Periaxin蛋白PDZ结构域结合在EphrinB2的C末端序列

发布时间：2018-12-16 13:26

【摘要】：轴周蛋白(Periaxin)是成髓鞘的施万细胞和晶状体纤维细胞中特异且大量表达的一种细胞骨架相关蛋白。Periaxin参与髓鞘中PDG复合物的形成,是Cajal条带存在的分子基础,一旦L-periaxin突变或缺失会引起过度髓鞘化或脱髓鞘现象。Periaxin的定位在髓鞘的形成过程中是不断变化的,从最初的细胞核到最后的远离轴突的质膜处。Periaxin可以编码含有PDZ结构域的两种蛋白亚型,分别为L-periaxin和S-periaxin。PDZ结构域在Periaxin定位和PDG复合物形成中具有重要作用,但与其结合的配体却未见报道。EphrinB2是受体酪氨酸激酶家族的一个成员。Ephrin B2配体可以和其对应的受体EphB2之间形成双向信号,这种信号可以直接指导轴突的生长、细胞的聚集和迁移、髓鞘的形成。EphrinB2配体包括胞外区,跨膜区和胞内区,在C端包含有PDZ结合基序。通过结合PDZ协调组织细胞膜上的蛋白复合物。本文对L-periaxin与EphrinB2间的相互作用进行了分析。首先,免疫荧光共定位实验结果表明了L-periaxin与EphrinB2在细胞质和细胞膜上有共定位现象。之后对L-periaxin与EphrinB2之间的相互作用进一步验证,结果显示L-periaxin与EphrinB2之间存在相互作用。为了确定EphrinB2与L-periaxin的互作的具体区域,对L-periaxin的四个不同的结构域分别利用双分子荧光互补实验、海肾荧光素酶互补实验及免疫共沉淀实验去分析与EphrinB2间的相互作用,结果表明L-periaxin的PDZ结构域与EphrinB2之间存在着相互作用。将EphrinB2的PDZ结合基序去掉之后,这种相互作用消失。S-periaxin与L-periaxin有着同样的PDZ结构域,通过双分子荧光互补实验(BiFC)、海肾荧光素酶互补实验、免疫共沉淀实验、GST Pull-down实验的检测,也证实S-periaxin的PDZ结构域与EphrinB2的相互作用。实验室前期研究揭示L-periaxin可以通过其分子内NLS2,3结构域与酸性结构域的相互作用形成首尾自环结构,而DRP2与L-periaxin的NLS2,3的结合会减弱L-periaxin的分子内的首尾相互作用。本文通过DRP2的干扰实验,体外NLS3多肽孵育施万细胞,结合双分子荧光互补实验,揭示DRP2及NLS3合成多肽片段可以竞争性结合L-periaxin的NLS结构域,从而破坏L-periaxin之间的分子内的相互作用,免疫荧光定位揭示L-periaxin的分子由闭环到开环的状态改变,引起L-periaxin细胞膜定位数量的增加。最后,为了检测L-periaxin是否影响RSC96细胞的增殖,利用MTT法及流式细胞术对敲除L-periaxin及过表达L-periaxin后对RSC96细胞的增殖的影响进行了研究。结果显示敲除了L-periaxin基因的细胞增殖速度减慢,G1期细胞的比例比正常细胞增加了18.16%,S期减少了20.62%。说明了L-periaxin会促进RSC96细胞的增殖。
[Abstract]:(Periaxin) is a kind of cytoskeleton-associated protein expressed in Schwann cells and lens fibroblasts. Periaxin is involved in the formation of PDG complex in myelin and is the molecular basis of the existence of Cajal bands. Once L-periaxin mutation or deletion causes excessive myelination or demyelination, the localization of Periaxin is constantly changing during myelin formation. Periaxin encodes two protein subtypes containing PDZ domain, L-periaxin and S-periaxin.PDZ domains play an important role in Periaxin localization and PDG complex formation. EphrinB2 is a member of the receptor tyrosine kinase family. Ephrin B2 ligand can form a bidirectional signal with its corresponding receptor EphB2, which can direct the growth of axons. EphrinB2 ligands include extracellular domain, transmembrane region and intracellular region, and contain PDZ binding motifs at the C-terminal. Protein complexes on tissue membranes are coordinated by binding to PDZ. The interaction between L-periaxin and EphrinB2 is analyzed in this paper. First, immunofluorescence co-localization results showed that L-periaxin and EphrinB2 were co-located in cytoplasm and cell membrane. The interaction between L-periaxin and EphrinB2 is further verified. The results show that there is interaction between L-periaxin and EphrinB2. In order to determine the specific region of interaction between EphrinB2 and L-periaxin, four different domains of L-periaxin were analyzed by bimolecular fluorescence complementation test, sea kidney luciferase complementary test and immunoprecipitation test to analyze the interaction with EphrinB2. The results show that there is interaction between PDZ domain and EphrinB2 of L-periaxin. After the PDZ binding motif of EphrinB2 was removed, the interaction disappeared. S-periaxin and L-periaxin shared the same PDZ domain. The (BiFC), sea kidney luciferase complementation test and the immunoprecipitation assay were used to detect the binding motifs of S-periaxin and L-periaxin. The GST Pull-down test also confirmed the interaction between PDZ domain of S-periaxin and EphrinB2. Early laboratory studies have shown that L-periaxin can form a self-ring structure through the interaction between its intramolecular NLS2,3 domain and acidic domain, while the binding of DRP2 to L-periaxin NLS2,3 weakens the intramolecular head-and-tail interaction of L-periaxin. In this paper, by the interference experiment of DRP2, incubating Schwann cells with NLS3 polypeptide in vitro and combining with bilayer fluorescence complementary experiment, it is revealed that DRP2 and NLS3 synthetic polypeptide fragments can competitively bind to the NLS domain of L-periaxin. In order to destroy the intramolecular interaction between L-periaxin, the immunofluorescence localization revealed the change of the state of L-periaxin from closed loop to open loop, which resulted in the increase of the number of L-periaxin cell membrane localization. Finally, in order to determine whether L-periaxin affects the proliferation of RSC96 cells, the effects of L-periaxin knockout and L-periaxin overexpression on the proliferation of RSC96 cells were studied by MTT and flow cytometry. The results showed that the proliferation rate of the cells knockout L-periaxin gene decreased, the proportion of G1 phase cells increased by 18.16% than the normal cells, and the S phase decreased 20.62%. The results suggest that L-periaxin can promote the proliferation of RSC96 cells.
【学位授予单位】：山西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

【参考文献】