Role of amelogenin phosphorylation in regulating enamel formation in vivo

釉原蛋白磷酸化在调节体内牙釉质形成中的作用

• 批准号: 8636648
• 负责人: HENRY C MARGOLIS
• 金额: $ 24.64万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8636648
• 关键词: Affect; Alanine; Area; Biological; Biomechanics; Chimera organism; Data; Dental Enamel; Dental caries; Development; Digestion; Electrons; Enamel Formation; Engineering; Future; Goals; Hardness; High Prevalence; Histology; Housing; In Vitro; Indium; Investigation; Knock-in Mouse; Laboratories; Lead; Length; Measurement; Methods; Minerals; Modification; Morphology; Mus; Mutant Strains Mice; Mutation; Natural regeneration; Phase; Phosphorylation; Phosphorylation Site; Play; Point Mutation; Procedures; Process; Production; Property; Proteins; Proteolysis; Regulation; Research Design; Reverse Transcriptase Polymerase Chain Reaction; Role; Serine; Site; Staging; Structure; Testing; Tissues; Tooth structure; Western Blotting; Wild Type Mouse; Work; amelogenin; base; calcium phosphate; enamel matrix proteins; improved; in vivo; insight; mineralization; minimal risk; mouse model; mutant; nanoparticle; novel; novel strategies; public health relevance; repaired; restriction enzyme; tissue regeneration; tool; vector

DESCRIPTION (provided by applicant): This R21 application focuses on providing critical new insights into the mechanism by which the predominant extracelluar enamel matrix protein, amelogenin, regulates initial enamel mineral formation and tissue organization. Based on extensive prior findings, our overall hypothesis is that phosphorylation of serine-16 (S-16) modulates the structure and hierarchical assembly of native (phosphorylated) full-length amelogenin, resulting in its greatly enhanced capacity to stabilize amorphous calcium phosphate (ACP) nanoparticles. However, this hypothesis is based solely on in vitro findings and the importance of the role of amelogenin phosphorylation in vivo is unknown and essentially unexplored. The goal of this R21 application is to explore this understudied area and to develop necessary tools to test this hypothesis directly in vivo and provide the basis and means for further studies on the specific role of amelogenin phosphorylation in regulating enamel formation. Two (2) multi-dimensional specific aims have been proposed to achieve these goals. Specifically, we propose: in Aim 1. To develop a novel mouse model in which amelogenin phosphorylation does not occur, to be used to assess the functional consequences of amelogenin phosphorylation in enamel development in vivo. A knock-in (KI) mouse model will be developed through the construction of a targeting vector in which a point mutation of the sole S-16 phosphorylation site for alanine is engineered in a constitutive manner; and in Aim 2. To assess the effect of altered amelogenin on the phase, morphology and structural organization of developing enamel mineral in KI mice that generate non-phosphorylated amelogenin, in comparison to that produced in WT littermates. These latter studies will be carried out to test the hypothesis that phosphorylation of the native full-length amelogenin is required to promote the stabilization, proper alignment and transformation of ACP to ordered apatitic crystals, as seen in vivo. Multiple approaches will be used to characterize the KI mouse, including routine histology, PCR followed by digestion with restriction enzymes, RT-PCR, and Western blot analyses. Developing teeth in KI and WT mice will be characterized using TEM, selected area electron diffraction, Raman microspectroscopy, SEM, micro-CT and via micro-hardness measurements. The proposed studies are designed to provide fundamental insight into how matrix proteins, like amelogenin, control mineralization and structure in mineralized tissues. As a long-term goal, our findings should aid in the development of novel approaches for the regeneration and repair of diseased or damaged dental enamel. Given the high prevalence of dental caries, there is a tremendous need for restorative procedures that are superior to those presently available. Findings obtained from studies proposed in this R21 application will serve as a basis for future investigations on the regulation of enamel formation in vivo.

描述（由申请人提供）：此R21申请专注于提供关键的新见解，以对主要的外部牙釉质基质蛋白Amelogenin，调节初始搪瓷矿物质形成和组织组织。基于大量的先前发现，我们的总体假设是丝氨酸16（S-16）的磷酸化调节天然（磷酸化的）全长酰胺蛋白的结构和层次组装，从而极大地增强了其稳定无脑钙（ACP）Nananoparticletic的能力。然而，该假设仅基于体外发现，并且体内氨基蛋白蛋白磷酸化的作用的重要性尚不清楚且基本上未探索。该R21应用的目的是探索该研究的领域，并开发必要的工具，直接在体内检​​验该假设，并为进一步研究氨基蛋白蛋白磷酸化在调节搪瓷形成中的特定作用提供基础和手段。已经提出了两（2）个多维特定目标来实现这些目标。具体而言，我们建议：在AIM 1中。要开发一种新的小鼠模型，其中未发生amelegen蛋白磷酸化，用于评估体内搪瓷发育中氨基蛋白蛋白磷酸化的功能后果。通过构建靶向载体，将开发出敲入（Ki）的模型，在该靶向载体中，丙氨酸的唯一S-16磷酸化位点的点突变以组成型方式进行设计。在AIM 2中，与WT同胞中产生的相比，在Ki小鼠中开发非磷酸化蛋白质蛋白的牙釉质矿物质的阶段，形态和结构组织的影响，形态和结构组织。这些后者将进行测试 如在体内所见，需要假设天然全长氨基蛋白的磷酸化需要促进ACP稳定，适当的比对和转化为有序的Apatitic晶体。多种方法将用于表征Ki小鼠，包括常规的组织学，PCR，然后使用限制性酶，RT-PCR和Western印迹分析进行消化。使用TEM，选定的区域电子衍射，拉曼微光谱，SEM，Micro-CT和通过微硬度测量值来表征Ki和WT小鼠中发育的牙齿。拟议的研究旨在提供有关基质蛋白（如氨基蛋白酶）如何控制矿化组织中的矿化和结构的基本见解。作为一个长期目标，我们的发现应有助于开发新的方法，用于再生和修复患病或受损的牙釉质。鉴于龋齿的盛行率很高，需要对恢复性程序的巨大需求优于目前可用的程序。从本R21应用中提出的研究中获得的发现将作为对体内搪瓷形成调节的未来研究的基础。