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

• 批准号: 7447327
• 负责人: RICHARD A STEINMAN
• 金额: $ 20.05万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 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 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7447327
• 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

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（NHR）通过结合小的亲脂性配体调节转录，并对正常细胞功能和发育产生深远影响。异常的NHR功能经常发生在癌症中。这促进了靶向NHR受体的治疗性配体的开发（例如乳腺癌中的他莫昔芬，早幼粒细胞白血病中的ATRA）。核受体的新的天然或合成配体可能包括有用的抗癌剂。我们提出了一种创新的，高通量的方法来确定核受体的功能配体。我们假设体外核小体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。我们的工作的总体目标是开发能力，通过使用这样的生物传感器，快速筛选大型化学库的功能性配体已知或孤儿核受体。我们开发了独特的工具，以促进我们实现这些目标。这些包括试剂和仪器，使我们能够在单分子和群体水平上实时跟踪核小体重塑。该R21应用程序侧重于雌激素受体（ER）信号通路作为模型，以证明该检测的能力和灵敏度。在目的一中，我们构建了一个核小体，其中含有雌激素反应元件（ERE）的荧光团标记的DNA包裹在组蛋白周围，使能量从供体荧光（Cy 3）转移到受体荧光（Cy 5）（荧光共振能量转移，或FRET）。在目标二，我们测量雌激素受体激动剂的能力，特别是改造ERE含有核小体，表现为FRET的损失。核小体重塑将作为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进行研究。一个细胞提取物免费的生物传感器也将开发仅利用重组蛋白。在目标三中，我们将调整核小体生物传感器的高通量格式筛选库和验证系统使用ER激动剂，拮抗剂和非配体。在这个项目中开发的生物传感器应该能够快速筛选库的推定配体选择的核受体。此外，它将能够快速分解NHR途径的蛋白质或DNA组分。这种生物传感器应该比基于细胞的报告基因测定更快速和通用，并且比仅测量配体/受体结合的测定更有信息量。