SMAD Proteins in TGF-Beta Signaling - structural studies

描述（由申请人提供）：本申请专注于TGF-β信号传导中Smad蛋白的晶体学研究。TGF-β从细胞膜到细胞核的信号传导由Smad蛋白家族（Smads）介导，其中Smad 2和Smad 4已被鉴定为人类的肿瘤抑制因子。我们在当前资金周期内对Smad蛋白的结构研究揭示了对TGF-β信号传导的一般机制的重要见解。本论文的主要工作集中在TGF-β信号转导中尚未解决的基本问题：（1）磷酸化Smad 2-Smad 4复合物的结构和生化分析。磷酸化的Smad异聚体复合物是TGF-β信号传导中的最终功能单位。磷酸化Smad 2-Smad 4复合物的结构将通过分子置换来确定。深入了解将获得破译Smad激活的机制。(2)Smad 2-CBP和Smad 2-SnoN复合物的结构和生物化学分析。一般的转录辅激活因子CBP/p300和原癌蛋白SnoN/Ski是Smads最重要的核伙伴。了解它们与Smads的相互作用将揭示Smad介导的转录调控机制的重要见解。(3)Smad 7-Smurf 2和Smad 2-Smurf 2复合物的结构和生物化学分析。Smad蛋白通常被Smad泛素化调节因子（Smurfs）降解，并且仅响应于TGF-β信号传导而稳定。Smad- Smurf复合物的结构信息将有助于阐明Smad蛋白如何降解以及Smurf如何识别Smad与其泛素化偶联。(4)与纤溶酶原激活物抑制剂-1（派-1）启动子结合的Smad 3/Smad 4/TFE-3复合物的结构和生化分析。这种结构将最终解决TGF-β信号传导中的一个关键问题;即Smad蛋白如何与其他核伙伴合作来调节TGF-β反应基因的转录。特别是，我们将能够检查致瘤突变对活性转录复合物组装的影响。

DESCRIPTION (provided by applicant): This application focuses on the crystallographic studies of the Smad proteins in TGF-beta signaling. TGF-beta signaling from cell membrane to the nucleus is mediated by the Smad family of proteins (Smads), of which Smad2 and Smad4 have been identified as tumor suppressors in humans. Our structural studies on the Smad proteins during the current funding cycle have revealed significant insight into the general mechanisms of TGF-beta signaling. The work proposed here focuses on the fundamental unresolved issues in TGF-beta signaling: (1) Structural and biochemical analyses of a phosphorylated Smad2-Smad4 complex. A phosphorylated Smad heteromeric complex is the ultimate functional unit in TGF-beta signaling. The structure of a phosphorylated Smad2-Smad4 complex will be determined by molecular replacement. Insights will be gained to decipher the mechanism of Smad activation. (2) Structural and biochemical analyses of the Smad2-CBP and Smad2-SnoN complexes. The general transcriptional co-activator CBP/p300 and the proto-oncoprotein SnoN/Ski are among the most important nuclear partners of Smads. Understanding their interactions with Smads will reveal important insights into the mechanisms of Smad-mediated transcriptional regulation. (3) Structural and biochemical analyses of the Smad7-Smurf2 and Smad2-Smurf2 complexes. Smad proteins are routinely degraded by the Smad ubiquitination regulatory factors (Smurfs) and only stabilized in response to TGF-beta signaling. Structural information on the Smad- Smurf complex will help elucidate how Smad proteins are degraded and how Smad recognition by Smurfs is coupled to its ubiquitination. (4) Structural and biochemical analyses of Smad3/Smad4/TFE-3 complexes bound to the plasminogen activator inhibitor-1 (PAI-1) promoter. This structure will ultimately address a pivotal issue in TGF-beta signaling; that is, how Smad proteins cooperate with other nuclear partners to modulate transcription of the TGF-beta-responsive genes. In particular, we will be able to examine the effects of the tumorigenic mutations on the assembly of an active transcriptional complex.