朱筱娟；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2002， 硕士

【摘要】 哺乳动物Disco-interacting protein 2（DIP2）蛋白家族包括DIP2A、DIP2B和DIP2C三位成员,氨基酸序列含有三个高度保守的功能结构域:DMAP1结合结构域、CaiC结构域和AMP结合结构域。DMAP1结合结构域与DNA甲基化和细胞增殖分化有关。CaiC结构域和AMP结合结构域调节酰基辅酶A代谢,参与神经轴突生长。人类DIP2B基因启动子区域CGG重复序列的过度甲基化可能与染色体脆性部位FRA12A造成的智力障碍相关。本实验室前期工作表明,小鼠中DIP2B的缺失会导致神经元轴突过度生长,通过对DIP2B的三个功能结构域进行功能恢复研究发现,CaiC结构域能够抑制轴突生长,补救DIP2B功能缺陷。因此,我们利用GST-DIP2B CaiC融合蛋白与鼠脑蛋白孵育,通过GST-pull down联合质谱技术筛选与DIP2B结合的蛋白。质谱鉴定共有252个DIP2B结合蛋白,我们利用PANTHER^TM数据库对这些蛋白进行GO分析,结合蛋白归类为GTP酶活性、ATP酶活性、激酶活性以及磷酸酶活性蛋白。由于本实验室前期工作已经发现,Dip2b纯合敲除的神经元突触前递质释放损害,因此,我们统计了参与囊泡循环的蛋白。综合分析表明,Dynamin-1既有GTPase催化活性,又参与突触囊泡内吞过程,调节轴突发育并与递质分泌有关。我们应用GST-pull down技术和免疫共沉淀技术进一步证实了DIP2B与Dynamin-1的相互作用关系。为了探究DIP2B与Dynamin-1二者之间的作用关系,我们通过Cre-Lox P系统构建了DIP2B条件性敲除小鼠,即Dip2b^flox/flox;NEX-Cre小鼠。Western blot检测结果表明,DIP2B的缺失会引起Dynamin-1磷酸化水平增加,提示其磷酸化异常。这些结果为进一步研究DIP2B在突触囊泡回收机制中的作用提供了重要的理论依据,同时,也为DIP2家族高度保守的DIP2A、DIP2B和DIP2C三种蛋白的研究提供了帮助。更多还原

【Abstract】 The mammalian Disco-interacting protein 2（DIP2）family includes three members:DIP2A,DIP2B and DIP2C.Their amino acid sequences have three highly conserved domains:DMAP1 binding domain,CaiC domain,and AMP binding domain.The DMAP1 binding domain is associated with DNA methylation and cell proliferation/differentiation.The CaiC domain and the AMP binding domain are supposed to regulate acyl-Co A metabolism,which mediate neuronal axon growth.Studies have shown that hypermethylation of the CGG-repeat sequences at the promoter region of DIP2B gene was related to fragile FRA12A mental retardation.Previous work in our laboratory has shown that the loss of DIP2B in mice led to excessive axon growth.Functional rescue studies showed the CaiC domain along inhibited the excessive axon growth of DIP2B deleted neurons,and remedy DIP2B functional defects.Therefore,we used the GST-DIP2B CaiC fusion protein to interact with mouse brain proteins,and screened proteins that interacted with DIP2B by GST-pull down combined mass spectrometry.We identified 252 binding partners of DIP2B CaiC domain.We then performed GO analysis via PANTHER^TM database.Most of the binding proteins belong to GTPase,ATPase,kinase and phosphatases protein families.Previous study in our lab showed Dip2b deletion impaired presynaptic transmission.Therefore,we focused on synaptic vesicle recycling related proteins.Among the binding partners,Dynamin-1 draw our particular attention.Dynamin-1 has GTPase activity,and was shown to regulate synaptic vesicle endocytosis,axon growth and neurotransmitter release.GST-pull down and co-immunoprecipitation technology were used to confirm the interaction between DIP2B and Dynamin-1.In order to investigate the relationship between DIP2B and Dynamin-1,we constructed DIP2B conditional knockout mice via the Cre-Lox P system.Western blot results showed that the loss of DIP2B caused an increase in the level of phosphorylation of Dynamin-1,which indicated that its phosphorylation was abnormal.These data provide an important theoretical basis for further studying the role of DIP2B in the process of synaptic vesicle endocytosis.Meanwhile,results in this study also provide critical information for the function of the conserved DIP2 protein family.更多还原