BR22, A Novel Protein Interacting with TTF-1

描述(申请人提供)：表面活性物质是一种脂类和 肺泡表面的蛋白质对气体交换起着重要作用。 表面活性蛋白B(SP-B)是表面活性物质的重要蛋白质组分。 表达于肺中的II型细胞和Clara细胞。SP-B表达式为 主要受甲状腺转录因子-1和肝细胞调控 核因子-3。在此之前，我们发现了一种新的多肽BR22，一种TTF-1 与人SP-B启动子结合的相关蛋白(分子量26 kD)(TAP26) 包含TTF-1结合位点的近端区域的序列并相互作用 用TTF-1激活SF-B启动子。这种蛋白质还表现出直接的 与TTF-1在体内和体外的蛋白质-蛋白质接触。在这项提案中，我们 将证明TAP26是TTF-1调节SP-B的辅助因子 肺中的启动子活性。为了了解这一机制 TAP26调节启动子活性，将采取以下方法 已执行：1)表征TAP26和TTF-1的触点域模块 更好地了解详细的蛋白质复合体结构。删除和 突变分析将用于表征。2)确定 TAP26与TTF-1共同调控SP-B启动子的机制 活动。H441细胞中的TAP26将被隔离以展示其影响 对启动子活性的影响。此外，地塞米松、cAMP和 佛波酯对SP-B启动子活性的影响将在不存在的情况下进行检查 TAP26以确定调控是否通过TAP26介导。3)建立 TAP26与SPB表达的关系。TAP26报文数量 和蛋白质水平将在SF-B 在体内，表达会发生变化。4)TAP26和TAP26的DNA结合活性 将检测SP-B启动子上的TAP-26/TTF-1复合体。联谊会 该蛋白复合体和体内SP-B启动子将通过染色质进行检测 交联剂和免疫沉淀(CHIP)分析。5)检查蜂窝 TAP26和TTF-1在表达SF-B的肺细胞中的定位双色或 H441细胞及肺切片内源性蛋白的三色染色 以检测它们在SP-B产生细胞中的表达。

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.