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: 10677921
• 负责人: ELIA BENIASH
• 金额: $ 7.8万
• 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/10677921
• 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.

这个新的R 01建议旨在阐明单个氨基酸的磷酸化在蛋白质合成中的重要作用。 最丰富的釉基质蛋白质釉原蛋白在釉形成的调节中起作用。提出 研究建立在我们广泛的新发现基础上，这些发现表明，在天然细胞中，单个丝氨酸位点（S-16）的磷酸化 釉原蛋白对于高度有序的釉质结构的形成是关键的。使用新型基因敲入（KI）小鼠 在我们实验室开发的模型中，S16被丙氨酸取代，阻止釉原蛋白磷酸化， 我们现在已经首次在体内证实了釉原蛋白磷酸化在 釉质形成的分泌和成熟阶段。对发育中的釉质组织进行了广泛的分析， KI、杂合子（HET）和野生型（WT）同窝出生的小鼠揭示KI小鼠表现出不同的釉质表型， 包括釉质棒结构的丧失，哺乳动物釉质的标志性特征，许多表面缺陷， 较短的釉质晶体，发育不全和钙化不全。特别值得注意的是，发现HET釉质是马赛克状的， 在自然界中具有也包含正常棱柱结构的区域，如在WT釉质中所见。我们还发现 KI成釉细胞缺乏Tomes'过程，并表现出成釉细胞层组织的丧失和严重的 通过分泌阶段逐渐形成的细胞病理学。这些发现，沿着其他最近的 来自我们实验室的证据，引导我们发展新的工作假设， 釉原蛋白磷酸化对釉质矿化的调节及维持成釉细胞完整性的作用 并在釉质形成过程中发挥作用。与矿化有关的拟议职能活动反映了 天然磷酸化的全长釉原蛋白和其主要磷酸化的 裂解产物，以稳定矿物相前体，作为控制矿化的手段 釉质形成我们进一步假设釉原蛋白磷酸化的缺乏导致细胞- 基质相互作用和釉质基质蛋白的运输。提出了四（4）个具体目标： 确定釉原蛋白如何引导釉质晶体的线性并置生长和组织; 确定KI突变体中阶段特异性异常釉质发育的基础;确定S-16是否 釉原蛋白磷酸化是釉原蛋白与其它必需釉基质蛋白相互作用所必需的 并阐明釉原蛋白磷酸化在维持釉质形成中的重要性。 成釉细胞的完整性和功能。拟议的研究旨在提供 对磷酸化釉原蛋白调节形成的机制的基本认识 高度有序的牙釉质组织。从长远来看，我们的发现应该有助于我们理解遗传性 釉质疾病和影响龋齿易感性的因素。这项工作的顺利完成 也将为开发牙齿釉质再生的改进方法提供新的见解。 鉴于龋齿的高流行率，有必要提高对这些领域的认识。