Essential role of amelogenin phosphorylation in tooth enamel formation

牙釉质磷酸化在牙釉质形成中的重要作用

• 批准号: 10458671
• 负责人: ELIA BENIASH
• 金额: $ 45.1万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-14 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458671
• 关键词: Acidity; Affect; Alanine; Ameloblasts; Amelogenesis; Amino Acids; Area; Autophagocytosis; Biochemical; Cell Death; Cell physiology; Cells; Cellular biology; Crystal Formation; Crystallization; Defect; Dental Caries Susceptibility; Dental Enamel; Dental caries; Deposition; Development; Disease; Electron Microscopy; Enamel Formation; Exhibits; Extracellular Matrix Proteins; Goals; Growth; High Prevalence; In Vitro; Incisor; Inherited; Knock-in; Knock-in Mouse; Laboratories; Lead; Length; Mass Spectrum Analysis; Methods; Minerals; Molecular Biology; Mosaicism; Mus; Mutant Strains Mice; Mutate; Mutation; Nature; Pathology; Pathway interactions; Pattern; Phase; Phenotype; Phosphorylation; Play; Predisposition; Process; Proteins; Recombinant Proteins; Regulation; Resolution; Rod; Role; Serine; Site; Stress; Structure; Surface; Techniques; Testing; Time; Tissues; Tooth regeneration; Tooth structure; Transmission Electron Microscopy; Variant; Work; ameloblastin; amelogenin; base; biomineralization; bone; design; driving force; electron diffraction; electron tomography; enamel matrix proteins; enamelin; endoplasmic reticulum stress; fascinate; improved; in vivo; insight; mineralization; mouse model; mutant; novel; photoemission; prevent; trafficking; transcriptome; transcriptome sequencing

This new R01 proposal is designed to elucidate the essential role the phosphorylation of a single amino acid in the most abundant enamel matrix protein, amelogenin, plays in the regulation of enamel formation. Proposed studies build on our extensive new findings that show that phosphorylation of a single serine site (S-16) in native amelogenin is critical for the formation of the highly-ordered enamel structure. Using a novel knock-in (KI) mouse model developed in our laboratory with a S16 to alanine substitution that prevents amelogenin phosphorylation, we have now for the first time demonstrated in vivo that amelogenin phosphorylation plays an essential role in both the secretory and maturation stages of amelogenesis. Extensive analyses of developing enamel tissues from KI, heterozygous (HET) and wild-type (WT) littermates reveal that KI mice exhibit distinct enamel phenotypes, including, the loss of enamel rod structure, the hallmark feature of mammalian enamel, numerous surface defects, shorter enamel crystals, hypoplasia and hypocalcification. Of particular note, HET enamel was found to be mosaic in nature with regions that also contain normal prismatic structures as seen in WT enamel. We have also found that KI ameloblasts lack Tomes' processes and exhibit a loss of organization of the ameloblast layer and severe cell pathology that builds gradually through the secretory stage. These findings, along with other recent evidence from our laboratories, have lead us to develop new working hypotheses regarding the role of amelogenin phosphorylation in the regulation of enamel mineralization and in maintaining ameloblast integrity and function during amelogenesis. Proposed functional activities with respect to mineralization reflect the enhanced capacity of both native phosphorylated full-length amelogenin and its predominant phosphorylated cleavage products to stabilize mineral phase precursors, as a means to control mineralization throughout amelogenesis. We further hypothesize that lack of amelogenin phosphorylation leads to disruption of cell- matrix interactions and trafficking of enamel matrix proteins. Four (4) specific aims have been proposed: to determine how amelogenin guides the linear appositional growth and organization of enamel crystals; to determine the basis for stage-specific abnormal enamel development in the KI mutants; to determine if S-16 amelogenin phosphorylation is required for amelogenin interactions with other essential enamel matrix proteins during enamel formation; and to elucidate the importance of amelogenin phosphorylation in maintaining ameloblast integrity and function throughout amelogenesis. The proposed studies are designed to provide fundamental insight into the mechanism by which phosphorylated amelogenin serves to regulate the formation of the highly-ordered dental enamel tissue. Long-term, our findings should aid in our understanding of inherited enamel diseases and factors that influence dental caries susceptibility. The successful completion of this work will also provide new insights for the development of improved methods for the regeneration of tooth enamel. Given the high prevalence of dental caries, there is need for improved understanding in these noted areas.

该新的R01建议旨在阐明单个氨基酸在 最丰富的搪瓷基质蛋白Amelogenin在牙釉质形成的调节中发挥作用。建议的 研究基于我们广泛的新发现，这些发现表明，本机中单个丝氨酸位点的磷酸化（S-16） 氨基蛋白蛋白对于形成高度有序的搪瓷结构至关重要。使用新颖的敲入（Ki）鼠标 在我们的实验室中开发的模型S16对丙氨酸取代，可防止氨甲蛋白酶磷酸化， 现在，我们首次在体内证明了蛋白蛋白磷酸化在 分泌阶段和成熟阶段。从开发搪瓷组织的广泛分析 Ki，杂合（HET）和野生型（WT）同窝仔表明，Ki小鼠表现出不同的搪瓷表型， 包括搪瓷杆结构的丧失，哺乳动物搪瓷的标志性特征，许多表面缺陷， 较短的牙釉质晶体，发育不全和低钙化。特别值得注意的是，发现Het搪瓷是马赛克 在自然界中，区域也包含正常的棱柱结构，如WT搪瓷所示。我们也发现 ki成成木缺乏TOMES的过程，并且表现出不足的组织层和严重的组织损失 通过分泌阶段逐渐建立的细胞病理。这些发现，以及其他最近的发现 我们实验室的证据已导致我们提出有关作用的新工作假设 在调节搪瓷矿化和维持成成细胞完整性方面的氨基蛋白蛋白磷酸化 和作用期间的功能。提出的有关矿化的功能活动反映了 天然磷酸化的全长氨基蛋白及其主要磷酸化的磷酸化的能力增强 切割产物稳定矿物相前体，作为控制矿化的一种手段 醒目。我们进一步假设缺乏氨基蛋白酶磷酸化导致细胞破坏 基质相互作用和搪瓷基质蛋白的运输。已经提出了四（4）个特定目标： 确定阿米尔生成蛋白如何指导牙釉质晶体的线性同位生长和组织；到 确定Ki突变体中特异性异常搪瓷发育的基础；确定S-16是否 氨基蛋白磷酸化是与其他必需牙釉质基质蛋白相互作用所必需的 在搪瓷形成期间；并阐明氨基蛋白蛋白磷酸化在维持的重要性 整个没有余生的成熟细胞完整性和功能。拟议的研究旨在提供 对磷酸化氨基蛋白用于调节形成的机制的基本见解 高度有序的牙釉质组织。长期，我们的发现应有助于我们对继承的理解 搪瓷疾病和影响龋齿敏感性的因素。这项工作的成功完成 还将为开发改进的牙釉质再生方法提供新的见解。 鉴于龋齿的高流行，在这些著名领域需要改善理解。