Structural determinants of amelogenin function in regulating enamel formation

描述(申请人提供)：这项新的申请侧重于确定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。基于大量以前的发现，我们的总体假设是丝氨酸-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(ACP)纳米颗粒的能力大大增强。我们进一步假设，随后需要对天然全长釉原蛋白进行蛋白水解性修饰，以促进ACP的排列和转化为有序的磷灰石晶体束，如在釉质发育的分泌阶段所看到的那样。我们认为釉质溶素在这一转化过程中起着关键作用。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。假说将在体外进行验证，主要是使用含有单一磷酸化位点的天然釉原蛋白，并在体内使用釉裂蛋白缺失的小鼠。将使用多种互补的方法实现四个具体目标，包括：动态光散射、透射电子显微镜、电子衍射、傅立叶变换红外光谱、拉曼显微光谱、小角X射线散射和冷冻显微镜。具体地说：目的1.确定磷酸化对天然全长釉原蛋白逐步分级组装的影响，检验磷酸化影响釉原蛋白低聚体的形成和结构以及随后的高级组装的假说；目的2.确定磷酸化的天然全长釉原蛋白有效稳定ACP纳米粒的机制，检验单个丝氨酸-16位点的磷酸化影响蛋白质构象和结构变化，从而独特地增强全长釉原蛋白与形成ACP纳米粒相互作用的能力的假说；目的3.进行体外研究，以验证体内可见的ACP纳米颗粒向有序的羟基磷灰石(HA)晶体的排列和随后的转化是由天然全长釉质原蛋白的特定蛋白水解性修饰诱导的假说；以及目的4.通过检验无序的ACP样矿物将在体内持续存在的假说，在体内验证釉溶素在调节排列的ACP颗粒到有序的HA晶体束的转变中起重要作用。

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.