显性致聋突变GJB2 p.D179N影响缝隙连接蛋白26寡聚平衡状态

The dominant deafness point mutation GJB2 p.D179N affects the oligomeric equilibrium of connexin 26

骆华鸿;黄国辉;高云鸽;杨涛;吴皓;张治华

1:上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科

2:上海市耳鼻疾病转化医学重点实验室

3:上海交通大学医学院耳科学研究所

4:复旦大学附属华东医院神经外科

摘要
目的 探究缝隙连接蛋白β2(GJB2)基因显性致聋点突变p.D179N对缝隙连接蛋白26(Cx26)寡聚平衡状态的影响。方法 通过在HEK293F细胞表达Cx26野生型(Cx26-WT-GFP)及突变体融合蛋白(Cx26-D179N-GFP,Cx26-D179C-GFP);通过荧光尺寸排阻色谱法(fluorescence-detection size-exclusion chromatography, FSEC)及尺寸排阻色谱法(size-exclusion chromatography, SEC)分别在溶膜纯化条件下根据分子量分析目的蛋白的寡聚状态;通过冷冻电镜(cryo-electron microscopy, Cryo-EM)单颗粒分析(single particle analysis, SPA)根据目的蛋白颗粒2D分类形态结果来分析目的蛋白的寡聚状态。结果 在离体条件下,野生型Cx26蛋白(Cx26-WT)几乎均为十二聚体,Cx26致聋点突变p.D179N蛋白(Cx26-D179N)同时存在十二聚体及六聚体,Cx26人为改造点突变p.D179C蛋白(Cx26-D179C)不存在十二聚体。结论 显性致聋突变GJB2 p. D179N可减弱其六聚体蛋白间的对接能力,影响其六聚体、十二聚体之间的平衡状态。
关键词
致聋突变;缝隙连接蛋白26;蛋白纯化;尺寸排阻色谱法;冷冻电镜单颗粒分析
基金项目(Foundation):
上海市耳鼻疾病转化医学重点实验室专项基金(14DZ2260300);; 黄浦区产业扶持基金(XK2019011)
作者
骆华鸿;黄国辉;高云鸽;杨涛;吴皓;张治华
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