本文關鍵詞： HERG鉀通道 蛋白酪氨酸磷酸酶非受體型12 免疫共沉淀 GST pull-down Assay 熒光免疫組織化學 膜片鉗技術　出處：《汕頭大學》2011年碩士論文　論文類型：學位論文

【摘要】：背景：由人類果蠅相關基因—HERG (Human ether-a-go-go-related gene)編碼的心臟HERG鉀通道屬于電壓依賴性鉀通道,介導快速激活延遲整流鉀電流(rapidly activated delayed rectifier potassium currents, Ikr)。HERG鉀通道是心臟正常電活動的基礎,HERG基因突變及藥物阻斷該通道后可導致長QT綜合征(the long QT syndrome, LQTS)。目前已發(fā)現(xiàn)10個與LQTS相關基因。其中,HERG突變可以導致第二型的LQTS(LQT2),是LQTS的第二常見原因。而蛋白質(zhì)-蛋白質(zhì)相互作用是很多細胞功能的重要基礎,信號轉(zhuǎn)導、細胞周期調(diào)控、RNA轉(zhuǎn)錄、DNA復制、蛋白質(zhì)翻譯、蛋白質(zhì)翻譯后加工及修飾等功能的完成都依賴于蛋白質(zhì)-蛋白質(zhì)的相互作用。近年已報道一些蛋白質(zhì)與HERG鉀通道存在相互作用并調(diào)控該通道蛋白的表達、轉(zhuǎn)運及功能。本課題組前期研究應用酵母菌雙雜交技術初步篩選出與HERG鉀通道蛋白氨基末端存在相互作用的15個蛋白質(zhì),包括：蛋白酪氨酸磷酸酶非受體型12 (Tyrosine-protein phosphatase non-receptor type 12, PTPN12)、caveolin-1、FHL2、Myotrophin蛋白等,其中的PTPN12可能對HERG鉀通道具有重要的調(diào)控作用,進一步證明PTPN12與心臟HERG鉀通道存在相互作用,并闡明PTPN12對HERG鉀通道功能是否存在調(diào)控作用,為開發(fā)治療HERG鉀通道異常所致心律失常的蛋白質(zhì)藥物奠定基礎。 目的：鑒定HERG鉀通道的相互作用蛋白,研究PTPN12對HERG鉀通道功能的調(diào)控作用。 方法：(1)應用免疫共沉淀技術進一步驗證酵母雙雜交所篩選蛋白與HERG之間的相互作用：將抗HERG特異性抗體和總蛋白進行混合,再加入Protein A/G Plus-Agarose進行混合,離心沉淀,將沉淀物進行電泳,隨后應用抗PTPN12抗體進行Western Blot分析。 (2) GST pull-down分析：應用GST-HERGT-NT融合蛋白和谷光苷肽-瓊脂糖4B小球從大鼠心肌裂解物中沉淀蛋白質(zhì),應用抗PTPN12抗體對沉淀物進行Western Blot分析；(3)免疫熒光組織化學分析：培養(yǎng)HEK293細胞,應用Lipofectamine2000將pcDNA3.1-PTPN12質(zhì)粒和pcDNA3.0-HERG質(zhì)粒共轉(zhuǎn)染HEK293細胞,轉(zhuǎn)染48小時后固定,應用抗PTPN12和抗HERG的抗體和熒光標記二抗顯示PTPN12及HERG的亞細胞定位,應用激光共聚焦顯微鏡觀察。(4)應用膜片鉗技術,研究PTPN12對HERG鉀通道功能的影響。 結(jié)果：(1)免疫共沉淀分析發(fā)現(xiàn)抗HERG的抗體能夠沉淀HERG和PTPN12復合物；(2)應用GST pull-down分析發(fā)現(xiàn)GST-HERG-NT能夠?qū)TPN12沉淀,而GST蛋白則不能沉淀PTPN12;(3)免疫熒光組織化學分析：用倒置熒光顯微鏡觀察可見在細胞膜上PTPN12蛋白和HERG蛋白分別發(fā)出綠色熒光和紅色熒光,二者熒光重疊部分(黃色熒光)提示這兩種蛋白在細胞上共定位,發(fā)現(xiàn)PTPN12和HERG兩個蛋白共定位的地方主要出現(xiàn)在細胞膜。(4)膜片鉗檢測發(fā)現(xiàn),PTPN12蛋白能降低HERG電流幅度。 結(jié)論：本實驗結(jié)果表明蛋白酪氨酸磷酸酶非受體型12 (PTPN12)與心臟HERG鉀通道的氨基末端存在相互作用,并通過這種相互作用發(fā)揮了調(diào)控HERG鉀通道功能的作用,使HERG鉀通道電流振幅降低。本結(jié)果提示PTPN12參與了HERG鉀通道功能的調(diào)控,其機制可能是通過改變HERG鉀通道磷酸化程度而發(fā)揮作用。這一發(fā)現(xiàn)將有助于進一步理解HERG通道功能的調(diào)控機制,為更深刻理解LQTS的發(fā)病機理及開發(fā)治療LQTS的蛋白質(zhì)藥物提供新的思路。
[Abstract]:Background: human related gene HERG from Drosophila melanogaster (Human ether-a-go-go-related gene) cardiac HERG potassium channel encoding belong to voltage dependent potassium channels, mediated by rapid activation of delayed rectifier potassium current (rapidly activated delayed rectifier potassium currents, Ikr).HERG potassium channel is the basis of normal cardiac electrical activity, HERG gene mutation and drug blocked the channel may cause long QT syndrome (the long QT syndrome, LQTS). The 10 LQTS related genes have been found. Among them, HERG mutations can lead to type second LQTS (LQT2), is the second most common cause of LQTS. Protein - protein interaction is an important basis, many functions of the cell signal transduction. The regulation of cell cycle, RNA transcription, DNA replication, protein translation, interaction processing and modification functions of protein after translation are dependent on the completion of protein - protein in recent years has been. The interaction between expression and regulation of the channel protein reported some protein and HERG potassium channel, and the transfer function. Ourprevious studies using yeast two hybrid technique screened 15 proteins that interact with HERG potassium channel protein N-terminal include: protein tyrosine phosphatase nonreceptor 