A Nucleosomal Biosensor for Identification and Isolation of Nuclear Hormone Recep

用于识别和分离核激素受体的核小体生物传感器

基本信息

批准号：
7193578
负责人：
RICHARD A STEINMAN
金额：
$ 16.71万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
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/7193578
关键词：
Acute Promyelocytic Leukemia Agonist Antineoplastic Agents Behavior Benchmarking Binding Biological Assay Biosensor Breast Cell Extracts Cell physiology Cells Complex Mixtures DNA DNA Sequence Dependence Detection Development Disease Disruption Dissection Doctor of Philosophy Elements Estrogen Receptors Estrogens Exhibits Fluorescence Resonance Energy Transfer Genetic Polymorphism Genetic Transcription Goals Histones Hormones In Vitro Individual Ionic Strengths Libraries Ligand Binding Ligands Malignant Neoplasms Measures Modeling Mutate Nuclear Nuclear Extract Nuclear Hormone Receptors Nuclear Orphan Receptor Nuclear Receptors Nucleosomes Numbers Orphan Ovarian Pathway interactions Placement Population Process Proteins Reagent Receptor Signaling Recombinant Proteins Recombinants Reporter Genes Response Elements Retinoid Receptor Screening procedure Sensitivity and Specificity Signal Pathway Signal Transduction Source Specificity Structure System Tamoxifen Testing Therapeutic Time Tissues Tretinoin Work base bone clinically significant cofactor cyanine dye 5 fluorophore high throughput screening innovation instrumentation malignant breast neoplasm mutant novel receptor receptor binding receptor function response single molecule small molecule libraries steroid hormone time use tool

项目摘要

DESCRIPTION (provided by applicant): Nuclear hormone receptors (NHRs) modulate transcription by binding small lipophilic ligands and have a profound impact on normal cellular function and on development. Aberrant NHR function occurs frequently in cancers. This has prompted the development of therapeutic ligands targeting NHR receptors (e.g. tamoxifen in breast cancer, ATRA in promyelocytic leukemia). New natural or synthetic ligands for nuclear receptors are likely to include useful anticancer agents. We propose an innovative, high-throughput approach to identify functional ligands of nuclear receptors. We hypothesize that an in vitro nucleosome- based biosensor can identify and distinguish ligands that activate or inhibit nuclear receptor activity. The overall goal of our work is to develop the capacity to screen large chemical libraries rapidly for functional ligands to known or to orphan nuclear receptors through the use of such a biosensor. We have developed unique tools to facilitate our pursuit of these goals. These include reagents and instrumentation that enables us to follow nucleosomal remodeling at both the single molecule and population level in real-time. This R21 application focuses on the estrogen receptor (ER) signaling pathway as a model to demonstrate the power and sensitivity of this assay. In Aim One, we construct a nucleosome in which fluorophore-tagged DNA containing an estrogen response element (ERE) is wrapped around histones so that energy is transferred from the donor fluor (Cy3) to the acceptor fluor (Cy5) (fluorescence resonance energy transfer, or FRET). In Aim Two, we measure the ability of estrogen receptor agonists to specifically remodel ERE-containing nucleosomes, manifested as a loss of FRET. Nucleosome remodeling will be studied as a function of ERE sequence, ligand (agonist versus antagonist) and tissue source of cell extracts. A cell extract-free biosensor will also be developed utilizing only recombinant proteins. In Aim Three, we will adapt the nucleosomal biosensor to a high throughput format for screening libraries and validate the system using ER agonists, antagonists and nonligands. The biosensor developed in this project should enable rapid screening of libraries of putative ligands for chosen nuclear receptors. Moreover, it will enable rapid dissection of protein or DNA components of NHR pathways. This biosensor should be more rapid and versatile than cell-based reporter gene assays and more informative than assays that measure only ligand/receptor binding.

描述（由申请人提供）：核激素受体（NHRS）通过结合小亲脂配体来调节转录，并对正常细胞功能和发育产生深远的影响。异常NHR功能在癌症中经常发生。这促使靶向NHR受体的治疗配体的发展（例如，乳腺癌中的他莫昔芬，在探针细胞性白血病中的ATRA）。用于核受体的新天然或合成配体可能包括有用的抗癌药。我们提出了一种创新的高通量方法，以鉴定核受体的功能配体。我们假设基于核小体的生物传感器可以识别和区分激活或抑制核受体活性的配体。我们工作的总体目标是通过使用这种生物传感器来迅速筛选出大型化学文库的能力，以迅速筛选出功能性配体的大型化学文库，以筛选出已知或孤儿核受体。我们开发了独特的工具来促进我们实现这些目标的追求。这些包括试剂和仪器，使我们能够实时遵循单分子和种群水平的核小体重塑。该R21应用集中在雌激素受体（ER）信号通路上，以证明该测定法的功率和灵敏度。在AIM ONE中，我们构建了一个核小体，其中包含含有雌激素反应元件（ERE）的荧光团标签的DNA周围围绕组蛋白包裹，以便将能量从供体荧光（CY3）转移到受体荧光（CY5）（CY5）（荧光共振能转移或FRET）。在目标两个中，我们测量了雌激素受体激动剂特异性重塑核小体的能力，这表现为FRET的损失。将研究核小体重塑作为ERE序列，配体（激动剂与拮抗剂）和细胞提取物的组织来源的函数。仅利用重组蛋白的无细胞提取物生物传感器也将开发。在AIM三中，我们将将核小体生物传感器调整为高吞吐量格式，以筛选文库，并使用ER激动剂，拮抗剂和非配体验证系统。在该项目中开发的生物传感器应能够快速筛选选定的核受体的假定配体库。此外，它将能够快速解剖NHR途径的蛋白质或DNA成分。该生物传感器应比基于细胞的报告基因测定法更快，更通用，并且比仅测量配体/受体结合的测定法更有用。