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: 6338748
• 负责人: JAMES P SIMMER
• 金额: $ 18.22万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6338748
• 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所固有的，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-蛋白质相互作用是牙釉质生物矿化不可或缺的一部分，并且了解这些相互作用对于理解牙釉质形成的分子机制至关重要。在此子程序中，使用酵母两种混合系统和免疫蛋白方法研究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，用于识别蛋白质之间潜在的相互作用。它非常适合克隆编码与所选蛋白质相互作用的未知蛋白质的 cDNA，或识别两种所选蛋白质之间的相互作用。提出了两个具体目标。 1) 分离和表征编码牙釉质基质蛋白的 cDNA，重点关注蛋白酶抑制剂。这一目标将通过识别牙釉质基质蛋白来实现，该蛋白通过牙釉质基质蛋白与发育中牙釉质基质中先前分离的成分的蛋白质-蛋白质相互作用来实现。通过酵母双杂交系统和亲和层析来鉴定与选定的或“诱饵”蛋白质相互作用的蛋白质。用作诱饵的釉质蛋白包括釉质素（MMP-20）、釉质基质丝氨酸蛋白酶1（EMSP1）、釉质蛋白、鞘蛋白和釉原蛋白。通过这些方法鉴定的蛋白质-蛋白质相互作用通过亲和印迹和/或免疫沉淀来证实。优先鉴定调节釉质素和 EMSP1 活性的釉质蛋白酶抑制剂。 2) 表征釉质形成过程中蛋白质-蛋白质相互作用的特征。这一目标将通过亲和方法和酵母双杂交系统来实现。与 SA1 中“诱饵”蛋白与未知蛋白文库杂交不同，SA2 中特定的牙釉质蛋白与其自身或与第二种牙釉质蛋白杂交。研究了五种潜在的蛋白质-蛋白质相互作用：1）牙釉蛋白和牙釉蛋白，2）鞘蛋白和鞘蛋白，3）牙釉蛋白和鞘蛋白，4）釉原蛋白和釉蛋白，以及5）釉原蛋白和鞘蛋白。