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泛素连接酶Mule泛素化Miz1及其对TNFα激活JNK信号通路的调节

作　者: 仰毅
导　师: 刘新垣；Jing Liu
学　校: 浙江理工大学
专　业: 生物化学与分子生物学
关键词: TNFα JNK 泛素化 Miz1 Mule 细胞凋亡
分类号: R363
类　型: 硕士论文
年　份: 2011年
下　载: 45次
引　用: 0次
阅　读: 论文下载

内容摘要

TNFα是TNF家族中研究最为广泛的多功能细胞因子,其在多种病理过程中起着重要的作用,诸如炎症、细胞增殖、凋亡等,因此与多种的人类疾病相关比如肿瘤,感染性休克、病毒复制、骨重形成、风湿关节炎、糖尿病以及其它一些炎症相关疾病。TNFα主要诱导两条重要的信号通路:NF-κB和JNK。特别是JNK信号通路,众多的研究表明其在许多人类疾病中发挥这十分重要和复杂的作用。然而,其一些重要的调控机制的研究并不是很明确。本实验组研究发现了Miz1通过选择性的结合TRAF2并抑制其K63型泛素化抑制TRAF2的活性进而抑制TNFα诱导JNK的激活及其引起的细胞凋亡。同时也发现在TNFα诱导下Miz1自身会发生K48型泛素化-蛋白酶体介导的降解从而解除对JNK激活的抑制。在本实验中,我们发现含HECT结构域的泛素连接酶Mule特异性的诱导Miz1的泛素化降解,沉默Mule的表达导致Miz1表达量增加因而抑制TNFα诱导的细胞凋亡,异源过表达Mule则显著降低Miz1表达水平。因此我们的研究揭示了Mule通过泛素化降解Miz1调节TNFα诱导的JNK激活信号通路及其诱导的细胞凋亡,这为更进一步了解TNFα-JNK信号通路及相关疾病的治疗策略提供了一定的参考价值。

全文目录

摘要  4-5
Abstract  5-8
Introduction  8-20
  1. The TNFα signaling pathway  8-15
    1.1 The TNFα induced NF-κB signaling pathway  9-10
    1.2 The JNK signaling pathway  10-14
      1.2.1. How does TNFα induce JNK activation?  10-11
      1.2.2. JNK induces apoptosis by several ways  11-14
        1.2.2.1 JNK induces apoptosis through mitochondrial pathway  11-13
        1.2.2.2 JNK induces apoptosis by non-mitocondrial pathway  13-14
    1.3 The crosstalk between NF-κB and JNK determines TNF-α cytotoxicity  14-15
  2. Miz1 regulates the JNK signaling pathway  15-17
    2.1 Miz1 protein and its well-known funtion  15-16
    2.2 Miz1 play a role in cytoplasm by combinding to TRAF2  16-17
  3. The ubiquitination system  17-20
    3.1 The ubiquitination pathway  17-18
    3.2. HECT-Type E3 ligases  18-20
      3.2.1 General Overview of the HECT-Type E3s  18-19
      3.2.2 Mule: an important member of the Single-HECT E3s  19-20
Materials and methods  20-30
  1. Materials  20-22
    1.1 Bacteria and Plasmids  20-21
    1.2 Proteins and Adenovirus  21
    1.3 Reagent and Antibody  21
    1.4 SiRNA synthesis ans sequence  21
    1.5 Cell lines  21-22
  2.Methods  22-30
    2.1 Mass Spectrometry  22
    2.2 Cell Culture and Apoptosis Assays  22
    2.3 Plasmids preparation  22-26
      2.3.1 Mini-prep procedure  22-23
      2.3.2 Large-prep procedure  23-26
    2.4 transfection  26
      2.4.1 Gene transfection  26
      2.4.2 SiRNA transfection  26
    2.5 western blot  26-27
      2.5.1 preparation of cell extraction (6-well plate)  26-27
      2.5.2 Running gel, transferring and blocking  27
    2.6 Coimmunoprecipitation  27-28
    2.7 Purification of GST-Xpress-Miz1 protein  28-29
    2.8 In Vitro Ubiquitination Assay  29
    2.9 In Vivo Ubiquitination Assay  29-30
    2.10 Cell Fractionation  30
    2.11 Purification of His-Tagged Xpress-Miz1  30
Results  30-41
  1. Mule Is Required for TNFα-Induced Miz1 Degradation  30-33
  2. Mule Ubiquitinates Miz1 in Vitro  33-35
  3. Mule Regulates Miz1 K48-Linked Polyubiquitination and Degradation in Vivo  35-36
  4. TNFα Promotes Interaction Between Mule and Miz1  36-38
  5. Mule Regulates TNFα-Induced JNK Activation by Targeting Miz1 for Degradation  38-39
  6. Mule Regulates TNFα-Induced Cell Death  39-41
Supplemental data  41-42
Discussion  42-45
Reference  45-50
Acknowledgement  50-51

泛素连接酶Mule泛素化Miz1及其对TNFα激活JNK信号通路的调节

内容摘要

全文目录

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