12 (Tyrosine-protein phosphatase non-receptor type 12, PTPN12), caveolin-1, FHL2, Myotrophin protein, in which the PTPN12 may play an important role in the regulation of HERG potassium channel, further evidence of the interaction between PTPN12 and cardiac HERG potassium channel, and to clarify the existence of PTPN12 regulation on HERG potassium channel function, lay the foundation for the development of protein drugs for the treatment of HERG caused by the abnormal potassium channel arrhythmia.
Objective: to identify the interaction proteins of HERG potassium channels and to study the role of PTPN12 in the regulation of potassium channel function of HERG.
Methods: (1) technology is used to validate the yeast two hybrid screening of the interaction between the protein and the application of HERG co immunoprecipitation: Anti HERG specific antibodies and total protein were mixed, then add Protein A/G Plus-Agarose mixed, centrifugal sedimentation and sediment electrophoresis, then the application of anti PTPN12 antibodies by Western Blot analysis.
(2) GST pull-down analysis: application of GST-HERGT-NT fusion protein and glutathione Sepharose 4B beads to precipitate protein from rat myocardium lysate, application of anti PTPN12 antibody to precipitate Western Blot analysis; (3) immunofluorescence histochemistry analysis in cultured HEK293 cells, the application of Lipofectamine2000 pcDNA3.1-PTPN12 plasmid and pcDNA3.0-HERG plasmid were transfection of HEK293 cells 48 hours after transfection, the application of anti PTPN12 and anti HERG antibody and fluorescently labeled two antibody showed the subcellular localization of PTPN12 and HERG, using confocal laser microscope. (4) using the patch clamp technique to study effects of PTPN12 on HERG potassium channel function.
Results: (1) analysis showed that anti HERG antibody precipitated HERG and PTPN12 complexes co immunoprecipitation; (2) the application of GST pull-down analysis showed that GST-HERG-NT PTPN12 can be precipitated, while GST protein was not precipitated PTPN12; (3) immunofluorescence histochemistry analysis: using inverted microscope observation showed PTPN12 in the cell membrane protein and HERG protein were green fluorescence and red fluorescence, two fluorescence overlap (yellow fluorescence) that CO localization of these two proteins in cells, PTPN12 and HERG two protein co localization where the main cell membrane. Now (4) patch detection showed that PTPN12 protein can reduce the HERG current amplitude.
Conclusion: These results suggest that protein tyrosine phosphatase nonreceptor type 12 (PTPN12) interaction between N-terminal and cardiac HERG potassium channel, and regulation of HERG potassium channel function through this interaction, the amplitude of HERG currents decreased. The results suggest that PTPN12 participates in the regulation of HERG potassium channel function, the mechanism may play a role by changing the HERG potassium channel phosphorylation and regulation mechanism. This discovery will help to further understand the function of HERG channels, to provide new ideas for the development of protein drugs and pathogenesis of a more profound understanding of the treatment of LQTS LQTS.

【學位授予單位】：汕頭大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R331

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