Biophysical Characterization of PDX1 interactions with DNA and other transcriptio

描述（由申请人提供）：本项目旨在从生物药理学上表征胰腺十二指肠同源框1（PDX 1）与启动子序列和其他转录因子的相互作用。PDX 1的结构是嵌入尾部的球状同源结构域（HD），缺乏二级结构，通常研究不足。该项目的总体假设是，无序尾部在PDX 1的转录调控机制中起着核心作用;确定这些区域的作用将定义源自PDX 1突变体的糖尿病表型的分子起源。PDX 1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，并且还参与各种细胞特异性基因的调节。无序的尾巴参与介导蛋白质-蛋白质相互作用;然而，1，2它们的直接 在基因调控中的作用是不确定的。PDX 1的11个突变体导致2型3-8型糖尿病; 9个位于紊乱的尾部。这些区域已经在细胞生物学水平和通过启动子活化测定合理地表征; 导致糖尿病的PDX 1突变体的这些区域或分子细节仍然不确定。 核磁共振（NMR）光谱将用于原子表征PDX 1和突变体中所有区域的结构。NMR是由于其无序区域的高度动态特性而选择的技术。同时，等温滴定量热法（ITC）将提供灵敏和完整的热力学信息，定义PDX 1与DNA和其他蛋白质的相互作用。我们有完整的1骨架分配的未结合和结合形式的HD，这是使用传统的H 15 9-12 13和N的方法产生的。将采用新的C检测方法来表征无序区域。 15将通过测量N自旋弛豫和弛豫分散来研究所有变体和突变体的构象动力学。NMR还将用于研究PDX 1变体和突变体与DNA或其他蛋白质的相互作用。ITC将研究PDX 1与来自5个基因的11个基因启动子元件相互作用以及与其他蛋白质相互作用的热力学。基于所有ITC数据，将生成结合亲和力、化学计量、结合熵、反应自由能、热容变化和表观结合焓的目录。滴定到含有核心位点5 '-TAAT-3'和胰岛素启动子元件A3的序列中的PDX 1-HD的初步结合研究表明，在接近14，15的生理pH和与体外研究一致的Kd值下，结合化学计量为1：1。初步NMR显示HD结合形式的高质量光谱，HD的ITC数据产生定量结合参数。这里提出的全面的结构和结合研究需要更充分地表征PDX 1功能的无序区域的影响，得出的结论应该很容易推广应用到其他转录因子包含嵌入在无序的尾巴HD。

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.