Enamelysin Processing Mechanisms in Amelogenesis

描述（由申请人提供）：本申请的重点是定义珐琅质蛋白（MMP-20）如何在体外和体内处理珐琅质蛋白，并确定这种处理如何允许适当的珐琅质发育。提出的研究利用了珐琅质领域的一个独特发展：产生一个珐琅质蛋白敲除小鼠。釉质溶素（-/-）小鼠表现出严重的釉质表型，包括发育不全的釉质和消失的棒状图案，没有其他可检测到的组织畸形。敲除小鼠证明了牙釉质基质蛋白的蛋白水解是牙釉质形成的关键部分，并使我们能够在体内测试有关牙釉质蛋白裂解功能影响的特定假设。这些假设是：1)在小鼠成釉发育的分泌阶段，釉蛋白酶是唯一能切割釉质蛋白的蛋白酶；2)在敲除釉蛋白酶的小鼠中，釉质基质蛋白不会被切割，因此仍保持其分泌形式；3)釉原蛋白和/或成釉酶的切割产物是釉质棒组织所必需的；4)全长釉质蛋白的组装催化晶体伸长的增加，一旦完成，随后是蛋白质水解，分解结构，为另一轮晶体延伸让路。通过对釉质酶敲除和野生型小鼠发育中的牙釉质基质进行蛋白质组学表征（Aim 1），将获得关于各种釉质蛋白切割和替代剪接产物的相对丰度的重要新信息。通过确定釉质蛋白酶的底物特异性（目的2），我们将了解釉质蛋白酶裂解位点偏好的相对贡献以及优选裂解位点的可及性（即，蛋白质折叠是否起作用？）在确定釉质基质的组成中。为了验证我们假设的有效性，我们建议利用基因敲除小鼠进行转基因实验，以确定成釉素和/或成釉素切割产物是否对釉质棒组织（目的3）是必要的，并确定全长釉质蛋白的蛋白质水解是否对釉质晶体的延伸是必要的（目的4）。该应用程序利用独特的工具（釉质溶素敲除小鼠）和最新的高度先进的技术，包括用于蛋白质分离的ProteoSep系统和用于底物特异性分析的基于混合物的定向肽库，以帮助确定蛋白质加工如何允许适当的珐琅质发育。

DESCRIPTION (provided by applicant): The focus of this application is to define how enamelysin (MMP-20) processes enamel proteins in vitro and in vivo, and to determine how such processing allows for proper enamel development. The proposed research takes advantage of a unique development in the enamel field: the generation of an enamelysin knockout mouse. The enamelysin (-/-) mouse displays a severe enamel phenotype consisting of hypoplastic enamel and an obliterated rod pattern with no other detectable tissue malformations. The knockout mouse proves that proteolysis of enamel matrix proteins is a critical part of Dental enamel formation and allows us to test specific hypotheses concerning the functional effects of enamelysin cleavages in vivo. These hypotheses are: 1) enamelysin is the only protease that cleaves enamel proteins during the secretory stage of mouse amelogenesis, 2) enamel matrix proteins characterized from the enamelysin knockout mouse will be uncleaved, and therefore still in their secreted forms, 3) amelogenin and/or ameloblastin cleavage products are necessary for enamel rod organization, and 4) assemblies of full-length enamel proteins catalyze an increment of crystal elongation that, once completed, is followed by proteolysis to disassemble the structure and make way for another round of crystal extension. By performing a proteomic characterization of the enamel matrix of developing teeth obtained from the enamelysin knockout and from wild-type mice (Aim 1), important new information will be gained on the relative abundance of various enamel protein cleavage and alternative splice products. By determining the substrate specificity of enamelysin (Aim 2) we will understand the relative contributions of enamelysin cleavage site preferences and the accessibility of preferred cleavage sites (i.e., does protein folding play a role?) in determining the make-up of the enamel matrix. To test the validity of our hypotheses, we propose to utilize the knockout mice for transgenic experiments that determine if amelogenin and/or ameloblastin cleavage products are necessary for enamel rod organization (Aim 3) and to determine if proteolysis of full-length enamel proteins is necessary for enamel crystallite elongation (Aim 4). This application utilizes a unique tool (enamelysin knockout mouse) and recent, highly advanced technologies including, the ProteoSep system for protein isolation and mixture based oriented peptide libraries for substrate specificity analysis, to help determine how protein processing allows for proper enamel development.