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北京大学学报（医学版） ›› 2019, Vol. 51 ›› Issue (2): 197-205. doi: 10.19723/j.issn.1671-167X.2019.02.001

• 论著 •    下一篇

小鼠卡英酸颞叶癫痫慢性发作期的磷酸化蛋白组学研究

孙智明1,陈倩1,李明华1,马维宁2,赵旭阳1,3,∆(),黄卓1,∆()   

  1. 1. 北京大学药学院分子与细胞药理学系 天然药物与仿生药物国家重点实验室 系统生物医学研究所, 北京 100191
    2. 中国医科大学附属盛京医院神经外科, 沈阳 110000
    3. 北京大学基础医学院北京肿瘤系统生物学重点实验室, 北京 100191
  • 收稿日期:2017-04-27 出版日期:2019-04-18 发布日期:2019-04-26
  • 通讯作者: 赵旭阳,黄卓 E-mail:zhao-xu-yang@163.com;huangz@hsc.pku.edu.cn
  • 基金资助:
    国家重点基础研究发展计划(973 Program);国家重点基础研究发展计划(2015CB559200);国家自然科学基金(81371432);国家自然科学基金(31400695)

Chronic phosphoproteomic in temporal lobe epilepsy mouse models induced by kainic acid

Zhi-ming SUN1,Qian CHEN1,Ming-hua LI1,Wei-ning MA2,Xu-yang ZHAO1,3,∆(),Zhuo HUANG1,∆()   

  1. 1. Institute of Systems Biomedicine, State Key Laboratory of Natural and Biomimetic Drugs, Department of Molecular and Cellular Pharmacology, Peking University School of Pharmaceutical Science, Beijing 100191, China
    2. Department of Neurosurgery, Sheng Jing Hospital affiliated to China Medical University, Shenyang 110000, China
    3. Beijing Key Laboratory of Tumor Systems Biology, Peking University School of Basic Medical Science, Beijing 100191, China
  • Received:2017-04-27 Online:2019-04-18 Published:2019-04-26
  • Contact: Xu-yang ZHAO,Zhuo HUANG E-mail:zhao-xu-yang@163.com;huangz@hsc.pku.edu.cn
  • Supported by:
    the National Basic Research Program of China(973 Program);the National Basic Research Program of China(2015CB559200);National Science Foundation of China(81371432);National Science Foundation of China(31400695)

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摘要：

目的: 探究小鼠颞叶癫痫慢性发作期蛋白质功能和信号通路的改变。方法: (1)制备小鼠卡英酸颞叶癫痫模型,行为学达到Racine分级4分判定为造模成功。28 d后,取对照组和实验组小鼠海马组织进行磷酸化蛋白组学实验;(2) 选取检出密度大于10 6的数据进行统计分析;(3)利用GO(Gene Ontology)数据库、KEGG(Kyoto Encyclopedia of Genes and Genomes)数据库和STRING数据库对磷酸化蛋白组学数据进行统计分析;(4)结合文献对组学结果进行分析。 结果: (1)质谱共检测出12 697个蛋白质磷酸化位点,其中159个位点变化差异具有统计学意义(P<0.05);(2)在蛋白质功能层面,磷酸化水平显著性变化的蛋白质的分子功能主要是结合(39.5%)和催化活性(35.7%), 这些蛋白质参与细胞交流(20.8%)、初级代谢和含磷酸盐化合物代谢等生化过程;(3)在信号通路层面,这些蛋白质参与10条信号转导通路,包括谷氨酸能突触信号通路、Ras信号通路、长时程增强信号通路等;(4)在蛋白质相互作用层面,这些蛋白质形成以Grin1和Dlg3为核心,以Arhgef 2、Arhgap33和Tiam1为核心与以Spnb1/3/4、Add3和Ank2为核心的蛋白质相互作用网;(5)磷酸化蛋白组学数据显示,Grin1、Arhgef2、Arhgap33、Tiam1、Sptbn1/2/4和Ank2等磷酸化水平在癫痫慢性发作期显著升高。结论: 磷酸化蛋白组学的结果从蛋白质功能、信号通路和蛋白质相互作用3个层面阐明了小鼠颞叶癫痫慢性发作期海马体蛋白质的变化,验证了磷酸化蛋白组学研究的可靠性,并提示多巴胺功能和Kir3.1钾通道功能可能与癫痫发生相关。

