兰州大学机构库 >生命科学学院
Neuronostatin对痛觉、胃肠运动和情绪的影响及脂基化蛋白LC3-II的半合成
Alternative TitleEffects of Neuronostatin on Pain,Gastrointestinal Motility and Depression and Semisynthesis of Lipidated Protein LC3-II
杨爱民
Thesis Advisor陈强
2013-05-30
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
KeywordNeuronostatin 痛觉 胃肠运动 情绪 细胞自噬 脂基化蛋白 LC3-II 半合成 表达蛋白连接 膜融合
Abstract在小鼠甩尾实验中,侧脑室注射neuronostatin能诱导镇痛效应;在福尔马林实验中,侧脑室注射neuronostatin能诱导痛敏效应;在强迫游泳动物模型中,侧脑室注射neuronostatin诱导抑郁样行为;侧脑室注射neuronostatin抑制了胃肠运动。进一步的研究结果显示,中枢黑皮素系统、阿片系统和GABA受体参与介导neuronostatin诱导的这些生理功能。相反,未酰胺化neuronostatin不能显著影响小鼠痛觉、胃肠运动和情绪。总之,本研究表明,neuronostatin是一种具有重要生理功能的多肽,在调节痛觉、情绪和胃肠运动等方面发挥着重要的作用。Neuronostatin的C末端酰胺化修饰对它的生物活性可能是必需的。 采用了多肽合成技术和表达蛋白连接(EPL)的方法,合成了具有生物活性的脂基化蛋白LC3-II,并探究了它的膜融合特性。选择LC3蛋白中Ala114<上标!>-Ser115<上标!>为连接位点,通过重组的方法得到了MBP-LC31-114<上标!>蛋白硫酯,利用有机化学方法合成了N末端为半胱氨酸的脂肽LC3115-120<上标!>-PE,通过天然化学连接(NCL)方法,半合成得到了脂基化蛋白LC3-II。进一步的研究结果显示,Atg4B也可切除LC3-II末端的PE,这说明半合成得到的脂基化蛋白LC3-II具有生物活性。体外脂质体实验表明,脂基化蛋白MBP-LC3-II能诱导膜牵引和膜融合。总之,本研究利用化学生物学方法,成功合成了具有生物活性的脂基化蛋白LC3-II,这将成为今后研究细胞自噬的强有力的工具。
Other AbstractOur results showed that i.c.v. administrated neuronostatin produced antinociceptive effect in acute pain tail immersion assay and induced hyperalgesia in tonic pain formalin test, and delayed gastric emptying and gastrointestinal transit in mice. The pain reponse and gastrointestinal inhibitory effect were mediated by the central melanocortin system and opioid system. I.c.v. administrated neuronostatin also induced depression-like behavior in the forced swim test of mice, and the effect was dependent upon the central melanocortin system and GABA receptor. However, we found that non-amidated neuronostatin had no effect on pain response, gastrointestinal motility and depression. In summary, neuronostatin may play a role in the regulation of pain response, gastrointestinal motility and depression, and C-terminal amidation modification of neuronostatin is essential to exert its activity. In the present study, LC3-PE was synthesized by the combination of expressed protein ligation (EPL) and organic synthesis. Ala114<上标!>-Ser115<上标!> was chosed as a possible ligation site, MBP-LC31-114<上标!> thioester was prepared by intein strategy and lipidated peptide was synthesized by organic chemical techniques. LC3-PE was obtained after native chemical ligation and purification steps. The LC3-PE was completely cleaved by Atg4B, suggesting that semisynthetic LC3-PE protein is functionally active and can be further used for other studies on autophagy. Liposome assay showed semisynthetic LC3-PE induced membrane tethering and fusion. In conclusions, active LC3-PE is successful prepared by EPL, we envisage that this approach will open a new avenue in the autophagy research field.
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221925
Collection生命科学学院
Recommended Citation
GB/T 7714
杨爱民. Neuronostatin对痛觉、胃肠运动和情绪的影响及脂基化蛋白LC3-II的半合成[D]. 兰州. 兰州大学,2013.
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