蛋白酪氨酸磷酸酶非受体型12与心脏HERG钾通道相互作用并调控通道功能的研究

发布时间：2018-02-23 21:05

本文关键词： HERG钾通道蛋白酪氨酸磷酸酶非受体型12 免疫共沉淀 GST pull-down Assay 荧光免疫组织化学膜片钳技术　出处：《汕头大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景：由人类果蝇相关基因—HERG (Human ether-a-go-go-related gene)编码的心脏HERG钾通道属于电压依赖性钾通道,介导快速激活延迟整流钾电流(rapidly activated delayed rectifier potassium currents, Ikr)。HERG钾通道是心脏正常电活动的基础,HERG基因突变及药物阻断该通道后可导致长QT综合征(the long QT syndrome, LQTS)。目前已发现10个与LQTS相关基因。其中,HERG突变可以导致第二型的LQTS(LQT2),是LQTS的第二常见原因。而蛋白质-蛋白质相互作用是很多细胞功能的重要基础,信号转导、细胞周期调控、RNA转录、DNA复制、蛋白质翻译、蛋白质翻译后加工及修饰等功能的完成都依赖于蛋白质-蛋白质的相互作用。近年已报道一些蛋白质与HERG钾通道存在相互作用并调控该通道蛋白的表达、转运及功能。本课题组前期研究应用酵母菌双杂交技术初步筛选出与HERG钾通道蛋白氨基末端存在相互作用的15个蛋白质,包括：蛋白酪氨酸磷酸酶非受体型12 (Tyrosine-protein phosphatase non-receptor type 12, PTPN12)、caveolin-1、FHL2、Myotrophin蛋白等,其中的PTPN12可能对HERG钾通道具有重要的调控作用,进一步证明PTPN12与心脏HERG钾通道存在相互作用,并阐明PTPN12对HERG钾通道功能是否存在调控作用,为开发治疗HERG钾通道异常所致心律失常的蛋白质药物奠定基础。目的：鉴定HERG钾通道的相互作用蛋白,研究PTPN12对HERG钾通道功能的调控作用。方法：(1)应用免疫共沉淀技术进一步验证酵母双杂交所筛选蛋白与HERG之间的相互作用：将抗HERG特异性抗体和总蛋白进行混合,再加入Protein A/G Plus-Agarose进行混合,离心沉淀,将沉淀物进行电泳,随后应用抗PTPN12抗体进行Western Blot分析。 (2) GST pull-down分析：应用GST-HERGT-NT融合蛋白和谷光苷肽-琼脂糖4B小球从大鼠心肌裂解物中沉淀蛋白质,应用抗PTPN12抗体对沉淀物进行Western Blot分析；(3)免疫荧光组织化学分析：培养HEK293细胞,应用Lipofectamine2000将pcDNA3.1-PTPN12质粒和pcDNA3.0-HERG质粒共转染HEK293细胞,转染48小时后固定,应用抗PTPN12和抗HERG的抗体和荧光标记二抗显示PTPN12及HERG的亚细胞定位,应用激光共聚焦显微镜观察。(4)应用膜片钳技术,研究PTPN12对HERG钾通道功能的影响。结果：(1)免疫共沉淀分析发现抗HERG的抗体能够沉淀HERG和PTPN12复合物；(2)应用GST pull-down分析发现GST-HERG-NT能够将PTPN12沉淀,而GST蛋白则不能沉淀PTPN12;(3)免疫荧光组织化学分析：用倒置荧光显微镜观察可见在细胞膜上PTPN12蛋白和HERG蛋白分别发出绿色荧光和红色荧光,二者荧光重叠部分(黄色荧光)提示这两种蛋白在细胞上共定位,发现PTPN12和HERG两个蛋白共定位的地方主要出现在细胞膜。(4)膜片钳检测发现,PTPN12蛋白能降低HERG电流幅度。结论：本实验结果表明蛋白酪氨酸磷酸酶非受体型12 (PTPN12)与心脏HERG钾通道的氨基末端存在相互作用,并通过这种相互作用发挥了调控HERG钾通道功能的作用,使HERG钾通道电流振幅降低。本结果提示PTPN12参与了HERG钾通道功能的调控,其机制可能是通过改变HERG钾通道磷酸化程度而发挥作用。这一发现将有助于进一步理解HERG通道功能的调控机制,为更深刻理解LQTS的发病机理及开发治疗LQTS的蛋白质药物提供新的思路。
[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.

【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R331

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