Essential role of amelogenin phosphorylation in tooth enamel formation

这一新的R01提案旨在阐明单一氨基酸的磷酸化在 最丰富的釉质基质蛋白，釉原蛋白，在釉质形成的调节中发挥作用。建议 研究基于我们广泛的新发现，表明单个丝氨酸位点(S-16)的磷酸化在天然 釉原蛋白是形成高度有序的釉质结构的关键。使用一种新型的敲入(KI)小鼠 我们实验室开发的模型，具有阻止釉原蛋白磷酸化的S16到丙氨酸的取代， 我们现在首次在活体内证明了釉原蛋白的磷酸化在 釉质发生的分泌期和成熟期。人牙釉质组织发育的广泛分析 KI、杂合子(HET)和野生型(WT)产仔表明KI小鼠表现出不同的釉质表型， 包括牙釉质杆状结构的丧失，哺乳动物牙釉质的特征，大量的表面缺陷， 釉质晶体较短，发育不良，钙化程度低。特别值得注意的是，HET牙釉质被发现是镶嵌的 在自然界中，如WT牙釉质中所见，其区域也包含正常的棱柱状结构。我们还发现了 KI成釉细胞缺乏Tomes‘s突起，表现为成釉细胞层组织丧失，严重 通过分泌期逐渐形成的细胞病理学。这些发现，以及最近的其他 来自我们实验室的证据，已经引导我们开发了关于 釉原蛋白磷酸化在调控釉质矿化和维持成釉细胞完整性中的作用 并在釉质形成过程中发挥作用。与矿化有关的拟议功能活动反映了 天然磷酸化全长釉原蛋白及其优势磷酸化蛋白的能力增强 稳定矿物相前体的解理产物，作为一种在整个过程中控制矿化的手段 成釉作用。我们进一步假设，缺乏釉原蛋白磷酸化会导致细胞- 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同位生长和组织； 确定KI突变体牙釉质发育阶段性异常的基础；确定S-16 釉原蛋白与其他必需的釉质基质蛋白相互作用需要釉原蛋白的磷酸化 在釉质形成过程中；并阐明釉原蛋白磷酸化在维持牙釉质形成过程中的重要性 成釉细胞在成釉过程中的完整性和功能。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 釉质疾病和影响龋病易感性的因素。这项工作的圆满完成 这也将为改进牙釉质再生方法的发展提供新的见解。 鉴于龋齿的高患病率，有必要提高对这些著名领域的认识。

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.