Nuclear Transcription Factor Translocation Assay

核转录因子易位测定

• 批准号: 6689115
• 负责人: PYARE L KHANNA
• 金额: $ 10万
• 依托单位: DISCOVERX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689115
• 关键词: HeLa cells; beta galactosidase; catalyst; chimeric proteins; enzyme activity; enzyme mechanism; flow cytometry; fluorimetry; intracellular transport; nuclear factor kappa beta; protein transport; technology /technique development; transcription factor; transfection

DESCRIPTION (provided by applicant): Transcription factors (TF) are key regulators of gene expression. Most TFs reside in the cytosol and when activated translocate to the nucleus to stimulate gene activity. A therapeutically important TF is NFkappaB, which stimulates genes involved in cell cycling, proliferation and inflammatory responses and is an important molecular target for the development of anti-cancer and anti-inflammatory drugs. Since nuclear translocation is a critical event in NFkappaB's ability to regulate gene expression, technologies that measure NFkB translocation could be used to screen for novel anti-neoplastic and anti-inflammatory agents that block the translocation process. However, there are few technologies to measure the movement of TFs from the cytosol to the nucleus and none that is amenable for high throughput drug discovery. DiscoveRx has developed an enzyme fragment complementation (EFC) technology that could be used to detect the translocation of TFs using an extremely sensitive beta-galactosidase (beta-gal) complementation assay. This method utilizes two genetically engineered fragments of E. coli beta-gal. The larger fragment, termed Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, termed Enzyme Donor (ED), contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, it can recombine with ED to form an active enzyme that can catalyze the formation of a fluorescent product detected photometrically as a visually amplified response. We propose to develop a direct assay for NFkB translocation using EFC technology. We will engineer ED into NFkB and target EA to the nucleus. When ED-NFkappaB translocates to the nucleus it will be able to recombine with EA to generate an active B-gal that can catalyze the formation of fluorescent products. As a complementary approach to measure NFkB translocation, we will develop a novel NFkappaB reporter gene assay using our EFC technology in which an NFkB response element drives the production of a GST-ED fusion protein whose expression will be detected with EA in a simplified cell based fluorescent assay. These studies will develop the first NFkB translocation for drug discovery. In the future, we will adapt this assay as a general technology for drug discovery against any TF to open up an entirely new field of transcription factor Dharmacoloav and therapeutics.

描述（由申请人提供）：转录因子（TF）是基因表达的关键调节因子。大多数转录因子存在于细胞质中，当被激活时会转位到细胞核以刺激基因活性。 NFkappaB 是治疗上重要的 TF，它刺激参与细胞周期、增殖和炎症反应的基因，是开发抗癌和抗炎药物的重要分子靶点。由于核转位是 NFkappaB 调节基因表达能力的关键事件，因此测量 NFkB 转位的技术可用于筛选阻断转位过程的新型抗肿瘤和抗炎药物。然而，很少有技术可以测量转录因子从细胞质到细胞核的运动，也没有适合高通量药物发现的技术。 DiscoveRx 开发了一种酶片段互补 (EFC) 技术，可用于使用极其灵敏的 β-半乳糖苷酶 (beta-gal) 互补测定来检测 TF 的易位。该方法利用了大肠杆菌 beta-gal 的两个基因工程片段。较大的片段称为酶受体 (EA)，包含氨基末端附近的缺失，而较小的片段称为酶供体 (ED)，包含 EA 缺失的氨基末端序列。 EA 本身并不活跃。然而，它可以与 ED 重组形成一种活性酶，该酶可以催化荧光产物的形成，通过光度计检测为视觉放大响应。我们建议使用 EFC 技术开发 NFkB 易位的直接检测方法。我们将把 ED 设计成 NFkB，并将 EA 定位到细胞核。当 ED-NFkappaB 转位到细胞核时，它将能够与 EA 重组，生成活性 B-gal，可以催化荧光产物的形成。作为测量 NFkB 易位的补充方法，我们将使用我们的 EFC 技术开发一种新型 NFkappaB 报告基因测定，其中 NFkB 响应元件驱动 GST-ED 融合蛋白的产生，该融合蛋白的表达将在简化的基于细胞的荧光测定中用 EA 进行检测。这些研究将开发第一个用于药物发现的 NFkB 易位。未来，我们将采用这种测定作为针对任何 TF 的药物发现的通用技术，以开辟转录因子 Dharmacoloav 和治疗学的全新领域。