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基因调控的潜在机制。人孕激素受体（PR），配体激活的转录因子的核受体超家族的成员，作为一个模型系统。PR作为两种功能不同的同种型共存：83 kD A受体和99 kD B受体。这两种亚型是相同的，除了B-受体在其N-末端具有额外的164个氨基酸。这两种蛋白质之间的功能差异可能是由于与转录共激活蛋白如AIB 1（乳腺癌扩增-1）的差异相互作用。我们的研究表明，B-受体独特的残基变构调节AIB 1的启动子复合物的招聘，从而调节亚型特异性功能。此外，这些相互作用与结构稳定性的急剧增加相耦合，这是由于大规模的无序到有序的折叠转变。这项建议的目标是进行PR亚型和共激活蛋白AIB 1之间的相互作用的生物物理和结构解剖，PR调节的启动子。目标1a：我们将使用定量DNA酶足迹滴定来解析与亚型特异性AIB 1募集相关的微观自由能贡献。目标1b：我们将通过评估受体-辅激活因子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、辅激活因子或受体-辅激活因子复合物的详细结构组成尚不清楚。我们将确定受体-辅激活因子-启动子复合物服从高分辨率的结构方法，使用有限的蛋白水解，质谱和分析超离心。目标2b：将使用NMR光谱学和晶体学方法在结构上评估已鉴定的复合物。总之，这些研究将有助于直接连接结构和能量的长期目标，以产生定量预测模型的功能。该提案的重点是了解人类蛋白质（孕酮受体）调节基因表达的基本机制。这些机制的阐明应最终允许开发药物，可以调节受体功能。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.