崔子期 1林学洋 2孙逸贤 2杨睿娇 2李奉安 2杨玉凤 2郎思敏 2孙岚 2骆媛2
作者信息
- 1. 国家安全特需药品全国重点实验室,北京 100039||解放军总医院海南医院老年医学科,海南 三亚 572013
- 2. 国家安全特需药品全国重点实验室,北京 100039
- 折叠
摘要
Abstract
OBJECTIVE To investigate the mechanism by which E3 ubiquitin-protein ligase CBL(CBLB)regulates hypoxia inducible factor-1α(HIF-1α)through ubiquitination under hypoxic conditions,and induces neuronal injury via HIF-1α-glucose transporter 1(GLUT1)-mediated metabolic reprogram-ming.METHODS Two types of neuronal hypoxia models,namely primary hippocampal neurons and SH-SY5Y cells,were established.Key differential proteins(CBLB,HIF-1α,and GLUT1)were screened and identified using proteomic mass spectrometry analysis,RT-qPCR and Western blotting.By performing HIF-1α knockdown and GLUT1 overexpression experiments,the survival status,metabolic changes,and physiological functions of neuronal cells under hypoxic conditions were evaluated.The interactions and ubiquitination modification between CBLB and HIF-1α were verified through computer-aided virtual molecular docking,co-immunoprecipitation(Co-IP),and Western blotting.Cells were trans-fected with plasmids overexpressing CBLB and treated with the proteasome inhibitor(MG132).The effects of CBLB on the ubiquitination level and expression level of HIF-1α were analyzed via Western blotting.RESULTS Proteomic analysis and verification showed that hypoxia significantly decreased the expression of ubiquitin ligase CBLB,but markedly increased the expressions of HIF-1α and GLUT1 in primary hippocampal neurons,resulting in enhanced glycolysis,elevated lactic acid levels,as well as neuronal apoptosis and damage.Compared with the hypoxic control group,HIF-1α knockdown signifi-cantly reduced glycolysis in hypoxic neuronal cells,increased neurotransmitters,improved cell survival,and decreased cell apoptosis,which,however,could be reversed by overexpression of GLUT1 based on HIF-1α knockdown,suggesting that HIF-1α regulated neuronal function through GLUT1.Subse-quent studies demonstrated that CBLB could directly interact with HIF-1α,and mediated the degrada-tion of HIF-1α through the ubiquitin-proteasome system,thereby promoting the protein accumulation of HIF-1α in hypoxic neurons.CONCLUSION Under hypoxic conditions,the expression level of CBLB in neuronal cells is significantly decreased,reducing its ubiquitination-mediated degradation of HIF-1α and leading to the nuclear accumulation of HIF-1α.HIF-1α then upregulates the expression of its down-stream target GLUT1,which enhances glycolysis and increases lactic acid levels.These changes consequently trigger metabolic disorders and exacerbate neuronal damage.关键词
E3泛素蛋白连接酶CBL/低氧诱导因子1α/葡萄糖转运蛋白1/低氧/泛素化/神经元损伤Key words
E3 ubiquitin-protein ligase CBL/hypoxia inducible factor-1α/glucose transporter 1/hypoxia/ubiquitination/neuronal injury分类
医药卫生
