The Role of LRAP in Enamel Mineral Formation

LRAP 在牙釉质矿物形成中的作用

• 批准号: 8588500
• 负责人: Megan Kardon Pugach
• 金额: $ 23.9万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588500
• 关键词: Address; Affect; Ameloblasts; Amelogenesis Imperfecta; Apatites; Area; Binding; Calcium Binding; Calcium-Binding Proteins; Cattle; Cell Culture Techniques; Child; Data; Defect; Dental Enamel; Development; Disease; Enamel Formation; Generations; Genes; Goals; Image Analysis; In Vitro; Individual; Lead; Length; Mass Spectrum Analysis; Mentors; Minerals; Molecular Biology; Molecular Weight; Mus; Mutation; Nanosphere; Phase; Phenotype; Process; Property; Protein Biochemistry; Protein Isoforms; Proteins; Proteolytic Processing; RNA Splicing; Recombinant Proteins; Recombinants; Research; Role; Signal Transduction; Site; Structure; Testing; Tissues; Tooth structure; Training; Transcript; Transfection; Transgenic Mice; Work; abstracting; amelogenin; improved; in vivo; interest; leucine-rich amelogenin peptide; mineralization; mouse model; nanoindentation; nanomechanical; overexpression; research study

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.

项目概要/摘要 牙釉蛋白突变会导致牙釉质缺陷，称为牙釉质生成不全（AI）。更远 了解不同的牙釉质同工型在牙釉质基质中是如何加工的 分泌物将有助于阐明AI疾病机制。拟议研究的目标是调查 富含亮氨酸的牙釉蛋白肽 (LRAP) C 末端加工的功能重要性 牙釉质形成，以及 LRAP 如何与全长牙釉蛋白 (M180) 相互作用形成牙釉质棱柱。我 选择了分子生物学和牙釉蛋白AI小鼠生成专家Carolyn Gibson 模特，作为我的主要导师，以及帕梅拉·登贝斯滕（Pamela DenBesten），牙釉质蛋白质生物化学和 成釉细胞培养作为我的导师。对于 K99 指导阶段，第一个具体目标是确定 富含亮氨酸的牙釉蛋白肽 (LRAP) 的 C 末端如何参与牙釉质棱柱的形成。 使用过度表达 LRAP 和 M180 且 C 末端截短的转基因小鼠模型，我将 分析牙釉质表型并接受矿物分析和成像方面的额外培训以确定 LRAP的C端是否可以挽救缺乏全长小鼠的非棱柱牙釉质表型 牙釉蛋白 C 末端 (CTRNC)。为了使用体外方法实现这一目标，我将接受以下方面的培训： 成釉细胞培养和转染、质谱、重组蛋白生成、钙 结合、蛋白质组装和矿物质诱导。使用之前的训练和我在 K99 阶段的训练， 第二个目标将在 R00 阶段执行，是确定是否处理 Mmp20 的 LRAP C 末端在分泌性牙釉质形成过程中是必需的。使用转基因小鼠 模型过度表达 LRAP 但缺乏 Mmp20，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 Mmp20 裂解是否发生突变 LRAP C 末端的位点可以影响矿物质的结合和形成或纳米球组装。最后， 将在独立 R00 阶段执行的第三个具体目标是确定 LRAP、 M180 及其裂解产物共同作用，在分泌过程中引导牙釉质矿物质的形成 牙釉质的形成。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 M180、LRAP 和截短 M180 和 LRAP 一起可以改善体内牙釉质形成，或蛋白质组装和矿物质诱导 体外。从所提出的实验中生成的数据可以提出一种机制的建议 牙釉质同工型及其裂解产物参与牙釉质矿化。