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

基本信息

批准号：
10904334
负责人：
ELIA BENIASH
金额：
$ 6.47万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-14 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10904334
关键词：
Acidity Affect Alanine Ameloblasts Amelogenesis Amino Acids Area Autophagocytosis Biochemical Cell Death Cell physiology Cells Cellular biology Crystal Formation Defect Dental Caries Susceptibility Dental Enamel Dental caries Deposition Development Disease Electron Microscopy Enamel Formation Exhibits Extracellular Matrix Proteins Goals Growth Heterozygote High Prevalence In Vitro Incisor Inherited Knock-in Knock-in Mouse Laboratories Length Mass Spectrum Analysis Methods Minerals Molecular Biology 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 biomineralization bone design driving force electron diffraction electron tomography enamel matrix proteins enamelin endoplasmic reticulum stress fascinate improved in vivo insight mineralization mosaic mouse model mutant novel photoemission prevent trafficking transcriptome transcriptome sequencing

项目摘要

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.

这项新的 R01 提案旨在阐明单个氨基酸磷酸化在最丰富的牙釉质基质蛋白，釉原蛋白，在牙釉质形成的调节中发挥作用。建议的研究建立在我们广泛的新发现的基础上，这些新发现表明天然植物中单个丝氨酸位点 (S-16) 的磷酸化釉原蛋白对于高度有序的牙釉质结构的形成至关重要。使用新型敲入 (KI) 小鼠我们实验室开发的模型将 S16 替换为丙氨酸，可防止釉原蛋白磷酸化，我们现在首次在体内证明牙釉蛋白磷酸化在釉质发生的分泌阶段和成熟阶段。对发育中的牙釉质组织进行广泛分析 KI、杂合子 (HET) 和野生型 (WT) 同窝小鼠显示 KI 小鼠表现出不同的牙釉质表型，包括，哺乳动物牙釉质标志性特征——牙釉质棒结构的丧失、众多表面缺陷、牙釉质晶体较短、发育不全和钙化不足。特别值得注意的是，HET 珐琅被发现是马赛克的自然界中也含有正常棱柱结构的区域，如 WT 牙釉质中所示。我们还发现 KI 成釉细胞缺乏 Tomes 突起，并表现出成釉细胞层组织的丧失，并且严重通过分泌阶段逐渐建立的细胞病理学。这些发现以及最近的其他发现来自我们实验室的证据，引导我们提出关于作用的新工作假设釉原蛋白磷酸化在釉质矿化调节和维持成釉细胞完整性中的作用和在釉质形成过程中的功能。拟议的矿化功能活动反映了天然磷酸化全长牙釉蛋白及其主要磷酸化的能力增强裂解产物以稳定矿物相前体，作为控制整个矿化的一种手段釉质发生。我们进一步假设缺乏牙釉蛋白磷酸化会导致细胞破坏基质相互作用和牙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牙釉质晶体的线性对位生长和组织；到确定 KI 突变体阶段特异性牙釉质发育异常的基础；确定是否为S-16 牙釉原蛋白磷酸化是牙釉蛋白与其他必需牙釉质基质蛋白相互作用所必需的在牙釉质形成过程中；并阐明釉原蛋白磷酸化在维持成釉细胞在整个成釉细胞形成过程中的完整性和功能。拟议的研究旨在提供对磷酸化牙釉蛋白调节形成机制的基本了解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对遗传的理解牙釉质疾病和影响龋齿易感性的因素。本次工作的顺利完成还将为开发改进牙釉质再生方法提供新的见解。鉴于龋齿的高患病率，需要加深对这些领域的了解。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice.

釉原蛋白磷酸化缺陷小鼠中牙釉质矿化的生物控制丧失。

DOI：
发表时间：
2022-06
期刊：
影响因子：
3
作者：
Stifler, Cayla A;Yamazaki, Hajime;Gilbert, Pupa U P A;Margolis, Henry C;Beniash, Elia
通讯作者：
Beniash, Elia

Identification of stages of amelogenesis in the continuously growing mandiblular incisor of C57BL/6J male mice throughout life using molar teeth as landmarks.

使用臼齿作为标志，识别 C57BL/6J 雄性小鼠一生中持续生长的下颌切牙的釉质形成阶段。

DOI：
发表时间：
2023
期刊：
影响因子：
0
作者：
Bui, Ai Thu;Lukashova, Lyudmila;Verdelis, Kostas;Vasquez, Brent;Bhogadi, Lasya;Gabe, Claire M;Margolis, Henry C;Beniash, Elia
通讯作者：
Beniash, Elia