核磁共振波谱研究转录抑制复合物Sin3A-TGIF1-Smad2组装的分子机制

转化生长作用因子TGIF1是一种重要的转录调控蛋白，对TGF-β等信号传导中的响应基因起转录抑制作用，其突变会诱发胎儿脑部发育缺陷以及多种癌症。TGIF1的转录抑制功能依赖于其C端RD2结构域与Sin3A和Smad2等蛋白的相互作用。RD2主要为无序结构，离体蛋白样品在溶液中构象并不均一，且易聚集或降解，严重阻碍了构效关系研究，TGIF1介导转录抑制或诱发疾病的分子机制目前仍不清楚。本项目将应用液体NMR方法深入研究TGIF1-RD2分别与Sin3A-PAH2和Smad2-MH2所形成的复合物结构，以及互作时构象变化特征和动力学性质，探索三元复合物Sin3A-TGIF1-Smad2的组装机理，并表征其整体结构。本项目的研究结果，将加深对于TGF-β信号中转录抑制作用的分子机理的认识，为理解TGIF1相关疾病的诱发机制提供理论依据，为相应疾病的药物筛选或设计提供靶标蛋白的结构基础。

Transforming growth-interacting factor 1 (TGIF1) is a key transcription factor that represses the expression of responsive genes induced by multiple signaling pathways, such as TGF-β. TGIF1 mutations in human embryos were found to be associated with developmental defects of fetal brain and face, and abnormal high expression of TGIF1 could lead to cancers. The repressive role of TGIF1 on the transcription of TGF-β-responsive genes depends on its C-terminal repressive domain 2 (RD2), which can bind with Smad2, the core transcription regulator of TGF-β signaling, and recruit Sin3A, the general transcription co-repressor. Our study has evidenced that TGIF1-RD2 is disordered in solution, and exists in more than one conformation. Due to the lack of structural information of TGIF1-RD2, the molecular mechanisms of TGIF1-mediated transcriptional repression and TGIF1-associated diseases are still largely unclear. In this proposal, NMR spectroscopy will be used to investigate the solution structure of TGIF1-RD2, and to solve the structures of TGIF1-RD2 in complex with Sin3A-PAH2 and Smad2-MH2 domains, as well multimeric state, conformational change and exchange rate of TGIF1-RD2 when it binds to Sin3A-PAH2 and Smad2-MH2. Furthermore, the interactions among TGIF1-RD2, Sin3A-PAH2 and Smad2-MH2 in their ternary complex, as well as the entire structure of the ternary complex will be explored. The expected results from this proposal will elucidate the assembly mechanism of the transcriptional repressive complex Sin3A-TGIF1-Smad2, lead to better understanding for the repressive role of TGIF1 in TGF-β signaling, and will provide information for the induction of TGIF1-associated diseases and structural basis for drug development targeting Sin3A-TGIF1-Smad2 complex.