Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1
Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1
复制标题
蛋白酶 Omi 通过裂解转录因子 E2F1 促进小鼠神经母细胞瘤 N2a 细胞的神经突生长
DOI：
10.1038/aps.2015.48
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发表时间：
2015-08-01
影响因子：
8.2
通讯作者：
Wang, Guang-hui
中科院分区：
文献类型：
--
作者：
Ma, Qi;Hu, Qing-song;Wang, Guang-hui
目的：OMI是一种不依赖于ATP的丝氨酸蛋白酶，是神经元功能和生存所必需的。本研究的目的是探讨OMI对小鼠神经母细胞瘤细胞分化的调控作用，并确定参与该过程的底物。方法：以小鼠神经母细胞瘤N2a细胞和OMI蛋白酶缺陷的mnd2小鼠为研究对象。为调节Omi和E2F1的表达，将表达载体、shRNA和siRNA分别导入N2a细胞。用免疫印迹法检测蛋白质水平。用免疫沉淀法、GST下拉试验和体外切割试验研究了Omi与E2F1的相互作用。用20MU/L维甲酸(RA)和1%胎牛血清诱导N2a细胞突起生长，并用Image J软件进行检测。结果：OMI基因敲除细胞和mnd2小鼠脑裂解产物中E2F1显著增加，而野生型OMI高表达细胞中E2F1明显减少，而非活性OMI S276C细胞中E2F1表达无明显变化。在mnd2小鼠脑裂解产物中，内源性E2F1与内源性Omi共沉淀。体外切割实验表明，Omi直接切割E2F1。用维甲酸处理N2a细胞后，N2a细胞分化明显，轴突生长明显，OMi显著增加，E2F1水平显著降低，而OMi特异性抑制剂UCF-101可抑制这一作用。N2a细胞中Omi基因的敲除抑制了RA诱导的轴突生长，这种作用可被E2F1基因敲除部分恢复。结论：蛋白水解酶Omi通过裂解分化的神经母细胞瘤细胞中的转录因子E2F1促进轴突生长，E2F1是Omi的底物。
Aim: Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process.Methods: Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 mu mol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software.Results: E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1.Conclusion: Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.