Structural determinants of amelogenin function in regulating enamel formation

描述（由申请人提供）：这项新申请的重点是确定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。基于广泛的先前发现，我们的总体假设是丝氨酸-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。我们进一步假设，在珐琅质发育的分泌阶段，需要对天然全长淀粉原蛋白进行随后的蛋白水解修饰，以促进ACP的排列和向有序的磷灰质晶体束的转变。我们认为，珐琅素在这一转变过程中起着至关重要的作用。拟议的研究旨在为基质蛋白（如淀粉原蛋白）如何控制矿化组织中的矿化和结构提供基本见解。作为一个长期的目标，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。鉴于龋齿的高发率，对比现有的修复方法更好的修复方法的需求是巨大的。假设将在体外进行测试，主要使用含有单个磷酸化位点的天然淀粉原蛋白，在体内使用无釉蛋白酶小鼠。四个具体目标将采用多种互补的方法，包括：动态光散射、透射电子显微镜、电子衍射、傅里叶变换红外光谱、拉曼微光谱、小角度x射线散射和低温显微镜。具体来说：目标1。确定磷酸化对天然全长淀粉原蛋白的阶梯级组装的影响，验证磷酸化影响淀粉原蛋白低聚物的形成和结构及其随后的高阶组装的假设；目标2。为了确定磷酸化的天然全长淀粉原蛋白有效稳定ACP纳米颗粒的机制，为了验证单个丝氨酸-16位点的磷酸化影响蛋白质构象和结构变化的假设，从而独特地增强了全长淀粉原蛋白与形成ACP纳米颗粒相互作用的能力；目标3。进行体外研究，以验证ACP纳米颗粒在体内排列和随后转化为有序的羟基磷灰石（HA）晶体束的假设，这是由天然全长淀粉原蛋白的特定蛋白水解修饰诱导的；和Aim 4。为了在体内验证珐琅素在调节排列的ACP颗粒向有序的HA晶体束的转变中起着至关重要的作用，通过测试与野生型小鼠相比，在缺乏珐琅素的情况下，无序的ACP样矿物质将在体内持续存在的假设。

DESCRIPTION (provided by applicant): This new application focuses on the determination of 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 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. We further hypothesize that subsequent proteolytic modification of native full-length amelogenin is required to promote the alignment and transformation of ACP to ordered bundles of apatitic crystals, as seen in the secretory stage of enamel development. We propose that enamelysin plays a critical role in this transformation process. 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. Hypotheses will be tested in vitro, primarily using native amelogenins that contain a single phosphorylated site, and in vivo, using enamelysin null mice. Four specific aims will be carried out using multiple complementary approaches, including: dynamic light scattering, transmission electron microscopy, electron diffraction, Fourier-transform infrared spectroscopy, Raman microspectroscopy, small angle x-ray scattering, and cryomicroscopy. Specifically: Aim 1. To determine the effect of phosphorylation on the step-wise hierarchical assembly of native full-length amelogenin, to test the hypothesis that phosphorylation affects the formation and structure of amelogenin oligomers and their subsequent higher-order assembly; Aim 2. To determine the mechanism by which phosphorylated native full-length amelogenin effectively stabilize nanoparticles of ACP, to test the hypothesis that phosphorylation of the single serine-16 site affects protein conformation and structural changes that uniquely enhance the capacity of the full-length amelogenin to interact with forming ACP nanoparticles; Aim 3. To conduct in vitro studies to test the hypothesis that the alignment and subsequent transformation of ACP nanoparticles seen in vivo to ordered bundles of hydroxyapatite (HA) crystals is induced by specific proteolytic modifications of native full-length amelogenin; and Aim 4. To verify in vivo that enamelysin plays an essential role in regulating the transformation of aligned ACP particles to ordered bundles of HA crystals, by testing the hypothesis that disordered ACP-like minerals will persist in vivo, in the absence of enamelysin, in contrast to what is seen in wild type mice.