Role of amelogenin phosphorylation in regulating enamel formation in vivo

描述(由申请人提供)：这份R21申请专注于提供关键的新见解，了解主要的细胞外釉质基质蛋白，釉原蛋白，通过其调节初始牙釉质矿物形成和组织结构的机制。基于大量的研究结果，我们的总体假设是，丝氨酸-16(S-16)的磷酸化调控了天然(磷酸化的)全长釉原蛋白的结构和分级组装，导致其稳定无定形磷酸钙纳米颗粒的能力大大增强。然而，这一假说完全基于体外研究结果，体内釉原蛋白磷酸化作用的重要性尚不清楚，基本上也没有被探索过。此R21应用的目的是探索这一未被研究的领域，并开发必要的工具来直接在体内验证这一假说，并为进一步研究釉原蛋白磷酸化在调控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，已经提出了两(2)个多维度的具体目标。具体地说，我们建议：目的1.建立一种新的小鼠模型，在该模型中，釉原蛋白不发生磷酸化，用于评估釉原蛋白磷酸化在体内釉质发育中的功能后果。通过构建定点突变S-16丙氨酸磷酸化位点的靶向载体，建立敲入(KI)小鼠模型；在目标2中，评估改变的釉原蛋白对KI小鼠发育中产生非磷酸化釉原蛋白的釉质矿物的时相、形态和结构组织的影响，并与WT产仔小鼠进行比较。后两项研究将会进行，以测试 假设天然全长釉原蛋白的磷酸化是必需的，以促进ACP的稳定、适当的排列和转化为有序的磷灰石晶体，如在体内所见。将使用多种方法来鉴定KI小鼠，包括常规组织学、聚合酶链式反应、限制性内切酶消化、RT-PCR和蛋白质印迹分析。KI和WT小鼠发育中的牙齿将用透射电子显微镜、选区电子衍射、拉曼显微光谱、扫描电子显微镜、显微CT和VIA显微硬度测量来表征。拟议的研究旨在为基质蛋白(如釉原蛋白)如何控制矿化组织中的矿化和结构提供基本的见解。作为一个长期目标，我们的发现应该有助于开发新的方法来再生和修复患病或受损的牙釉质。鉴于龋齿的高患病率，非常需要比目前可用的修复程序更好的修复程序。在这项R21应用中提出的研究结果将作为未来研究体内釉质形成调节的基础。

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.