Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro.
Bcl-2-dependent upregulation of autophagy by sequestosome 1/p62 in vitro.
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体外隔离体 1/p62 对 Bcl-2 依赖性自噬的上调
DOI：
10.1038/aps.2013.12
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发表时间：
2013-05
影响因子：
8.2
通讯作者：
Wang, Guang-hui
中科院分区：
文献类型：
--
作者：
Zhou, Liang;Wang, Hong-feng;Ren, Hai-gang;Chen, Dong;Gao, Feng;Hu, Qing-song;Fu, Chen;Xu, Ran-jie;Ying, Zheng;Wang, Guang-hui
目的：探讨在泛素-蛋白酶体系统和自噬-溶酶体系统中起重要作用的转运蛋白-螯合体1/p62（sequestosome 1/p62，p62）是否能直接调控细胞自噬。随后对细胞或细胞裂解物进行免疫荧光测定、免疫沉淀测定或免疫印迹分析。结果：p62过表达可显著增加HEK 293细胞和HeLa细胞自噬的基础水平，而p62敲低可显著降低HEK 293细胞自噬的基础水平。体外pulldown实验显示p62与Bcl-2直接相互作用。在HeLa细胞中观察到p62与Bcl-2共定位。结论：p62通过破坏Bcl-2与Beclin 1的结合而诱导自噬。大细胞自噬（以下简称自噬）主要参与底物蛋白或细胞器如线粒体2的降解。自噬的第一步是形成称为自噬体的双膜结构（包括起始和囊泡成核），第二步是自噬体的扩张和关闭，最后一步是自噬体与溶酶体融合。最终，底物在溶酶体中降解3、4、5。以前的报告表明，自噬与肿瘤发生，衰老和许多与衰老相关的疾病有关6，7。抑制神经元细胞中的自噬可导致小鼠神经变性8，9。此外，刺激自噬的药物，如雷帕霉素酯替西罗莫司，可以改善患有脊髓小脑共济失调3型的小鼠的运动表现10。
Aim:To investigate whether sequestosome 1/p62 (p62), a key cargo adaptor protein involved in both the ubiquitin-proteasome system and the autophagy-lysosome system, could directly regulate autophagy in vitro.Methods:HEK 293 cells or HeLa cells were transfected with p62-expressing plasmids or siRNA targeting p62. The cells or the cell lysates were subsequently subjected to immunofluorescence assay, immunoprecipitation assay, or immunoblot analysis. In vitro pulldown assay was used to study the interaction of p62 with Bcl-2.Results:Overexpression of p62 significantly increased the basal level of autophagy in both HEK 293 cells and HeLa cells, whereas knockdown of p62 significantly decreased the basal level of autophagy. In vitro pulldown assay showed that p62 directly interacted with Bcl-2. It was observed in HeLa cells that p62 co-localized with Bcl-2. Furthermore, knockdown of p62 in HEK 293 cells significantly increased the amount of Beclin 1 that co-immunoprecipitated with Bcl-2.Conclusion:p62 induces autophagy by disrupting the association between Bcl-2 and Beclin 1.IntroductionThe ubiquitin-proteasome system and autophagy-lysosome system are the two major degradation systems for cell homeostasis 1. Macroautophagy (hereafter referred to as autophagy) is primarily involved in the degradation of substrate proteins or organelles such as mitochondria 2. The first step of autophagy is the formation of a double-membrane structure called the autophagosome (including initiation and vesicle nucleation), the second step is autophagosome expansion and closure, and the final step is autophagosome fusion with the lysosome. Ultimately, substrates are degraded in the lysosome 3, 4, 5. Previous reports have suggested that autophagy is implicated in tumorigenesis, aging and many aging-associated diseases 6, 7. Suppression of autophagy in neuronal cells can cause neurodegeneration in mice 8, 9. Additionally, drugs that stimulate autophagy, such as rapamycin ester temsirolimus, can improve the motor performance of mice with spinocerebellar ataxia type 3 10.
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影响因子：
4.7
作者：
Babu, JR;Geetha, T;Wooten, MW
通讯作者：
Wooten, MW
p62/SQSTM1形成自噬降解的蛋白质聚集体，并对亨廷顿蛋白诱导的细胞死亡具有保护作用。
影响因子：
7.8
作者：
Bjorkoy, Geir;Lamark, Trond;Brech, Andreas;Outzen, Heidi;Perander, Maria;Overvatn, Aud;Stenmark, Harald;Johansen, Terje
通讯作者：
Johansen, Terje
影响因子：
4.7
作者：
Gal J;Ström AL;Kwinter DM;Kilty R;Zhang J;Shi P;Fu W;Wooten MW;Zhu H
通讯作者：
Zhu H
影响因子：
7.7
作者：
Lee, Sang Jun;Pfluger, Paul T.;Moscat, Jorge
通讯作者：
Moscat, Jorge
影响因子：
16
作者：
Korolchuk, Viktor I.;Mansilla, Alicia;Menzies, Fiona M.;Rubinsztein, David C.
通讯作者：
Rubinsztein, David C.