Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

• 批准号: 7638611
• 负责人: JOHN D BARTLETT
• 金额: $ 51.17万
• 依托单位: ADA FORSYTH INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7638611
• 关键词: Alternative Splicing; Amelogenesis; Cleaved cell; Dental Enamel; Dental Enamel Hypoplasia; Dentin; Development; Electrons; Enamel Formation; Figs - dietary; Fracture; Generations; Genes; Human; In Vitro; Incisor; Knock-out; Knockout Mice; Length; MMP-20; Messenger RNA; Molecular; Mus; N-terminal; Nature; Pattern; Peptide Hydrolases; Peptide Library; Phenotype; Play; Process; Protein Analysis; Protein Isoforms; Proteins; Proteolysis; Proteolytic Processing; Proteomics; Qualifying; RNA Splicing; Relative (related person); Research; Research Personnel; Role; Scanning; Site; Staging; Structure; Substrate Specificity; System; Technology; Testing; Thick; Tissues; Tooth structure; Transcript; Transgenic Organisms; Translating; Wild Type Mouse; amelogenin; base; disease-causing mutation; enamel matrix proteins; enamelysin; in vivo; insight; malformation; preference; programs; protein folding; research study; retinal rods; tool

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.

描述（由申请人提供）：本申请的重点是定义在体外和体内的令人印象（MMP-20）处理搪瓷蛋白的过程，并确定这种处理如何允许正确的搪瓷开发。拟议的研究利用了搪瓷领域的独特发展：一群令人着迷的淘汰小鼠的产生。迷名的（ - / - ）小鼠显示出严重的搪瓷表型，该表型由腹膜牙釉质和闭塞棒图案组成，没有其他可检测的组织畸形。敲除小鼠证明，牙釉质基质蛋白的蛋白水解是牙齿牙釉质形成的关键部分，使我们能够检验有关体内namelysin裂解功能效应的特定假设。 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全长的搪瓷蛋白会催化晶体伸长的增量，后者一旦完成，然后进行蛋白水解以拆卸结构，并为另一轮晶体延伸而腾出空间。通过执行从名敲除和野生型小鼠获得的发育牙齿的蛋白质组学表征（AIM 1），将获得有关各种搪瓷蛋白裂解和替代剪接产物的相对丰度的重要新信息。通过确定Inamelysin的底物特异性（AIM 2），我们将了解令人着迷的裂解位点偏好的相对贡献，以及首选裂解位点的可及性（即蛋白质折叠起作用起作用？为了测试我们的假设的有效性，我们建议利用敲除小鼠进行转基因实验，以确定搪瓷杆组织（AIM 3）是否必要确定是否需要蛋白质的蛋白质分解，并确定是否需要蛋白质的蛋白质，以确定是否需要全长的搪瓷蛋白来确定源源是烯酰胺蛋白的必要条件（源是烯酰胺蛋白是烯酰胺蛋白是必需的，源是源代烯酰胺蛋白是必需的。该应用程序利用了独特的工具（令人着迷的敲除小鼠）和最新高级的技术，包括用于蛋白质隔离的蛋白质系统和基于混合的基于底物特异性分析的基于混合的定向肽库，以帮助确定蛋白质加工如何允许蛋白质加工。