Quantitative Analysis of AIB-1 Recruitment

描述（由申请人提供）：本研究的长期目标是确定真核生物基因调控的机制。人孕激素受体（PR）是配体激活转录因子核受体超家族的成员，是一个模型系统。PR以两种功能不同的亚型共存：83 kD a受体和99 kD b受体。这两种同工异构体是相同的，除了b受体在其n端有额外的164个氨基酸。两种蛋白之间的功能差异可能是由于与转录共激活蛋白（如AIB1）的不同相互作用（在乳腺癌中扩增1）。我们的研究表明，b受体特有的残基可变地调节AIB1向启动子复合体的募集，从而调节异构体特异性功能。此外，由于大规模无序到有序折叠转变，这些相互作用与结构稳定性的急剧增加相耦合。本研究的目标是对PR调控启动子上的PR同工异构体与共激活蛋白AIB1之间的相互作用进行生物物理和结构解剖。目的1a：我们将使用定量dna酶足迹滴定法解决与亚型特异性aib1募集相关的微观自由能贡献。目标1b：我们将通过评估对受体-辅激活剂组装的物理和化学贡献来确定负责招募的分子力。目的2a：任何完整的受体、辅激活剂或受体-辅激活剂复合物的详细结构组成是未知的。我们将使用有限的蛋白水解、质谱分析和超离心分析来鉴定适合高分辨率结构方法的受体-辅激活剂-启动子复合物。目的2b：鉴定的配合物将使用核磁共振波谱和晶体学方法进行结构评估。总之，这些研究将有助于直接连接结构和能量学的长期目标，以产生功能的定量预测模型。本建议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允许开发能够调节受体功能的药物。这些药物应该对激素依赖性疾病如乳腺癌或子宫内膜癌有影响。

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.