Enamelysin Processing Mechanisms in Amelogenesis

描述(申请人提供)：本申请的重点是确定釉质溶素(基质金属蛋白酶-20)如何在体外和体内处理釉质蛋白，并确定这种处理如何允许釉质的正常发育。这项拟议的研究利用了釉质领域的一项独特发展：产生了一种釉质溶蛋白基因敲除小鼠。釉质溶素(-/-)小鼠表现出严重的釉质表型，包括发育不良的釉质和消失的杆状图案，没有其他可检测到的组织畸形。这只基因敲除的小鼠证明了釉质基质蛋白的蛋白分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釉质溶蛋白裂解的功能效应的特定假设。这些假设是：1)釉溶素是在小鼠成釉过程的分泌阶段唯一能分解釉蛋白的水解酶，2)来自釉溶素基因敲除小鼠的釉基质蛋白将不被切割，因此仍然以其分泌的形式存在，3)釉原蛋白和/或成釉蛋白切割产物是釉质棒组织所必需的，以及4)全长釉蛋白的组装催化晶体延长，一旦完成，随后的蛋白水解物将分解结构，为下一轮晶体延伸让路。通过对从釉质溶蛋白基因敲除和野生型小鼠(目标1)获得的发育中牙齿的釉质基质进行蛋白质组学分析，将获得关于各种釉质蛋白切割和替代剪接产物的相对丰富的重要新信息。通过确定釉解素的底物专一性(目标2)，我们将了解釉解素切割位点偏好和偏好切割位点的可及性的相对贡献(即，蛋白质折叠起作用吗？)在确定牙釉质基质的组成方面。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，以确定釉质原蛋白和/或成釉蛋白裂解产物是否对釉质棒组织是必需的(目标3)，并确定全长釉质蛋白的蛋白质分解是否对釉质微晶的伸长是必要的(目标4)。这项应用利用一种独特的工具(釉质蛋白基因敲除小鼠)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多肽库，以帮助确定蛋白质处理如何允许适当的釉质发育。

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.