TIP39KDA AND TOOTH FORMATION

TIP39KDA 和牙齿形成

• 批准号: 6678002
• 负责人: Michael Lansdell Paine
• 金额: $ 36.45万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678002
• 关键词: MDCK cell; ameloblasts; amelogenin; antibody; antisense nucleic acid; binding proteins; dental development; enamel organ; functional /structural genomics; genetically modified animals; green fluorescent proteins; laboratory mouse; messenger RNA; oligonucleotides; organ culture; peptide library; posttranscriptional RNA processing; protein isoforms; protein localization; protein structure function; proteomics; ribozymes; transfection /expression vector; yeast two hybrid system

DESCRIPTION (provided by applicant): Understanding the physiologic relationship between genomics and proteomics is a common theme and focus for investigation. The use of cell models and organ systems remains a fundamental approach to achieving such an understanding. Enamel is a complex bioceramic tissue that is a reflection of its unique molecular and cellular formation. From their ectodermal origins, ameloblasts exist for the sole purpose of producing a mineralized shell. The shell is almost entirely devoid of protein and is deposited over the "bone-like" dentine. We have recently discovered TIP39kDa, a protein that favors the secretory surface of ameloblasts (Tomes' processes). Antibodies to TIP39kDa react to secretory vesicles within Tomes' processes, and also the immature enamel matrix. TIP39kDa expression is not unique to ameloblasts, but preliminary data suggest that it may be unique to cells with a significant secretory function. This project will pursue an experimental strategy to identify a definitive function for TIP39kDa, and also explain the nature of the multiple messenger RNA isoforms observed for TIP39kDa. The first aim will test the hypothesis that TIP39kDa is involved with the cells secretory activity by down-regulating TIP39kDa expression within cell and organ cultures, and look at the subsequent levels of a secreted protein (amelogenin). For this I will employ both an antisense RNA and a ribozyme approach. The second aim will be to use the TIP39kDa gene promoter in an appropriate reporter system and produce transgenic animals to define organs for which TIP39kDa plays a role. The third aim will identify the subcellular localization of TIP39kDa. For this I will employ available TIP39kDa antibodies and green fluorescent protein tags. Regular database searches will be performed to assist in the identification of functional domains. The fourth aim will be to screen a yeast two-hybrid mouse embryonic library to look for TIP39kDa protein partners. This data will be used to help identify a physiological role for TIP39kDa. The fifth and final specific aim will seek to answer the question of how multiple messenger RNA isoforms of TIP39kDa are generated as evident from previously published data. These first four specific aims will assign a subcellular location and function to a TIP39kDa that, in the case of ameloblasts, has a preference for Tomes' processes, secretory vacuoles and the immature enamel matrix. The fifth specific aim will answer a very basic question related to how TIP39kDa is processed post-transcriptionally, and the potential significance of this.

描述（由申请人提供）：了解基因组学和蛋白质组学之间的生理关系是研究的共同主题和焦点。细胞模型和器官系统的使用仍然是实现这种理解的基本方法。牙釉质是一种复杂的生物陶瓷组织，反映了其独特的分子和细胞形成。成釉细胞起源于外胚层，其存在的唯一目的是产生矿化壳。外壳几乎完全不含蛋白质，沉积在“骨状”牙本质上。我们最近发现了 TIP39kDa，一种有利于成釉细胞分泌表面（Tomes 过程）的蛋白质。 TIP39kDa 抗体与 Tomes 突内的分泌囊泡以及未成熟的牙釉质基质发生反应。 TIP39kDa 表达并非成釉细胞所独有，但初步数据表明，它可能是具有显着分泌功能的细胞所独有的。该项目将采用实验策略来确定 TIP39kDa 的明确功能，并解释观察到的 TIP39kDa 多种信使 RNA 亚型的性质。第一个目标将测试 TIP39kDa 通过下调细胞和器官培养物中 TIP39kDa 表达参与细胞分泌活动的假设，并观察分泌蛋白（釉原蛋白）的后续水平。为此，我将采用反义 RNA 和核酶方法。第二个目标是在适当的报告系统中使用 TIP39kDa 基因启动子并产生转基因动物来定义 TIP39kDa 发挥作用的器官。第三个目标是确定 TIP39kDa 的亚细胞定位。为此，我将使用可用的 TIP39kDa 抗体和绿色荧光蛋白标签。将进行定期数据库搜索以协助识别功能域。第四个目标是筛选酵母双杂交小鼠胚胎文库，以寻找 TIP39kDa 蛋白伴侣。该数据将用于帮助确定 TIP39kDa 的生理作用。第五个也是最后一个具体目标将寻求回答 TIP39kDa 的多种信使 RNA 同工型是如何生成的问题，这一点从之前发表的数据中可以明显看出。前四个具体目标将为 TIP39kDa 分配亚细胞位置和功能，就成釉细胞而言，TIP39kDa 偏爱 Tomes 过程、分泌液泡和未成熟的牙釉质基质。第五个具体目标将回答一个非常基本的问题，涉及 TIP39kDa 如何进行转录后处理及其潜在意义。