The Role of LRAP in Enamel Mineral Formation

描述（由申请人提供）：淀粉原蛋白突变导致釉质缺陷，称为淀粉原性不完全性（AI）。进一步了解不同的淀粉原蛋白异构体是如何在分泌过程中在牙釉质基质中加工的，将有助于阐明AI的发病机制。本研究的目的是研究富含亮氨酸的淀粉原蛋白肽（LRAP） c端加工在牙釉质形成中的功能重要性，以及LRAP如何与全长淀粉原蛋白（M180）相互作用形成牙釉质棱镜。我选择了分子生物学专家Carolyn Gibson作为我的主要导师，并选择了珐琅蛋白生物化学和成釉细胞培养专家Pamela DenBesten作为我的共同导师。对于K99期，第一个具体目标是确定富含亮氨酸的淀粉原蛋白肽（LRAP）的c端如何参与釉质棱镜的形成。我将利用过表达LRAP和截断c端M180的转基因小鼠模型，分析牙釉质表型，并接受额外的矿物分析和成像训练，以确定LRAP的c端是否可以挽救缺乏全长淀粉原蛋白c端（CTRNC）的小鼠的非棱柱状牙釉质表型。为了利用体外方法实现这一目标，我将接受成釉细胞培养和转染、质谱分析、重组蛋白生成、钙结合、蛋白质组装和矿物质诱导方面的培训。利用之前的训练和我在K99期的训练，第二个目标将在R00期进行，是确定在分泌性牙釉质形成过程中是否需要Mmp20加工LRAP的c端。利用过表达LRAP但缺乏Mmp20的转基因小鼠模型，我将研究牙釉质结构和裂解产物。我还将使用重组蛋白和细胞培养来确定LRAP c端Mmp20切割位点的突变是否会影响矿物的结合和形成或纳米球的组装。最后，在独立的R00阶段进行的第三个具体目标是确定LRAP、M180及其解理产物在分泌性牙釉质形成过程中如何共同引导牙釉质矿物的形成。我将采用转基因小鼠模型、纳米压痕、重组蛋白和细胞培养的体内和体外方法来确定M180、LRAP以及截断的M180和LRAP是否能在体内改善牙釉质形成，或在体外改善蛋白质组装和矿物质诱导。这些实验数据有助于提出淀粉原异构体及其裂解产物参与牙釉质矿化的机制。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Mutations in amelogenins cause enamel defects referred to as amelogenesis imperfecta (AI). Improved understanding of the role of the genes involved in enamel formation can improve the prospects for treatment of children with this condition. The fundamental goal of this research is to determine how abundant amelogenin isoforms are processed and involved in enamel mineral formation, which will help elucidate AI disease mechanisms.