DEDD与Smad3相互作用负性调控TGF-β介导的生物学活性

发布时间：2018-01-10 12:00

本文关键词：DEDD与Smad3相互作用负性调控TGF-β介导的生物学活性　出处：《中国协和医科大学》2009年博士论文　论文类型：学位论文

更多相关文章： DEDD Smad3 TGF-β 相互作用 肿瘤

【摘要】： 转化生长因子β(TGF-β)是一类对细胞增殖、分化、迁移、凋亡以及细胞外基质形成、血管形成、免疫、发育均发挥调节作用的分泌型细胞因子。该家族调节异常可以导致各种疾病发生,例如肿瘤、纤维化、自身免疫性疾病以及血管性疾病等。TGF-β家族发挥其生物学功能主要通过Smads蛋白,Smads是TGF-β家族细胞内信号传导分子。目前在哺乳动物中已经发现八种Smads蛋白,根据功能不同可分为三类：受体激活型Smads、通用介质型Smads、抑制型Smads。TGF-β信号在整个传递过程中均受到不同层面的调节,因此TGF-β家族具有生物功能多样性。细胞内多种蛋白都可与Smads相互作用,这可以促进TGF-β信号与其他信号途径发生“整合”。死亡受体结构域(Death effector domains, DEDs)是蛋白发生相互作用的区域,含有DED的蛋白可以介导由死亡受体触发的程序性细胞死亡或凋亡。DED本身没有酶活性而是作为其他DEDs的结合区域,因而促进蛋白复合体形成。FADD、caspase8以及DEDD均属于含有DED结构域的蛋白。研究表明,DEDD通过活化caspase3或capase6在CD95介导的凋亡途径中发挥重要作用。近来发现DEDD也参与细胞周期调控并抑制有丝分裂进程。然而,关于DEDD是否通过与关键的蛋白相互作用参与其他信号转导途径以及DEDD本身生物学功能尚未清楚。我们实验室前期以全长的Smad3蛋白为“钓饵”,通过酵母双杂交系统筛选出DEDD与Smad3相互作用。本次研究中,我们应用免疫共沉淀和GST-pull down方法均证明DEDD与Smad3可以发生相互作用,这是首次报道Smad3与含有DED结构域蛋白之间的相互作用。我们发现TGF-β可以下调DEDD与Smad3的结合,而且DEDD与磷酸化的Smad3结合能力减弱。双荧光素酶报告基因实验证明DEDD通过其DED结构域可以抑制TGF-β/Smad3介导的转录活性,而且Smad3可以协同增强DEDD对NF-κB基因的转录。细胞免疫荧光染色和Western blotting实验证明DEDD可以抑制Smad3入核作用。免疫沉淀实验证明DEDD可以抑制Smad3磷酸化以及Smad3与Smad4复合体形成作用。Western blotting和RT-PCR实验证明DEDD抑制TGF-β靶基因的转录和翻译,而且TGF-β抑制DEDD蛋白表达和基因转录。细胞划痕实验和Transwell实验证明DEDD抑制TGF-β介导的细胞迁移和侵袭作用,而且DEDD本身也具有抑制细胞迁移和侵袭作用。Annexin V-FITC细胞凋亡检测实验和MTT实验证明DEDD本身可以诱导细胞凋亡和生长阻滞。最后,我们应用临床结肠癌病人组织标本进行免疫组化分析得到：与正常组织相比,DEDD蛋白表达在结肠癌病人组织下降。总之,我们的结果表明通过DEDD与Smad3的相互作用在TGF-β信号途径和CD95途径之间建立崭新的联系,DEDD成为TGF-β信号途径的抑制因素。
[Abstract]:Transforming growth factor 尾 (TGF- 尾) is a kind of cell proliferation, differentiation, migration, apoptosis and extracellular matrix formation, angiogenesis, immune. A secreted cytokine that plays a regulatory role in development. Abnormal regulation in this family can lead to various diseases such as tumours and fibrosis. Autoimmune diseases and vascular diseases. TGF- 尾 family play its biological function mainly through Smads protein. Smads is a signal transduction molecule of TGF- 尾 family. At present, eight Smads proteins have been found in mammals, which can be classified into three types according to their functions: receptor-activated Smads. The signal of Smads, Smads.TGF- 尾 is regulated by different levels during the whole process of transmission. Therefore, TGF- 尾 family has a variety of biological functions. Many proteins can interact with Smads, which can promote the "integration" of TGF- 尾 signal with other signaling pathways. Death effector domain (DEDs) is a region in which proteins interact with each other. Proteins containing DED can mediate programmed cell death or apoptosis triggered by death receptors. DED itself has no enzyme activity but acts as a binding region of other DEDs. Therefore, promoting the formation of protein complex. FADDD caspase8 and DEDD belong to the protein containing DED domain. DEDD plays an important role in CD95 mediated apoptosis by activating caspase3 or capase6. Recently, it has been found that DEDD also participates in cell cycle regulation and inhibits mitosis. ... but... It is not clear whether DEDD participates in other signal transduction pathways by interacting with key proteins and the biological function of DEDD itself. In our laboratory, the full-length Smad3 protein was used as "bait", and the interaction between DEDD and Smad3 was screened by yeast two-hybrid system. We used both immunoprecipitation and GST-pull down methods to prove that DEDD and Smad3 could interact with each other. This is the first report on the interaction between Smad3 and DED domain proteins. We found that TGF- 尾 can down-regulate the binding of DEDD to Smad3. Moreover, the binding ability of DEDD to phosphorylated Smad3 was weakened. Double luciferase reporter gene experiment showed that DEDD could inhibit transcriptional activity mediated by TGF- 尾 / Smad3 through its DED domain. Sex. Moreover, Smad3 can enhance the transcription of NF- 魏 B gene by DEDD. Cellular immunofluorescence staining and Western. Blotting assay showed that DEDD could inhibit the nucleation of Smad3, and immunoprecipitation assay showed that DEDD could inhibit the phosphorylation of Smad3 and the Smad3 / Smad4 complex. Forming action. Western. Blotting and RT-PCR experiments demonstrated that DEDD inhibited the transcription and translation of TGF- 尾 target genes. Moreover, TGF- 尾 inhibited DEDD protein expression and gene transcription. Cell scratch assay and Transwell assay demonstrated that DEDD inhibited TGF- 尾 -mediated cell migration and invasion. And DEDD itself can inhibit cell migration and invasion. Annexin. V-FITC cell apoptosis assay and MTT assay proved that DEDD itself can induce apoptosis and growth arrest. Finally. We used clinical colon cancer tissue samples for immunohistochemical analysis: compared with normal tissues, the expression of DDEDD protein in colon cancer patients decreased. Our results show that a new relationship between TGF- 尾 signaling pathway and CD95 pathway is established by the interaction of DEDD and Smad3. DEDD is a factor that inhibits TGF- 尾 signaling pathway.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R363

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