Periaxin蛋白PDZ結(jié)構(gòu)域結(jié)合在EphrinB2的C末端序列
發(fā)布時(shí)間：2018-12-16 13:26
【摘要】：軸周蛋白(Periaxin)是成髓鞘的施萬(wàn)細(xì)胞和晶狀體纖維細(xì)胞中特異且大量表達(dá)的一種細(xì)胞骨架相關(guān)蛋白。Periaxin參與髓鞘中PDG復(fù)合物的形成,是Cajal條帶存在的分子基礎(chǔ),一旦L-periaxin突變或缺失會(huì)引起過(guò)度髓鞘化或脫髓鞘現(xiàn)象。Periaxin的定位在髓鞘的形成過(guò)程中是不斷變化的,從最初的細(xì)胞核到最后的遠(yuǎn)離軸突的質(zhì)膜處。Periaxin可以編碼含有PDZ結(jié)構(gòu)域的兩種蛋白亞型,分別為L(zhǎng)-periaxin和S-periaxin。PDZ結(jié)構(gòu)域在Periaxin定位和PDG復(fù)合物形成中具有重要作用,但與其結(jié)合的配體卻未見(jiàn)報(bào)道。EphrinB2是受體酪氨酸激酶家族的一個(gè)成員。Ephrin B2配體可以和其對(duì)應(yīng)的受體EphB2之間形成雙向信號(hào),這種信號(hào)可以直接指導(dǎo)軸突的生長(zhǎng)、細(xì)胞的聚集和遷移、髓鞘的形成。EphrinB2配體包括胞外區(qū),跨膜區(qū)和胞內(nèi)區(qū),在C端包含有PDZ結(jié)合基序。通過(guò)結(jié)合PDZ協(xié)調(diào)組織細(xì)胞膜上的蛋白復(fù)合物。本文對(duì)L-periaxin與EphrinB2間的相互作用進(jìn)行了分析。首先,免疫熒光共定位實(shí)驗(yàn)結(jié)果表明了L-periaxin與EphrinB2在細(xì)胞質(zhì)和細(xì)胞膜上有共定位現(xiàn)象。之后對(duì)L-periaxin與EphrinB2之間的相互作用進(jìn)一步驗(yàn)證,結(jié)果顯示L-periaxin與EphrinB2之間存在相互作用。為了確定EphrinB2與L-periaxin的互作的具體區(qū)域,對(duì)L-periaxin的四個(gè)不同的結(jié)構(gòu)域分別利用雙分子熒光互補(bǔ)實(shí)驗(yàn)、海腎熒光素酶互補(bǔ)實(shí)驗(yàn)及免疫共沉淀實(shí)驗(yàn)去分析與EphrinB2間的相互作用,結(jié)果表明L-periaxin的PDZ結(jié)構(gòu)域與EphrinB2之間存在著相互作用。將EphrinB2的PDZ結(jié)合基序去掉之后,這種相互作用消失。S-periaxin與L-periaxin有著同樣的PDZ結(jié)構(gòu)域,通過(guò)雙分子熒光互補(bǔ)實(shí)驗(yàn)(BiFC)、海腎熒光素酶互補(bǔ)實(shí)驗(yàn)、免疫共沉淀實(shí)驗(yàn)、GST Pull-down實(shí)驗(yàn)的檢測(cè),也證實(shí)S-periaxin的PDZ結(jié)構(gòu)域與EphrinB2的相互作用。實(shí)驗(yàn)室前期研究揭示L-periaxin可以通過(guò)其分子內(nèi)NLS2,3結(jié)構(gòu)域與酸性結(jié)構(gòu)域的相互作用形成首尾自環(huán)結(jié)構(gòu),而DRP2與L-periaxin的NLS2,3的結(jié)合會(huì)減弱L-periaxin的分子內(nèi)的首尾相互作用。本文通過(guò)DRP2的干擾實(shí)驗(yàn),體外NLS3多肽孵育施萬(wàn)細(xì)胞,結(jié)合雙分子熒光互補(bǔ)實(shí)驗(yàn),揭示DRP2及NLS3合成多肽片段可以競(jìng)爭(zhēng)性結(jié)合L-periaxin的NLS結(jié)構(gòu)域,從而破壞L-periaxin之間的分子內(nèi)的相互作用,免疫熒光定位揭示L-periaxin的分子由閉環(huán)到開(kāi)環(huán)的狀態(tài)改變,引起L-periaxin細(xì)胞膜定位數(shù)量的增加。最后,為了檢測(cè)L-periaxin是否影響RSC96細(xì)胞的增殖,利用MTT法及流式細(xì)胞術(shù)對(duì)敲除L-periaxin及過(guò)表達(dá)L-periaxin后對(duì)RSC96細(xì)胞的增殖的影響進(jìn)行了研究。結(jié)果顯示敲除了L-periaxin基因的細(xì)胞增殖速度減慢,G1期細(xì)胞的比例比正常細(xì)胞增加了18.16%,S期減少了20.62%。說(shuō)明了L-periaxin會(huì)促進(jìn)RSC96細(xì)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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R741
本文編號(hào)：2382422
[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.
【學(xué)位授予單位】：山西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R741
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