Role of amelogenin phosphorylation in regulating enamel formation in vivo

描述（由申请人提供）：这项R21申请的重点是为主要的细胞外珐琅质基质蛋白，淀粉原蛋白，调节初始珐琅质矿物形成和组织组织的机制提供关键的新见解。基于广泛的先前发现，我们的总体假设是丝氨酸-16 （S-16）的磷酸化调节了天然（磷酸化）全长淀粉原蛋白的结构和分层组装，从而大大增强了其稳定无定形磷酸钙（ACP）纳米粒子的能力。然而，这一假设仅仅是基于体外研究结果，而淀粉原蛋白磷酸化在体内的重要性是未知的，基本上没有被探索过。本R21申请的目的是探索这一研究不足的领域，并开发必要的工具来直接在体内验证这一假设，为进一步研究淀粉原蛋白磷酸化在调节牙釉质形成中的具体作用提供基础和手段。为了实现这些目标，提出了两(2)个多维度的具体目标。具体而言，我们提出：在目标1中。建立一种新的小鼠模型，其中不发生淀粉原蛋白磷酸化，用于评估淀粉原蛋白磷酸化在体内牙釉质发育中的功能后果。一个敲入（KI）小鼠模型将通过构建一个靶向载体来开发，在这个载体中，丙氨酸唯一的S-16磷酸化位点的点突变以组成的方式被设计；在第二章中。为了评估改变的淀粉原蛋白对产生非磷酸化淀粉原蛋白的KI小鼠发育中的珐琅质矿物的阶段、形态和结构组织的影响，并与WT幼崽产生的淀粉原蛋白进行比较。后一项研究将用于测试

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.