关键词: 磷酸化蛋白组学, 颞叶癫痫, 癫痫发生

Abstract:

Objective: To investigate functions of proteins and signaling pathways involved in epileptogenesis during the chronic stage of temporal lobe epilepsy in mouse models.Methods: Kainic acid-induced temporal lobe epilepsy models were conducted, when reaching stage 4 using racine scale, the mice of experimental group were supposed to be successfully established. Pentobarbital sodium was injected to stop epileptic seizure in case of death. Twenty-eight days after the kainic acid injection, when the experimental group generally turned into chronic spontaneous seizures, mice hippocampal tissues were extracted from the control and the experimental groups respectively for phosphoproteomic. Enriched phosphorylated proteins were detected using mass spectrometry, only the proteins whose density was greater than 10 6 were analyzed by matching the Gene Ontology (GO) database, Kyoto Encyclopedia of Genes and Genomes (KEGG) database and STRING database to detect proteins involved in epileptogenesis in protein functions, signaling pathways and protein-protein interaction respectively. After that, literatures were reviewed about the key proteins.Results: (1) Total of 12 697 phosphorylation sites of enriched proteins were detected by mass spectrometry, and there were 159 sites whose phosphorylation levels were significantly different from the control (P<0.001). (2) GO database showed that 35.7% of the 159 sites were about “catalytic activity”, 39.5% were about “binding” and 20.8% were about “cell communication”, and the 159 proteins also participated in many biological processes, such as “primary metabolic process” “response to stimulus” “developmental process” “localization” and “phosphate-containing compound metabolic process”. (3) KEGG database showed that the 159 protein sites mainly involved in 10 signaling pathways: glutamatergic synapse, Ras signaling pathway, African trypanosomiasis, Cocaine addiction, Circadian entrainment, Amyotrophic lateral sclerosis (ALS), Long-term potentiation, Endocytosis, Gap junction, Nicotine addiction. (4) STRING database showed that the protein-protein interaction network formed by the 159 proteins was focused on Grin1/Dlg3, Arhgef 2/Arhgap33/Tiam1 and Sptnb1/3/4/Add3/Ank2 protein group respectively. (5) Phosphorylation levels of Grin1, Arhgef 2, Arhgap33, Tiam1, Sptbn1/2/4 and Ank2 in experimental group were significantly higher than in the control (P<0.001).Conclusion: Phosphoproteomic illustrated integral distribution of phosphorylated proteins at the chronic stage of temporal lobe epilepsy in the mouse model. Literatures showed that most key proteins were closely related to epileptogenesis, suggesting that some proteins or signaling pathways may play a role in epileptogenesis, such as dopamine and Kir3.1.

Key words: Temporal lobe epilepsy, Phosphoproteomic, Epileptogenesis

中图分类号: 

  • R393

表1

磷酸化蛋白组学检测数据汇总"

Items Phospho site Phospho protein Up-regulated phospho site Up-regulated phospho protein Down-regulated phospho site Down-regulated
phospho protein
Total 12 697 3 435 4 597 2 084 3 531 1 791
P<0.05 159 147 118 112 41 40

图1

GO数据库功能注释统计"

表2

信号转导通路汇总"

Signaling pathways Number of genes Percentage/% P value Benjamini
Glutamatergic synapse 7 4.7 <0.001 0.03
Ras signaling pathway 8 5.4 <0.001 0.11
African trypanosomiasis 4 2.7 <0.001 0.08
Cocaine addiction 4 2.7 <0.001 0.17
Circadian entrainment 5 3.4 <0.001 0.15
Amyotrophic lateral sclerosis (ALS) 4 2.7 <0.001 0.13
Long-term potentiation 4 2.7 <0.001 0.21
Endocytosis 7 4.7 <0.001 0.26
Gap junction 4 2.7 <0.001 0.31
Nicotine addiction 3 2 <0.001 0.35

图2

STRING数据库磷酸化蛋白相互作用网"

图3

蛋白磷酸化水平变化统计"

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