PROTEIN /PROTEIN INTERACTIONS IN THE ENAMEL MATRIX

蛋白质-蛋白质相互作用是几乎每一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，并且这些相互作用的知识对于理解牙釉质形成的分子机制至关重要。本项目采用酵母双杂交系统和免疫蛋白方法研究了釉质基质蛋白之间的蛋白-蛋白相互作用。酵母双杂交系统是鉴定蛋白质间潜在相互作用的分子生物学工具。它非常适合克隆编码未知蛋白质的与选定蛋白质相互作用的cdna，或用于鉴定两个选定蛋白质之间的相互作用。提出了两个具体目标。1)分离和表征编码釉质基质蛋白的cdna，重点研究蛋白酶抑制剂。这一目标将通过鉴定釉质基质蛋白与先前分离的釉质基质成分之间的蛋白质相互作用来实现。与选定的“诱饵”蛋白相互作用的蛋白通过酵母双杂交系统和亲和层析进行鉴定。作为诱饵的釉质蛋白包括搪瓷溶素（MMP-20）、搪瓷基质丝氨酸蛋白酶1 （EMSP1）、搪瓷蛋白、鞘蛋白和淀粉原蛋白。通过这些方法鉴定的蛋白-蛋白相互作用通过亲和印迹和/或免疫沉淀得到证实。目前研究的重点是确定能调节珐琅质蛋白酶和EMSP1活性的珐琅质蛋白酶抑制剂。2)表征淀粉形成过程中蛋白-蛋白相互作用。这一目的将通过亲和法和酵母双杂交系统来实现。与SA1中“诱饵”蛋白与未知蛋白库杂交不同，SA2中特异性的珐琅质蛋白与自身或与第二个珐琅质蛋白杂交。研究了五种潜在的蛋白质相互作用：1)漆包素和漆包素，2)鞘蛋白和鞘蛋白，3)漆包素和鞘蛋白，4)淀粉原蛋白和漆包素，5)淀粉原蛋白和鞘蛋白。

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.