泛素连接酶Mule泛素化Miz1及其对TNFα激活JNK信号通路的调节

论文摘要

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。

论文目录

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