The Role of LRAP in Enamel Mineral Formation

LRAP 在牙釉质矿物形成中的作用

• 批准号: 8270796
• 负责人: Megan Kardon Pugach
• 金额: $ 7.71万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8270796
• 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; amelogenin; improved; in vivo; interest; leucine-rich amelogenin peptide; mineralization; mouse model; nanoindentation; nanomechanical; overexpression; research study

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.

描述(申请人提供)：釉原蛋白的突变导致釉质缺陷，称为釉质发生不全(AI)。进一步了解不同的釉原蛋白亚型是如何在釉质基质中分泌的，将有助于阐明AI的发病机制。这项研究的目的是研究富含亮氨酸的釉原蛋白多肽(LRAP)C末端的加工在釉质形成中的功能重要性，以及LRAP如何与全长釉原蛋白(M180)相互作用形成釉质棱柱。我选择了分子生物学和一代造釉蛋白AI小鼠模型专家Carolyn Gibson作为我的主要导师，Pamela DenBesten作为我的共同导师，她是釉蛋白生物化学和成釉细胞培养方面的专家。对于K99，指导阶段，第一个特定的目标是确定富含亮氨酸的釉原蛋白多肽(LRAP)的C末端如何参与釉质棱柱的形成。使用高表达LRAP和M180的转基因小鼠模型，我将分析釉质表型，并接受矿物分析和成像方面的额外培训，以确定LRAP的C-末端是否可以挽救缺乏全长釉原蛋白C-末端(CTRNC)的小鼠的非棱柱状釉质表型。为了使用体外方法实现这一目标，我将接受成釉细胞培养和转基因、质谱学、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。利用先前的培训和我在K99阶段的培训，第二个目标将在R00阶段进行，目的是确定在分泌牙釉质形成过程中是否需要MMP20处理LRAP的C-末端。利用高表达LRAP但缺乏MMP20的转基因小鼠模型，研究釉质结构和切割产物。我还将使用重组蛋白和细胞培养来确定LRAP C末端MMP20裂解位点的突变是否会影响矿物结合和形成或纳米球组装。最后，在独立的R00阶段将实现的第三个具体目标是确定LRAP、M180和它们的切割产物如何在分泌性釉质形成过程中共同指导釉质矿物的形成。我将使用体内和体外转基因小鼠模型、纳米压痕、重组蛋白和细胞培养的方法来确定M180、LRAP和截短的M180和LRAP一起是否可以在体内促进釉质形成，或者在体外促进蛋白质组装和矿物质诱导。从拟议的实验中产生的数据可以导致关于釉原蛋白亚型及其切割产物如何参与牙釉质矿化的机制的命题。 公共卫生相关性：釉原蛋白的突变会导致釉质缺陷，称为釉质发生不全(AI)。提高对牙釉质形成过程中基因作用的了解，可以改善这种情况下儿童的治疗前景。这项研究的基本目标是确定丰富的釉原蛋白亚型是如何被加工并参与釉质矿物形成的，这将有助于阐明AI疾病的机制。