解剖学报

›› 2012, Vol. 43 ›› Issue (5): 588-593.doi: 10.3969/j.issn.0529-1356.2012.05.002

• 神经生物学 • 上一篇    下一篇

发动蛋白-1的脯氨酸和精氨酸富集域功能域与大鼠神经突触小体相互作用蛋白的筛选及鉴定

彭镜1; 张慈柳1; 李玉飞2; 吴蕾1; 何芳1; 邓小鹿1; 尹飞1*    

  1. 1.中南大学湘雅医院儿科，长沙410008; 2.中南大学湘雅医院骨科，长沙410008
  • 收稿日期:2011-12-06 修回日期:2012-05-02 出版日期:2012-10-06
  • 通讯作者: 尹飞

Screening and identification interaction proteins of dynamin-1 PRD functional domain in rat brain synaptosome

  1. 1. Department of Pediatrics, Xiang Ya Hospital of Central South University, Changsha 410008, China; 2.Department of Orthopedics, Xiang Ya Hospital of Central South University, Changsha 410008, China
  • Received:2011-12-06 Revised:2012-05-02 Online:2012-10-06
  • Contact: YIN Fei

关键词: 神经突触小体, 发动蛋白-1, 脯氨酸和精氨酸富集域, 突触囊泡循环, 液相质谱, 大鼠

Abstract: Objective Synaptic vesicles complete of synaptic vesicles cycle by exocytosis and endocytosis. The retrieval of synaptic vesicle membrane after exocytosis is essential for the maintenance of synaptic transmission in central nervous system synapses. Dynamin-1 is a 96kD multidomain GTPase enzyme that is crucial for the fission stage of synaptic vesicle recycling and vesicle traffick. Several models range from viewing Dynamin-1 strictly as a mechanochemical enzyme to considering it as a regulatory protein for the recruitment of the downstream binding partners responsible for scission. To address the role of Dynamin-1 and its interaction proteins in synaptic vesicle endocytosis, we screened and identified interaction proteins of Dynamin-1 PRD functional domain in rat brain synaptosomes. Methods pGEX-4T-2-PRD, a prokaryotic expression plasmid of PRD functional domain, was constructed into Dynamin-1. GST-PRD fusion proteins were obtained by Escherichia coli (E.coli) expression system combined glutathione agarose purified column. Rat brain synaptosomal fractions were isolated by ultracentrifugation. Glutathione S-transferase(GST) pull-down assay was employed to screen interaction proteins between rat brain synaptosome and GST-PRD fusion proteins. Subsequently, these proteins were identified using liquid chromatography spectroscopy (LC-MS). Result We successfully purified the GST-PRD fusion proteins and extracted rat brain synaptosome. Thirty-five interaction proteins of Dynamin-1 PRD functional domain in rat synaptosome were isolated and identified, consisting of synaptic vesicle-associated proteins, cytoskeletal proteins, metabolic enzymes and other proteins. Conclusion Here we reported a comprehensive set of candidate proteins that are closely related to synaptic vesicle recycling. The study has laid the foundation for clarifying the function, regulatory mechanism of Dynamin-1 and cracking the pathway/protein network of synaptic vesicle recycling.

Key words: Brain synaptosome, Dynamin-1, PRD functional domain, Synaptic vesicle recycling, Liquid chromatography, Rat

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