COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

doi:10.19586/j.2095-2341.2022.0053

生物技术进展 ›› 2022, Vol. 12 ›› Issue (6): 906-914.DOI: 10.19586/j.2095-2341.2022.0053

COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

赵云鹏(), 张浩林, 熊倩倩, 李雨婷, 王娟()

北京工业大学环境与生命学部，北京 100124

收稿日期:2022-04-11 接受日期:2022-05-16 出版日期:2022-11-25 发布日期:2022-11-30
通讯作者: 王娟
作者简介:赵云鹏 E-mail：zhaoyunpeng1997@163.com；
基金资助:
国家自然科学基金委重大研究计划培育项目(91854115);国家自然科学基金项目(31970044)

The Role of COPII Coat Protein SEC24A in Mammalian Macroautophagy

Yunpeng ZHAO(), Haolin ZHANG, Qianqian XIONG, Yuting LI, Juan WANG()

Department of Environment and Life Sciences，Beijing University of Technology，Beijing 100124，China

Received:2022-04-11 Accepted:2022-05-16 Online:2022-11-25 Published:2022-11-30
Contact: Juan WANG

摘要/Abstract

摘要：

细胞自噬是一种重要且保守的细胞内降解过程，通过形成双层膜的自噬体包裹细胞内容物进行降解。内质网来源的COPII囊泡被认为是饥饿诱导的应激过程中自噬体的膜源。探究了COPII囊泡衣被蛋白SEC24A在巨自噬通路中的作用。利用siRNA干扰技术敲低SEC24A的表达，EBSS饥饿处理对照组和SEC24A敲低组HeLa细胞2 h诱导自噬发生，经Western blot和免疫荧光实验检测自噬底物蛋白p62和自噬标志蛋白LC3-II的蛋白水平变化，以确定SEC24A是否参与自噬。通过RFP-GFP-LC3串联荧光检测自噬体和自噬溶酶体的数目，利用蛋白酶K保护实验验证自噬缺陷发生在自噬体闭合之前或者之后，利用免疫荧光实验检测敲低SEC24A对自噬通路上ATG复合物的影响，以确定SEC24A调控自噬通路的位点。通过免疫共沉淀实验验证SEC24A与自噬相关蛋白ATG9A是否存在相互作用。蛋白检测实验发现，饥饿条件下与对照细胞相比，敲低SEC24A细胞内自噬底物蛋白p62积累，而标志蛋白LC3-II减少。RFP-GFP-LC3串联荧光实验显示，敲低SEC24A后自噬体及自噬溶酶体的数目均减少。蛋白酶K保护实验显示，SEC24A敲低细胞中受膜结构保护的p62和GFP-LC3均减少,提示SEC24A作用位点在自噬体闭合之前。免疫荧光实验显示，敲低SEC24A的表达后ATG14L、ATG16L1点状结构减少，而ATG9A点状结构的数量没有明显变化，提示SEC24A作用于ATG14L、ATG16L1上游。免疫共沉淀实验显示SEC24A与ATG9A存在相互作用。研究结果不仅有助于深化对自噬体形成过程和分子机制的了解，也为全面解读COPII囊泡及其衣被蛋白在自噬中的重要作用提供了信息。

关键词: 细胞自噬, COPII囊泡, SEC24A蛋白, ATG蛋白

Abstract:

Autophagy is an important and conserved intracellular degradation process in which cellular contents are degraded by the formation of bilayered autophagosomes that encapsulate cellular contents. During starvation-induced stress， COPII vesicles are considered to be the membrane source of autophagosomes. The aim of this study was to explore the role of COPII coat protein SEC24A in mammalian macroautophagy pathway. SEC24A was knocked down by siRNA interference technology. Autophagy was induced by starvation in EBSS for 2 h. Western blot and immunofluorescence experiments were used to detect the levels of autophagy substrate protein p62 and autophagy marker protein LC3-II to determine whether SEC24A is involved in autophagy. The numbers of autophagosomes and autolysosomes were detected by RFP-GFP-LC3 tandem fluorescence assay. Proteinase K sensitivity assay was used to verify that autophagy defects occurred before or after autophagosome closure. Immunofluorescence experiments were used to detect the effect of knockdown of SEC24A on ATG complexes in the autophagy pathway to identify the site where SEC24A regulates the autophagy pathway. Co-immunoprecipitation assay verified the interaction between SEC24A and autophagy-related protein ATG9A. Under starvation conditions， knockdown of SEC24A resulted in accumulation of p62 and decreased LC3-II protein levels. RFP-GFP-LC3 tandem fluorescence experiments showed that the number of autophagosomes and autolysosomes decreased in SEC24A-depleted cells. Protease K protection assay showed reduced protection of p62 and GFP-LC3 in SEC24A-depleted cells. Immunofluorescence experiments showed that the punctate structures of ATG14L and ATG16L1 were reduced after knockdown of SEC24A， indicating that SEC24 acted on the upstream of ATG14L and ATG16L1. Co-immunoprecipitation assay showed that SEC24A interacted with ATG9A. This study not only deepened the understanding of the autophagosome formation process and molecular mechanism， but also provided information for a comprehensive interpretation of the important role of COPII vesicles and their coat proteins in autophagy.

