SMAD Proteins in TGF-Beta Signaling - structural studies

TGF-β 信号转导中的 SMAD 蛋白 - 结构研究

• 批准号: 6861021
• 负责人: YIGONG SHI
• 金额: $ 26.27万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6861021
• 关键词: DNA binding protein; SDS polyacrylamide gel electrophoresis; biological signal transduction; crystallization; genetic transcription; mass spectrometry; nuclear proteins; oncoproteins; phosphorylation; plasminogen activator inhibitors; protein degradation; protein protein interaction; protein purification; protein structure function; three dimensional imaging /topography; transcription factor; transforming growth factors

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.

描述(申请人提供)：本申请侧重于转化生长因子-β信号转导中Smad蛋白的结晶学研究。转化生长因子-β信号从细胞膜到细胞核是由Smad家族蛋白(Smad2和Smad4)介导的，其中Smad2和Smad4在人类中已被发现是肿瘤抑制因子。我们在当前资金周期中对Smad蛋白的结构研究揭示了对转化生长因子-β信号转导的一般机制的重要见解。本文的工作集中于转化生长因子-β信号转导中尚未解决的基本问题：(1)磷酸化的Smad2-Smad4复合体的结构和生化分析。磷酸化的Smad异构体复合体是转化生长因子-β信号转导的最终功能单位。磷酸化的Smad2-Smad4复合体的结构将通过分子置换来确定。我们将对Smad激活的机制进行深入的解读。(2)Smad2-CBP和Smad2-SnoN络合物的结构和生化分析。通用转录共激活因子CBP/p300和原癌蛋白SnoN/Ski是SMADS最重要的核合作伙伴。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)Smad7-SMurf2和Smad2-SMurf2复合体的结构和生化分析。Smad蛋白通常被Smad泛素化调节因子(SMurf)降解，并且只有在响应转化生长因子-β信号时才稳定下来。关于Smad-蓝精灵复合体的结构信息将有助于阐明Smad蛋白质是如何降解的，以及蓝精灵对Smad的识别是如何与其泛素化相结合的。(4)与纤溶酶原激活物抑制物-1(PAI-1)启动子结合的Smad3/Smad4/TFe-3复合体的结构和生化分析。这种结构最终将解决转化生长因子-β信号转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转化生长因子-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体组装的影响。