首页|p62体募集自噬相关蛋白WIPI2的机制研究

p62体募集自噬相关蛋白WIPI2的机制研究

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目的 探讨自噬接头蛋白(Sequestosome 1,SQSTM1/p62)与磷脂酰肌醇3-激酶(Phosphatidylinositide 3-kinase,PI3K)复合物的结合蛋白 WD重复结构域磷酸肌醇互作蛋白2(WD repeat domain phosphoinositide-interacting protein 2,WIPI2)在空间上定位的调控关系.方法 使用CRISPR-Cas9技术敲除正常大鼠肾上皮细胞(Normal rat kidney,NRK)中的自噬相关基因 2A(Autophagy-related gene,Atg2A)、自噬相关基因 2B(Autophagy-related gene,Atg2B)和p62 基因,建立 Atg2A 和 Atg2B 基因敲除的细胞系(Atg2ABdouble knockout,Atg2ABDKO)以及p62基因敲除细胞系(p62knockout,p62KO);透射电镜观察Atg2AB DKO细胞中p62体周围囊泡的定位;活细胞成像观察Atg2AB DKO细胞内p62体与 自噬相关蛋白WIPI2的定位变化;光漂白实验观察相变蛋白p62与WIPI2的荧光漂白恢复;通过免疫荧光观察WT、p62 KO和Atg2AB DKO细胞中WIPI2与p62的定位关系;通过蛋白免疫印记检测 WT和p62 KO细胞中WIPI2表达量的变化.结果 建立了 Atg2AB DK O和p62 KO细胞系;透射电镜显示Atg2AB DKO细胞中p62附近聚集了大量囊泡;在Atg2AB DKO中,通过活细胞成像观察到tdTomato-p62与WIPI2-GFP高度共定位;荧光漂白实验观察到 WIPI2具有流动性;通过免疫荧光观察到,与 WT细胞相比,在Atg2AB DKO中WIPI2点的数量明显增多(P<0.000 1);与 WT细胞相比,p62 KO细胞中WIPI2点的数量和表达量无明显变化(P>0.05).结论 无膜细胞器p62能够与具有流动性的WIPI2阳性囊泡动态融合,促进自噬体的形成.
Study on mechanism of recruitment of autophagyassociated protein WD repeat domain phosphoinositide-interacting protein 2(WIPI2)by p62
Objective To investigate the regulation of the spatial localization of autophagy junction protein(SQSTM1/p62)to the binding protein of the phosphatidylinositol 3-kinase complex(PI3K),WD repeat structural domain phosphosphoinositide-interacting protein 2(WIPI2).Methods CRISPR-Cas9 was used to knockout the autophagy-related gene 2A(Atg2A),autophagy related gene 2B(Atg2B)and p62 gene in normal rat kidney epithelial cells(NRK),and established cell lines of Atg2AB DKO and p62 KO.The lo-cation of vesicles around p62 in Atg2AB DKO cells was observedby transmission electron microscopy.The location changes of p62 bodies and autophagyrelated protein WIPI2 in Atg2AB DKO cells were observed by live cell imaging.The restoration of phase change protein p62 bodiesand WIPI2 by fluorescence bleach-ing was observed by photobleaching experiment.The location relationship between WIPI2 and p62 in WT,p62 KO and Atg2AB DKO cellswas observed by immunofluorescence.The expression of WIPI2 expression in WT and p62 KO cellswas detected by immunoimprinting.Results Atg2AB DKO and p62 KO cell lines were established;transmission electron microscopy showed that a large number of vesicles were ag-gregated near p62 in Atg2AB DKO cells;tdTomato-p62 was observed to be highly co-localized with WI-PI2-GFP by live-cell imaging in Atg2AB DKO;fluorescence bleaching assay observed that WIPI2 was mo-bile;a significant increase in the number of WIPI2 sites was observed by immunofluorescence in Atg2AB DKO compared to WT cells(P<0.000 1);there was no significant change in the number and expression of WIPI2 sites in p62 KO cells compared with WT cells(P>0.05).Conclusions The membrane-free or-ganelle p62 is able to dynamically fuse with WIPI2-positive vesicles with mobility to promote autophago-some formation.

autophagosomesequestosome 1(p62/SQSTM1)WD repeat domain phosphoinositide-interac-ting protein 2

张金佩、冯学召、刘梦薇、何心瞳、徐梦波、阿来依·买提卡比力、麦尔哈巴·达毛拉、衡锐、米娜

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新疆医科大学基础医学院生物化学与分子生物学教研室,乌鲁木齐 830017

新疆医科大学重点实验室,乌鲁木齐 830017

中山大学肿瘤防治中心,广东 510060

新疆医科大学基础医学院生物学教研室,乌鲁木齐 830017

新疆医科大学第一附属医院临床医学研究院,乌鲁木齐 830054

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自噬体 自噬接头蛋白 WD重复结构域磷酸肌醇互作蛋白2

新疆维吾尔自治区青年基金省部共建中亚高发病成因与防治国家重点实验室开放基金

2021D01C272SKL-HIDCA-2023-5

2024

新疆医科大学学报
新疆医科大学

新疆医科大学学报

CSTPCD
影响因子:0.76
ISSN:1009-5551
年,卷(期):2024.47(5)