Key words: autophagy, COPII vesicles, SEC24A protein, ATG protein

中图分类号:

Q952

赵云鹏, 张浩林, 熊倩倩, 李雨婷, 王娟. COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究[J]. 生物技术进展, 2022, 12(6): 906-914.

Yunpeng ZHAO, Haolin ZHANG, Qianqian XIONG, Yuting LI, Juan WANG. The Role of COPII Coat Protein SEC24A in Mammalian Macroautophagy[J]. Current Biotechnology, 2022, 12(6): 906-914.

图/表 5

图1 SEC24A敲低后细胞内p62和LC3变化情况A～B：qPCR和Western blot分别检测NC组和SEC24A敲低组HeLa细胞中SEC24A的转录和蛋白表达水平；C～E：Western blot检测NC组和SEC24A敲低组HeLa细胞内p62和LC3蛋白表达量，收集细胞裂解液前饥饿组细胞被EBSS处理2 h，p62（D）和LC3-II（E）表达量统计结果；F～H：细胞被饥饿处理2 h后，NC组和SEC24A敲低组HeLa细胞内p62和LC3免疫荧光结果；G～I：细胞内p62点状结构和LC3点状结构量化统计图。比例尺为5 μm，*和**分别表示在P<0.05和P<0.01水平差异具有统计学意义。

Fig. 1 Changes of intracellular p62 and LC3 after SEC24A knockdown

图2 敲低SEC24A后细胞中RFP-GFP-LC3荧光信号变化A~B：EBSS饥饿处理2 h后，对照组和SEC24A敲低组HeLa细胞中RFP-GFP-LC3荧光信号；C：细胞内隔离膜以及自噬体数量；D：细胞内自噬溶酶体数量。比例尺为5 μm，**表示差异在P<0.01水平有统计学意义。

Fig. 2 Changes in RFP-GFP-LC3 fluorescence signal in cells after knockdown of SEC24A

图3 敲低SEC24A的蛋白酶K保护实验结果A：饥饿处理2 h，Bafilomycin A1处理6 h后，敲低SEC24A蛋白酶K处理后p62被保护情况；B：被保护的p62蛋白量化统计结果；C：敲低SEC24A蛋白酶K处理后GFP-LC3被保护情况；D：被保护的GFP-LC3蛋白量化统计结果。PNS—上清液；P—沉淀；**表示差异在P<0.01水平上有统计学意义。

Fig. 3 Proteinase K assay results after knockdown of SEC24A

图4 敲低SEC24A后细胞内ATG9A、ATG14L和ATG16L1点状结构数量变化A～B：对照组（A）和SEC24A敲低组（B）HeLa细胞被EBSS饥饿处理2 h后，细胞内ATG9A、ATG14L和ATG16L1点状结构数量的变化；C～E：ATG9A（C）、ATG14L（D）和ATG16L1（E）点状结构数量统计。比例尺为5 μm, ns表示P>0.05，**表示差异在P<0.01水平上有统计学意义。

Fig. 4 Changes in the number of punctate structures of ATG9A, ATG14L and ATG16L1 in cells after SEC24A knockdown

图5 ATG9A与SEC24A免疫共沉淀结果

Fig. 5 Co-immunoprecipitation results of ATG9A and SEC24A

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COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

The Role of COPII Coat Protein SEC24A in Mammalian Macroautophagy

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