BR22, A Novel Protein Interacting with TTF-1

BR22，一种与 TTF-1 相互作用的新型蛋白质

• 批准号: 6478566
• 负责人: YIH-SHENG YANG
• 金额: $ 25.9万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6478566
• 关键词: chromatin; gene expression; genetic promoter element; histology; human tissue; immunoprecipitation; protein binding; protein localization; protein protein interaction; protein structure function; pulmonary surfactants; respiratory epithelium; thyroid hormone binding protein; tissue /cell culture; transcription factor; transfection; yeast two hybrid system

DESCRIPTION (provided by applicant): Surfactant is a mixture of lipids and proteins on the alveolar surface that plays an important role for gas exchange. Surfactant protein B (SP-B), an important protein component of surfactant, is expressed in type II cells and Clara cells in the lung. SP-B expression is mainly controlled by thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3. Previously, we uncovered a novel polypeptide, BR22, a TTF-1 associated protein (m. wt. 26 kD) (TAP26), which bound to human SP-B promoter sequence at the proximal region containing TTF-1 binding sites and interacted with TTF-1 to activate SF-B promoter. This protein also exhibits a direct protein-protein contact with TTF-1 in vivo and in vitro. In this proposal, we will demonstrate that TAP26 is a co-factor of TTF-1 to regulate the SP-B promoter activity in the lung. In order to understand the mechanism by which TAP26 modulates the promoter activity, the following approaches will be performed: 1) Characterize the contact domain modules of TAP26 and TTF-1 for a better understanding of the detailed protein complex structure. Deletion and mutagenesis analyses will be employed for the characterization. 2) Determine the mechanism by which TAP26 acts with TTF-1 to modulate the SP-B promoter activity. TAP26 in H441 cells will be sequestered to demonstrate its influence on the promoter activity. In addition, the effect of dexamethasone, cAMP, and phorbol ester on SP-B promoter activity will be inspected in the absence of TAP26 to determine if the regulation is mediated through TAP26. 3) Establish the relationship between TAP26 and SPB expression. The amounts of TAP26 message and protein level will be evaluated under conditions in which the SF-B expression is altered in vivo. 4) The DNA-binding activity of TAP26 and TAP-26/TTF-1 complex on the SP-B promoter will be inspected. The association of this protein complex and SP-B promoter in vivo will be examined by chromatin cross-linking and immunoprecipitation (CHIP) analysis. 5) Inspect cellular locations of TAP26 and TTF-1 in lung cells expressing SF-B. Two-color or triple-color staining of endogenous proteins in H441 cells or the lung sections will be performed to detect their expression in SP-B producing cells.

描述（由申请人提供）：表面活性剂是脂质和 肺泡表面的蛋白质，在气体交换中起重要作用。 表面活性剂蛋白质B（SP-B）是表面活性剂的重要蛋白质成分， 在肺中的II型细胞和Clara细胞中表达。SP-B表达是 主要由甲状腺转录因子-1（TTF-1）和肝细胞控制 核因子-3之前，我们发现了一种新的多肽，BR 22，一种TTF-1， 相关蛋白（M.重量26 kD）（TAP 26），其与人SP-B启动子结合 序列的近端区域含有TTF-1结合位点，并相互作用 TTF-1激活SF-B启动子。这种蛋白质还表现出直接的 体内和体外与TTF-1的蛋白质-蛋白质接触。在本提案中，我们 将证明TAP 26是TTF-1调节SP-B的辅因子 肺中的启动子活性。为了理解 TAP 26调节启动子活性，以下方法将被详细描述。 1）表征TAP 26和TTF-1的接触域模块， 更好地了解蛋白质复合物的详细结构。缺失和 诱变分析将用于表征。2)确定 TAP 26与TTF-1共同调控SP-B启动子的机制 活动H441细胞中的TAP 26将被隔离以证明其影响 对启动子活性的影响。此外，地塞米松、cAMP和 将在不存在下检查佛波醇酯对SP-B启动子活性的影响。 TAP 26来确定调节是否通过TAP 26介导。3)建立 TAP 26与SPB表达的关系。TAP 26报文量 和蛋白质水平将在SF-B 体内表达发生改变。4)TAP 26的DNA结合活性和 将检查SP-B启动子上的TAP-26/TTF-1复合物。联盟 该蛋白复合物和SP-B启动子在体内将通过染色质 交联和免疫沉淀（CHIP）分析。5)检查细胞 TAP 26和TTF-1在表达SF-B的肺细胞中的位置。双色或 H441细胞或肺切片中内源性蛋白的三色染色 检测它们在SP-B产生细胞中的表达。