Mechanistic Studies of Progesterone Receptor Function

黄体酮受体功能的机制研究

• 批准号: 6572902
• 负责人: DAVID L BAIN
• 金额: $ 22.85万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6572902
• 关键词: DNA binding protein; DNA footprinting; analytical ultracentrifugation; circular dichroism; eukaryote; fluorescence spectrometry; gene induction /repression; genetic promoter element; genetic regulation; hormone inhibitor; microcalorimetry; molecular assembly /self assembly; progesterone receptors; protein isoforms; protein protein interaction; receptor binding; sedimentation equilibrium; thermodynamics; transcription factor

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular mechanisms underlying eukaryotic gene regulation. Focus is centered on the mechanisms by which human progesterone receptors (PR) cooperatively bind to complex promoters, and the role of hormone agonists and antagonists in regulating these reactions. A further goal is to determine the principles by which the associated structural transitions are propagated to neighboring domains. PR co-exist as two functionally distinct isoforms: an 83 kD A-receptor and a 99 kD B-receptor. The two isoforms are identical, except that the B-receptor has an additional 164 amino acids at its N-terminus. The B-receptor often functions as a strong transcriptional activator, while the A-receptor generally acts as a weak activator. It is hypothesized that this difference arises through the ability of the B-receptor to bind cooperatively at PR-regulated promoters. Mechanistically, the B-unique residues impose a hormone-dependent conformational constraint upon the remainder of the receptor. This constraint causes changes in PR structure and stability changes that can include dramatic disorder-order transitions, resulting in cooperative DNA binding. It is proposed that a role of antagonists is to decouple these linkages by stabilizing ineffective conformations within the PR hormone-binding domain. This hypothesis, and the underlying mechanism, will be examined by carrying out the following studies: Aim 1 - The energetics of self-assembly for both isoforms in the presence and absence of progestin agonists and antagonists will be determined using analytical ultracentrifugation. Aim 2 - A rigorous thermodynamic analysis of the interactions of each PR isoform with the multi-site mouse mammary tumor virus promoter will be determined using quantitative DNAse footprinting. Aim 3 - The changes in isoform structure and stability associated with ligand and DNA-binding will be mapped using hydroxyl radical proteolytic footprinting, CD spectroscopy and microcalorimetry.

描述（由申请人提供）：本研究的长期目标是阐明真核基因调控的分子机制。重点关注人类孕酮受体 (PR) 与复杂启动子协同结合的机制，以及激素激动剂和拮抗剂在调节这些反应中的作用。进一步的目标是确定相关结构转变传播到相邻域的原理。 PR 作为两种功能不同的亚型共存：83 kD A 受体和 99 kD B 受体。这两种异构体是相同的，只是 B 受体的 N 末端多了 164 个氨基酸。 B 受体通常充当强转录激活剂，而 A 受体通常充当弱激活剂。据推测，这种差异是由于 B 受体协同结合 PR 调节的启动子的能力而产生的。从机制上讲，B-独特残基对受体的其余部分施加激素依赖性构象限制。这种限制会导致 PR 结构的变化和稳定性的变化，其中可能包括剧烈的无序转变，从而导致协同 DNA 结合。有人提出拮抗剂的作用是通过稳定 PR 激素结合域内的无效构象来解耦这些连接。将通过进行以下研究来检验这一假设及其潜在机制： 目标 1 - 将使用分析超速离心确定在存在和不存在孕激素激动剂和拮抗剂的情况下两种亚型的自组装能量学。目标 2 - 将使用定量 DNAse 足迹法确定每种 PR 同工型与多位点小鼠乳腺肿瘤病毒启动子相互作用的严格热力学分析。目标 3 - 将使用羟自由基蛋白水解足迹、CD 光谱和微量热法绘制与配体和 DNA 结合相关的异构体结构和稳定性的变化。