PROTEIN /PROTEIN INTERACTIONS IN THE ENAMEL MATRIX

蛋白质-蛋白质相互作用是几乎所有细胞过程所固有的，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-蛋白质相互作用是釉质生物矿化不可或缺的，这些相互作用的知识是了解釉质形成的分子机制所必需的。本子程序利用酵母双杂交系统和免疫蛋白质方法研究釉基质蛋白之间的相互作用。酵母双杂交系统是研究蛋白质间相互作用的分子生物学工具。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，或用于鉴定两种所选蛋白质之间的相互作用。提出了两个具体目标。1)目的分离和鉴定编码釉基质蛋白的cDNA，重点是蛋白酶抑制剂。这一目标将通过鉴定釉基质蛋白质，通过它们与先前分离的发育中的釉基质成分的蛋白质-蛋白质相互作用来实现。通过酵母双杂交系统和亲和层析鉴定与所选蛋白或“诱饵”蛋白相互作用的蛋白。用作诱饵的釉质蛋白包括釉质溶素（MMP-20）、釉质基质丝氨酸蛋白酶1（EMSP 1）、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。优先考虑鉴定调节溶釉素和EMSP 1活性的釉质蛋白酶抑制剂。2)研究釉质形成过程中蛋白质间的相互作用。这一目标将通过亲和方法和酵母双杂交系统来实现.与其中“诱饵”蛋白质与未知蛋白质文库杂交的SA 1相反，在SA 2中，特异性釉质蛋白质与其自身或与第二釉质蛋白质杂交。研究了五种潜在的蛋白质-蛋白质相互作用：1）釉蛋白和釉蛋白，2）鞘蛋白和鞘蛋白，3）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。

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.