The Role of LRAP in Enamel Mineral Formation

项目总结/摘要 釉原蛋白的突变导致称为釉质形成缺失（AI）的釉质缺陷。进一步 了解不同的釉原蛋白异构体是如何在釉质基质中加工的， 分泌将有助于阐明AI的发病机制。这项研究的目的是调查 富含亮氨酸的釉原蛋白肽（LRAP）的C-末端的加工在 釉质形成，以及LRAP如何与全长釉原蛋白（M180）相互作用以形成釉质棱柱。我 我选择了卡罗琳吉布森，在分子生物学和釉原蛋白AI小鼠一代专家 模特，作为我的主要导师，帕梅拉·登贝斯滕，一位釉质蛋白生物化学专家， 成釉细胞培养的共同导师对于K99，辅导阶段，第一个具体目标是确定 富含亮氨酸的釉原蛋白肽（LRAP）的C-末端如何参与釉质棱柱的形成。 使用过表达LRAP和具有截短C末端的M180转基因小鼠模型， 分析釉质表型，并接受矿物质分析和成像方面的额外培训，以确定 如果LRAP的C-末端可以拯救缺乏全长LRAP的小鼠中的非棱柱状釉质表型， 釉原蛋白C末端（CTRNC）。为了使用体外方法实现这一目标，我将接受以下培训： 成釉细胞培养和转染，质谱，重组蛋白生成，钙离子 结合、蛋白质组装和矿物质诱导。利用之前的培训和我在K99阶段的培训， 第二个目标将在R 00阶段执行，是确定是否处理 Mmp 20的LRAP的C-末端在分泌性釉质形成期间是必需的。使用转基因小鼠 模型过表达LRAP但缺乏Mmp 20，我将研究釉质结构和卵裂产物。 我还将使用重组蛋白和细胞培养来确定Mmp 20切割突变是否 LRAP的C-末端的位点可以影响矿物质结合和形成或纳米球组装。最后， 将在独立R 00阶段期间执行的第三个具体目标是确定LRAP如何， M180和它们的卵裂产物在分泌过程中共同指导釉质矿物质的形成 釉质形成我将使用转基因小鼠模型的体内和体外方法， 纳米压痕，重组蛋白和细胞培养，以确定是否M180，LRAP，和截短 M180和LRAP一起可以在体内促进釉质形成，或蛋白质组装和矿物质诱导 体外从所提出的实验中产生的数据可以导致提出一种机制， 釉原蛋白同种型及其裂解产物参与釉质矿化。

Project Summary/Abstract Mutations in amelogenins cause enamel defects referred to as amelogenesis imperfecta (AI). Further understanding of how the different amelogenin isoforms are processed in the enamel matrix during secretion will help elucidate the AI disease mechanism. The goal of the proposed research is to investigate the functional importance of processing of the C-terminus of leucine rich amelogenin peptide (LRAP) in enamel formation, and how LRAP interacts with full-length amelogenin (M180) to form enamel prisms. I have selected Carolyn Gibson, an expert in molecular biology and generation of amelogenin AI mouse models, as my primary mentor, and Pamela DenBesten, an expert in enamel protein biochemistry and ameloblast cell culture as my co-mentor. For the K99, mentored phase, the first specific aim is determine how the C-terminus of leucine rich amelogenin peptide (LRAP) is involved in enamel prism formation. Using a transgenic mouse model overexpressing LRAP and M180 with a truncated C-terminus, I will analyze the enamel phenotype and receive additional training in mineral analysis and imaging to determine if the C-terminus of LRAP can rescue the non-prismatic enamel phenotype in mice lacking the full-length amelogenin C-terminus (CTRNC). To address this aim using an in vitro approach, I will receive training in ameloblast cell culture and transfection, mass spectrometry, recombinant protein generation, calcium binding, protein assembly and mineral induction. Using prior training and my training from the K99 phase, the second aim, which will be carried out during the R00 phase, is to determine whether processing of the C-terminus of LRAP by Mmp20 is required during secretory enamel formation. Using transgenic mouse model overexpressing LRAP but lacking Mmp20, I will investigate enamel structure and cleavage products. I will also use recombinant proteins and cell culture to determine whether mutation of the Mmp20 cleavage site at the C-terminus of LRAP can affect mineral binding and formation or nanosphere assembly. Finally, the third specific aim which will be carried out during the independent R00 phase is determine how LRAP, M180 and their cleavage products work together to guide enamel mineral formation during secretory enamel formation. I will use the in vivo and in vitro approaches of transgenic mouse models, nanoindentation, recombinant proteins and cell culture to determine whether M180, LRAP, and truncated M180 and LRAP together can improve enamel formation in vivo, or protein assembly and mineral induction in vitro. The data generated from the proposed experiments can lead to proposition of a mechanism of how the amelogenin isoforms and their cleavage products are involved in enamel mineralization.