NEDD4样E3泛素蛋白连接酶通过介导果糖二磷酸A的泛素化修饰调控胶质母细胞瘤细胞铁死亡敏感性

doi:10.6040/j.issn.1671-7554.0.2025.0294

摘要/Abstract

摘要： 目的探讨神经前体细胞表达发育性下调蛋白4(neural precursor cell expressed developmentally down-regulated 4 like, NEDD4L)介导胶质母细胞瘤(glioblastoma, GBM)铁死亡的作用,揭示NEDD4L介导醛缩酶(aldolase A, ALDOA,又名果糖二磷酸 A)的泛素化修饰在铁死亡信号通路中的调控机制。方法对TCGA-GBM数据库中NEDD4L基因表达水平进行分析,利用shRNA/CRISPR-Cas9技术在GBM LN229细胞中敲低/除NEDD4L,通过细胞死亡和脂质过氧化实验观察敲低NEDD4L表达对GBM铁死亡敏感性的影响,并利用蛋白质免疫共沉淀(co-immunoprecipitation, CO-IP)、免疫印记等实验鉴定ALDOA是NEDD4L的潜在修饰底物,通过siRNA方法敲低NEDD4L或ALDOA后检测其对GBM细胞铁死亡及铁死亡通路的影响。所有数据均使用 GraphPad Prism 9.0 软件进行统计分析。结果敲除NEDD4L明显抑制IKE诱导的铁死亡发生,同时显著降低GBM细胞内的脂质过氧化水平(P<0.001),鉴定NEDD4L是ALDOA新的E3泛素化连接酶,并与ALDOA相互作用介导其单泛素化修饰,调控GBM细胞铁死亡易感性。敲低NEDD4L和ALDOA的表达均增强4EBP1和ACC的磷酸化水平以及上调GPX4蛋白的表达。结论 NEDD4L介导ALDOA的单泛素化修饰并可能通过影响mTORC1-4EBP1信号通路和脂质代谢促进GBM细胞的铁死亡敏感性。

关键词: 胶质母细胞瘤, 神经前体细胞表达发育性下调蛋白4, 果糖二磷酸A, 泛素化, 铁死亡

Abstract: Objective To investigate the role of the E3 ubiquitin ligase neural precursor cell expressed developmentally down-regulated 4 like(NEDD4L)-mediating ferroptosis in glioblastoma, aiming to elucidate molecular mechanism of NEDD4L-mediated ubiquitination of aldolase A(ALDOA)which regulates ferroptosis pathway. Methods The effects of knockdown of NEDD4L on ferroptosis in glioblastoma(GBM)were assessed through cell death and lipid peroxidation assays. ALDOA was identified as a potential substrate modified by NEDD4L using protein Western blotting and co-immunoprecipitation(Co-IP)techniques. The impact of NEDD4L or ALDOA on ferroptosis and ferroptosis-related pathways in GBM cells were further investigated using siRNA/shRNA or CRISPR-Cas9 technology. Results Knockdown of NEDD4L significantly inhibited IKE-induced ferroptosis and reduced lipid peroxidation levels in GBM cells. This study validated NEDD4L as a novel E3 ubiquitin ligase for ALDOA, demonstrating its interaction with ALDOA to mediate monoubiquitination, thereby modulating GBM cell susceptibility to ferroptosis. Additionally, the knockdown of both NEDD4L and ALDOA promoted phosphorylation of 4EBP1 and ACC, and increased the expression of GPX4 protein, which suggested that NEDD4L-mediated ubiquitination of ALDOA might regulate ferroptosis susceptibility in GBM cells through the mTORC1-4EBP1 signalling pathway and lipid metabolism. All data were statistically analyzed using GraphPad Prism 9.0 software. Conclusion NEDD4L has been shown to mediate monoubiquitination of ALDOA, with potential to promote ferroptosis sensitivity by affecting the mTORC1-4EBP1 signaling pathway and lipid metabolism in GBM cells.

Key words: Glioblastoma, Neural precursor cell expressed developmentally down-regulated 4 like, Aldolase A, Ubiquitination, Ferroptosis

中图分类号:

R574

聂秋成,李云峰,田静,刘辛靖,孙丽丽,魏义举. NEDD4样E3泛素蛋白连接酶通过介导果糖二磷酸A的泛素化修饰调控胶质母细胞瘤细胞铁死亡敏感性[J]. 山东大学学报 (医学版), 2025, 63(11): 8-17.

NIE Qiucheng, LI Yunfeng, TIAN Jing, LIU Xinjing, SUN Lili, WEI Yiju. NEDD4L regulates ferroptosis by mediating ubiquitination of ALDOA in glioblastoma cells[J]. Journal of Shandong University (Health Sciences), 2025, 63(11): 8-17.

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