Quantitative Analysis of AIB-1 Recruitment

AIB-1招募的定量分析

• 批准号: 7313402
• 负责人: DAVID L BAIN
• 金额: $ 19.25万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313402
• 关键词: Affect; Amino Acids; Biological Models; Breast; Chemicals; Complex; Coupled; DNA; DNA Sequence Rearrangement; Data; Deoxyribonuclease I; Deoxyribonucleases; Disease; Dissection; Endometrial Carcinoma; Free Energy; Gene Expression; Gene Expression Regulation; Goals; Hormones; Human; Ligands; Mass Spectrum Analysis; Microscopic; Molecular; NMR Spectroscopy; Nuclear Receptor Coactivator 3; Nuclear Receptors; Pharmaceutical Preparations; Progesterone; Progesterone Receptors; Protein Isoforms; Proteins; Proteolysis; Reaction; Recruitment Activity; Research; Resolution; Structure; Titrations; activating transcription factor; analytical ultracentrifugation; base; driving force; drug development; member; predictive modeling; promoter; receptor; receptor function

DESCRIPTION (provided by applicant): The long-term goal of this research is to determine the mechanisms underlying eukaryotic gene regulation. Human progesterone receptors (PR), members of the nuclear receptor superfamily of ligand-activated transcription factors, serve as a model system. 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. Functional differences seen between the two proteins may be due to differential interactions with transcriptional coactivating proteins such as AIB1 (Amplified in Breast Cancer-1). Our studies indicate that residues unique to the B-receptor allosterically modulate recruitment of AIB1 to the promoter complex, thus modulating isoform-specific function. Further, these interactions are coupled to dramatic increases in structural stability due to large-scale disorder to order folding transitions. The goal of this proposal is to carry out a biophysical and structural dissection of the interactions between PR isoforms and the coactivating protein, AIB1, on a PR-regulated promoter. Aim 1a: We will resolve the microscopic free energy contributions associated with isoform-specificAIB1 recruitment using quantitative DNase footprint titration. Aim 1b: We will determine the molecular forces responsible for recruitment by assessing the physical and chemical contributions to receptor-coactivator assembly. Aim 2a: The detailed structural makeup of any intact receptor, coactivator, or receptor-coactivator complex is unknown. We will identify receptor-coactivator-promoter complexes amenable to high-resolution structural approaches using limited proteolysis, mass spectrometry, and analytical ultracentrifugation. Aim 2b: Identified complexes will be assessed structurally using NMR spectroscopy and crystallographic approaches. Together, these studies will contribute to the long-term objective of directly connecting structure and energetics in order to generate quantitatively predictive models of function. This proposal is focused on understanding the fundamental mechanisms by which human proteins (progesterone receptors) regulate gene expression. Elucidation of these mechanisms should eventually allow for the development of drugs that can modulate receptor function. These drugs should then have an impact on hormone dependent diseases such as breast or endometrial cancer.

描述（由申请人提供）：本研究的长期目标是确定真核基因调控的机制。人孕酮受体 (PR) 是配体激活转录因子核受体超家族的成员，可作为模型系统。 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。这两种蛋白之间的功能差异可能是由于与转录共激活蛋白（例如 AIB1（在乳腺癌中扩增-1））的不同相互作用所致。我们的研究表明，B 受体特有的残基可以变构调节 AIB1 向启动子复合物的募集，从而调节异构体特异性功能。此外，由于大规模的无序到有序折叠转变，这些相互作用与结构稳定性的显着增加有关。该提案的目标是对 PR 亚型与 PR 调节启动子上的共激活蛋白 AIB1 之间的相互作用进行生物物理和结构剖析。目标 1a：我们将使用定量 DNase 足迹滴定来解决与异构体特异性 AIB1 招募相关的微观自由能贡献。目标 1b：我们将通过评估对受体-共激活剂组装的物理和化学贡献来确定负责募集的分子力。目标 2a：任何完整受体、共激活剂或受体-共激活剂复合物的详细结构组成尚不清楚。我们将使用有限的蛋白水解、质谱和分析超速离心来鉴定适合高分辨率结构方法的受体-共激活剂-启动子复合物。目标 2b：将使用核磁共振波谱和晶体学方法对已识别的配合物进行结构评估。总之，这些研究将有助于实现直接连接结构和能量学的长期目标，以生成功能的定量预测模型。该提案的重点是了解人类蛋白质（孕激素受体）调节基因表达的基本机制。这些机制的阐明最终将有助于开发能够调节受体功能的药物。这些药物应该会对激素依赖性疾病（例如乳腺癌或子宫内膜癌）产生影响。