PROTEIN /PROTEIN INTERACTIONS IN THE ENAMEL MATRIX

牙釉质基质中的蛋白质/蛋白质相互作用

• 批准号: 6656479
• 负责人: JAMES P SIMMER
• 金额: $ 18.22万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6656479
• 关键词: affinity chromatography; amelogenin; complementary DNA; dental development; extracellular matrix proteins; immunoprecipitation; peptide library; protease inhibitor; protein protein interaction; serine proteinases; tooth enamel; yeast two hybrid system

Protein-protein interactions are intrinsic to virtually every cellular process, including DNA replication, transcription, translation, splicing, secretion, cell cycle control, signal transduction, and intermediary metabolism. We hypothesize that protein-protein interactions are integral to enamel biomineralization and that knowledge of these interactions is essential for understanding the molecular mechanisms of enamel formation. In this Sub-program, protein-protein interactions among enamel matrix proteins are investigated using the yeast two hybrid system and immuno-protein methods. The yeast two-hybrid system is a molecular biology tool for identify potential interactions between proteins. It is ideally suited for cloning cDNAs encoding unknown proteins that interact with a chosen protein, or for identifying interactions between two chosen proteins. Two Specific Aims are propose. 1) To isolate and characterize cDNAs encoding enamel matrix proteins, with a focus on proteinase inhibitors. This aim will be accomplished by identifying enamel matrix proteins by their protein-protein interactions with previously isolated constituents of the developing enamel matrix. Proteins interacting with a chosen, or "bait" protein are identified by the yeast two-hybrid system and affinity chromatography. The enamel proteins used as bait include enamelysin (MMP-20), enamel matrix serine proteinase 1 (EMSP1), enamelin, sheathlin, and amelogenin. Protein- protein interactions identified by these methods are confirmed by affinity blotting, and/or immunoprecipitation. Priority is given to identifying enamel proteinase inhibitors that regulate the activities of enamelysin and EMSP1. 2) To characterize protein-protein interactions during amelogenesis. This aim will be accomplished using affinity methods and the yeast two- hybrid system. In contrast to SA1 where a "bait" protein is crossed with a library of unknown proteins, in SA2 specific enamel protein is crossed with itself, or with a second enamel protein. Five potential protein-protein interactions are investigated: 1) enamelin and enamelin, 2) sheathlin and sheathlin, 3) enamelin and sheathlin, 4) amelogenin and enamelin, and 5) amelogenin and sheathlin.

蛋白质 - 蛋白质相互作用几乎是每个细胞过程的固有的，包括DNA复制，转录，翻译，剪接，分泌，细胞周期控制，信号转导和中间代谢。我们假设蛋白质 - 蛋白质相互作用对于搪瓷生物矿化是不可或缺的，并且对这些相互作用的了解对于理解搪瓷形成的分子机制至关重要。在此子程序中，使用酵母两种杂种系统和免疫蛋白方法研究了牙釉质基质蛋白之间的蛋白质 - 蛋白质相互作用。酵母双杂交系统是一种分子生物学工具，用于鉴定蛋白质之间的潜在相互作用。它非常适合克隆cDNA，编码与所选蛋白质相互作用的未知蛋白或鉴定两种选择蛋白之间的相互作用的蛋白质。提出了两个具体目标。 1）分离和表征编码搪瓷基质蛋白的cDNA，重点是蛋白酶抑制剂。通过通过蛋白质蛋白质相互作用与发育中的牙釉质基质的先前分离的成分来鉴定其蛋白质蛋白质相互作用来实现此目标。与所选或“诱饵”蛋白相互作用的蛋白质通过酵母双杂交系统和亲和力色谱鉴定。用作诱饵的搪瓷蛋白包括Inamelysin（MMP-20），搪瓷基质丝氨酸蛋白酶1（EMSP1），搪瓷蛋白，Sheathlin和Amelogenin。通过这些方法鉴定出的蛋白质蛋白质相互作用通过亲和力印迹和/或免疫沉淀证实。优先考虑鉴定调节Nomelysin和Emsp1活性的牙釉质蛋白酶抑制剂。 2）表征在没有变性过程中蛋白质 - 蛋白质相互作用。该目标将使用亲和力方法和酵母两种混合系统来实现。与SA1相反，在SA2特异性搪瓷蛋白中，“诱饵”蛋白与未知蛋白的库交叉，与自身或第二个搪瓷蛋白交叉。研究了五种潜在的蛋白质 - 蛋白质相互作用：1）搪瓷蛋白和搪瓷素，2）Sheathlin和Sheathlin，3）搪瓷和Sheathlin，4）4）amelogenin and Enamelin，以及5）amelin and Sheathlin。