Biophysical Characterization of PDX1 interactions with DNA and other transcriptio

描述(申请人提供)：这个项目旨在从生物物理学的角度描述胰腺十二指肠同源框1(PDX1)与启动子序列和其他转录因子的相互作用。PDX1的结构是嵌入在缺乏二级结构的尾巴中的球形同源结构域(HD)，通常研究不足。这个项目的总体假设是，无序的尾巴在PDX1转录调控机制中起着中心作用；确定这些区域的作用将定义糖尿病表型的分子起源，这些表型起源于PDX1突变。PDX1是一种含有HD的转录因子，被发现是调节胰岛素转录的核心，也参与了各种细胞特异性基因的调节。无序的尾巴参与调节蛋白质-蛋白质的相互作用；然而，1，2它们的直接 基因调控中的角色是不确定的。11个PDX1突变导致2型3-8型糖尿病；9个位于无序的尾巴内。这些区域已经在细胞生物学水平上得到了合理的表征，并通过启动子激活分析； 导致糖尿病的PDX1突变的这些区域或分子细节仍未确定。核磁共振波谱将被用来对PDX1和突变体的所有区域的结构进行原子学表征。核磁共振是由于其无序区域的高度动态特征而被选择的技术。同时，等温滴定量热法(ITC)将提供敏感和完整的热力学信息，定义PDX1与DNA和其他蛋白质的相互作用。我们已经完成了HD的未绑定和绑定形式的1个主干任务，这些任务是使用传统的H 15 9-12 13和N方法生成的。将使用新的C检测方法来表征无序区域。15通过测量N自旋驰豫和弛豫色散来研究所有变体和突变体的构象动力学。核磁共振也将被用来研究PDX1变异体和突变体与DNA或其他蛋白质的相互作用。用ITC研究PDX1与5个基因的11个基因启动子元件的相互作用以及与其他蛋白质的相互作用的热力学。基于ITC的所有数据，将生成结合亲和力、化学计量比、结合熵、反应自由能、热容变化和表观结合热的目录。PDX1-HD滴定为含有核心部位5‘-Taat-3’的序列与胰岛素启动子元件A3的初步结合研究表明，在接近14，15生理pH和Kd值的情况下，结合化学计量比为1：1，与体外研究一致。初步的核磁共振显示了高质量的HD结合形式的光谱，并且HD的ITC数据得到了定量的结合参数。为了更全面地表征无序区对PDX1功能的影响，需要进行全面的结构和结合研究，所得出的结论应易于推广应用于其他含有HD的转录因子。

DESCRIPTION (provided by applicant): This project aims to biophysically characterize the interactions of pancreas duodenum homeobox 1 (PDX1) with promoter sequences and other transcription factors. The structure of PDX1 is that of a globular homeodomain (HD) embedded within tails that lack secondary structure and that are generally under-studied. The overall hypothesis of this project is that the disordered tails serve a central function in the mechanism o transcriptional regulation by PDX1; establishing the role of these regions will define the molecular origins of diabetic phenotypes originating from PDX1 mutants harbored within them. PDX1 is a HD-containing transcription factor found to be at the core of regulating insulin transcription, and that is also involved in the regulation of various ¿ -cell specific genes. The disordered tails are involved in mediating protein-protein interactions; however, 1,2 their direct roles in gene regulation are indeterminate. Eleven mutants of PDX1 result in a form of type 2 3-8 diabetes; 9 are located within the disordered tails. These regions have been reasonably characterized at the cell biology level and through promoter activation assays; the direct roles of these regions or the molecular details of PDX1 mutants that result in diabetes remain undefined. Nuclear Magnetic Resonance (NMR) spectroscopy will be used to the atomistically characterize the structure of all regions in PDX1 and mutants. NMR is the technique of choice due to the highly dynamic character of its disordered regions. Simultaneously, Isothermal Titration Calorimetry (ITC) will provide sensitive and complete thermodynamic information defining PDX1 interactions with DNA and other proteins. We have complete 1 backbone assignments of the unbound and bound forms of the HD, which were generated using traditional H 15 9-12 13 and N methods. Novel C detection methods will be employed to characterize the disordered regions. 15 Conformational dynamics of all variants and mutants will be studied by measuring the N spin relaxation and relaxation dispersion. NMR will also be utilized to study the interactions of PDX1 variants and mutants with DNA or other proteins. The thermodynamics of PDX1 interactions with 11 gene promoter elements from 5 genes and interactions with other proteins will be studied by ITC. Based on all ITC data, a catalogue of the binding affinity, stoichiometry, entropy of binding, free energy of the reaction, change in heat capacity and apparent binding enthalpy will be generated. Preliminary binding studies of the PDX1-HD titrated into a sequence containing the core site, 5'-TAAT-3', and the insulin promoter element A3 demonstrate a 1:1 binding stoichiometry at near 14,15 physiological pH and Kd values consistent with in vitro studies. Preliminary NMR revealed a high quality spectrum of the bound form of the HD and ITC data of the HD yielded quantitative binding parameters. The comprehensive structural and binding studies proposed here are needed to more fully characterize the impact of the disordered regions on PDX1 function and the conclusions drawn should be readily generalized for application to other transcription factors containing HD embedded in disordered tails.