瘢痕疙瘩中DAB-1抑制E3连接酶SIAH1对TIEG1泛素化介导TGF-β/Smads信号通路的研究

项目名称： 瘢痕疙瘩中DAB-1抑制E3连接酶SIAH1对TIEG1泛素化介导TGF-β/Smads信号通路的研究

项目编号： No.81471875

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 医药、卫生

项目作者： 朱家源

作者单位： 中山大学

项目金额： 72万元

中文摘要： TGF-β1/Smads与瘢痕疙瘩形成密切相关。我们既往工作证实高水平的TIEG直接抑制Smad7的表达，介导了TGF-β1/Smads通路的激活。然而，TIEG1上调的机制不清楚。前期工作观察到仅TIEG1蛋白水平上调，而mRNA无变化；进一步证实泛素降解系统异常导致TIEG1上调，并筛选到介导TIEG1泛素化的E3连接酶SIAH1；运用酵母双杂交技术筛查到一个SIAH1相互作用蛋白DAB-1；DAB-1在瘢痕疙瘩中高表达，过表达DAB-1可拮抗TIEG1与SIAH1相互作用。因此，我们推测：瘢痕疙瘩中DAB-1通过蛋白相互作用拮抗了SIAH-1与TIEG1结合，导致TIEG1泛素降解异常而蛋白水平上调，从而介导TGF-β1/Smads通路。本课题将采用缺失突变、FRET及免疫共沉淀等技术，探讨DAB-1通过抑制SIAH1对TIEG1泛素化，介导TGF-β/Smads通路激活的具体机制。

中文关键词： DAB-1；SIAH1；TIEG1；泛素化；TGF-β/Smads

英文摘要： The sustained activation of TGF-β1/Smads signal transduction pathway is the most important mechanism for the formation of keloids. Our study found that the expression of Smad7, which is the most important negative feedback signaling molecule was significantly down regulated by the transcription inhibitive factor TIEG1, which is up-regulated in keloid. However, the mechanism of TIEG1 up-regulated is not clear. Our study found that: Only the TIEG1 protein is up-regulated, the TIEG1 mRNA level stay in the same; We screened a new E3 ubiquitin ligase SIAH1 mediating the ubiquitination degration of TIEG1; Using Yeast-two hybrid technique and generating SIHA-1 plasmid as bait protein, we screened a interacted protein DAB-1 in the human fetal liver cDNA library; DAB-1 is up regulated in keloid. TIEG1 is up regulated by silencing DAB-1 gene via siRNA. As a result, we supposed that DAB-1 affects the activity of SIAH-1 through protein-protein interaction in keloid, suppressing the ubiquitination of TIEG1, resulting in the up-regulation of TIEG1.and the sustained activation of TGF-β1/Smads signal transduction pathway. In this study, we will take some molecular biology technique, such as RT-PCR, western blotting, immunofluorescence, GST pull-down to acquire evidence that DAB-1 inhibits the activity of ubiquitination of SIAH-1 through protein-protein interaction to suppress the degradation of TIEG1, and to observe the biological behaviour of keloid under DAB-1 control in vivo and vitro. So we could provide a new target for preventing and curing keloids, moreover, provide a new clue and theory.

英文关键词： DAB-1;SIAH1;TIEG1;ubiquitin;TGF-β/Smads