文章摘要
盛蕾,蒋凤仙,戴俊.脊髓性肌萎缩症中运动神经元生存蛋白缺失抑制p53泛素化降解致其积累的研究.骨科,2023,14(3): 281-287.
脊髓性肌萎缩症中运动神经元生存蛋白缺失抑制p53泛素化降解致其积累的研究
Survival Motor Neuron Deficiency Induces p53 Accumulation Through Inhibiting Ubiquitination Degradation in Spinal Muscular Atrophy
投稿时间:2023-02-26  
DOI:10.3969/j.issn.1674-8573.2023.03.016
中文关键词: 脊髓性肌萎缩症  运动神经元生存蛋白  p53  泛素化降解
英文关键词: Spinal muscular atrophy  Survival motor neuron  p53  Ubiquitination degradation
基金项目:国家自然科学基金青年项目(81902179);中国博士后基金(2020T130308)
作者单位E-mail
盛蕾 苏州大学附属第二医院骨科江苏苏州 215004  
蒋凤仙 苏州大学附属第二医院骨科江苏苏州 215004  
戴俊 苏州大学附属第二医院骨科江苏苏州 215004 daijun@suda.edu.cn 
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中文摘要:
      目的 探究脊髓性肌萎缩症(spinal muscular atrophy,SMA)中p53积累的分子机制。方法 构建运动神经元生存基因(Survival motor neuron,Smn)敲低的稳转小鼠运动神经元细胞系NSC34,采用蛋白质印迹和荧光定量PCR检测蛋白和基因的变化;用蛋白合成抑制剂放线菌酮(Cycloheximide,CHX)抑制蛋白合成,蛋白质印迹检测p53蛋白降解情况,采用免疫共沉淀检测p53与MDM2结合情况;采用免疫荧光检测小鼠脊髓组织运动神经元数量,运用流式细胞仪分析细胞周期的分布。结果 在Smn敲低的NSC34细胞中,p53蛋白水平显著增加,但是其基因水平未发生明显变化;当蛋白合成被抑制后,SMN缺失会抑制p53蛋白降解,p53积累后会调控下游MDM2表达显著增加,但是其与p53结合却显著减少;p-p53(Ser18)和acetyl-p53(K382)水平显著升高,K382乙酰化位点会影响p53泛素化结合,导致其泛素化被抑制;最终p53积累导致NSC34细胞和SMA小鼠脊髓组织中细胞周期阻滞和凋亡增加。结论 SMN蛋白缺失通过抑制p53泛素化降解途径导致其积累,进而引起细胞周期阻滞和凋亡。
英文摘要:
      Objective To explore the underlying mechanism of p53 accumulation in spinal muscular atrophy (SMA). Methods Stable cell line NSC34 with depleted survival motor neuron (Smn) was constructed. Western blotting and quantitative real-time PCR were used to detect protein and gene expression. Protein synthesis inhibitor (cycloheximide, CHX) was utilized to inhibit protein synthesis and p53 protein degradation was detected by Western blotting. Co-IP was performed to examine the interaction between p53 and MDM2. Immunofluorescence staining was used to observe motor neurons of spinal cord in SMA mouse model and p-p53 (Ser18) level in NSC34 cells. The distribution of cell cycle was analyzed by flow cytometry. Results Smn knockdown significantly increased p53 protein level, but not resulting from p53 transcriptional level changes. Smn deficiency inhibited p53 degradation using protein synthesis inhibitor CHX. Furthermore, MDM2 was significantly increased, whereas it was suppressed to interact with p53. Moreover, Smn ablation induced activation of the phosphorylation (Ser18) and acetylation (K382) of p53 which can be acetylated and ubiquitinated at the C terminus of p53. Finally, p53 accumulation led to cell cycle arrest and apoptosis in NSC34 cells and spinal cord of SMA mouse model. Conclusion Smn deficiency resulted in the accumulation of p53 by inhibiting the degradation pathway, leading to cell cycle arrest and apoptosis.
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