Transcriptional Activator/Coactivator Interactions

描述（由申请人提供）：最近发现人ARC（激活因子-募集辅因子）复合物是许多控制重要基因表达的转录激活因子的靶标和共激活因子。最初，发现ARC被转录激活因子SREBP-1a、VP-16和NF-κ B靶向，但最近的信息显示ARC也与其他转录激活因子相互作用，如β-连环蛋白、Smad家族成员和p53。随后，ARC辅激活因子复合物被认为是酵母介体的人类同源物。它介导的RNA聚合酶II（Pol-II）的特定基因的启动子区的结合，一方面，反式激活结构域的DNA结合的激活剂，另一方面，从事的C-末端结构域的大亚基Pol-II的招聘。该研究的目的是研究病毒和细胞转录激活因子与ARC的两个组分ARC 92和ARC 105的相互作用。这两种组分是主要转录激活因子的主要靶标，所述转录激活因子在从炎症、细胞凋亡和脂质生物合成到癌症和病毒感染的多种生物现象中发挥关键作用。 我们建议使用结构和生物化学的方法来阐明辅激活剂-激活剂招聘的分子机制。我们将讨论ARC相互作用的特异性和混杂性问题。计划中的研究将追求三个具体目标： 目标1.使用NMR光谱、细胞生物学方法和它们相互作用的小分子抑制剂对SREBP与ARC 105的相互作用进行结构和功能表征。 目标二。确定NF-κ B/ARC 105复合物的结构并用生化方法表征其意义，并寻找小分子抑制剂以测试相互作用的意义。 目标3.解析具有VP 16反式激活结构域的ARC 92复合物的结构。确定最小的ARC 92-连环蛋白结合结构域，并解决激活因子/辅激活因子复合物的结构。寻找ARC 92与VP 16和<$-连环蛋白的反式激活结构域相互作用的小分子抑制剂，并使用它们来阐明体外和体内实验中相互作用的意义。

DESCRIPTION (provided by applicant): The human ARC (activator-recruited cofactor) complex has recently been discovered as a target and co-activator of a number of transcriptional activators that control the expression of important genes. Initially, ARC was found to be targeted by the transcriptional activators SREBP-1a, VP-16 and NF-KB, but more recent information has revealed that ARC also interacts with other transcriptional activators, such as ¿-catenin, members of the Smad family and p53. Subsequently, the ARC coactivator complex was recognized as the human homolog of the yeast Mediator. It mediates recruitment of RNA polymerase II (Pol-II) to the promoter regions of specific genes by binding, on the one hand, to transactivation domains of DNA-bound activators, and on the other hand, engaging the C-terminal domain of the large subunit of Pol-II. The goal of the proposed research is to study interactions of viral and cellular transcriptional activators with two components of ARC, ARC92 and ARC105. These two components are the primary targets of prominent transcriptional activators that play pivotal roles in a wide variety of biological phenomena ranging from inflammation, apoptosis and lipid biosynthesis, to cancer and viral infection. We propose to use structural and biochemical methods for elucidating the molecular mechanisms of coactivator-activator recruitment. We will address questions of specificity and promiscuity of ARC interactions. The planned research will pursue three specific aims: Aim 1. Structural and functional characterization the SREBP interaction with ARC105 using NMR spectroscopy, cell biology approaches and small molecule inhibitors of their interaction. Aim 2. Determine the structure of the NF-KB/ARC105 complex and characterize its significance with biochemical methods and search for small-molecule inhibitors to test the significance of the interaction. Aim 3. Solve the structure of the ARC92 complex with the VP16 transactivation domain. Identify the minimal ARC92-binding domain of ¿-catenin and solve the structure of the activator/co-activator complex. Search for small-molecule inhibitors of the interaction of ARC92 with the transactivation domains of VP16 and ¿-catenin and use them to elucidate the significance of the interactions in in-vitro and in-vivo experiments.