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牙釉质素（MMP-20）如何在体外和体内处理牙釉质蛋白，并确定这种处理如何允许适当的牙釉质发育。拟议的研究利用了牙釉质领域的独特发展：牙釉质素基因敲除小鼠的产生。釉素 (-/-) 小鼠表现出严重的牙釉质表型，包括牙釉质发育不良和消失的杆状图案，没有其他可检测到的组织畸形。敲除小鼠证明牙釉质基质蛋白的蛋白水解是牙釉质形成的关键部分，并使我们能够测试有关体内牙釉质裂解的功能影响的具体假设。这些假设是：1) 釉质素是在小鼠釉质生成的分泌阶段切割釉质蛋白的唯一蛋白酶，2) 釉质素敲除小鼠特征的釉质基质蛋白将被未切割，因此仍处于其分泌形式，3) 釉原蛋白和/或成釉素切割产物对于釉质棒组织是必需的，4) 全长牙釉质蛋白催化晶体延伸的增加，一旦完成，随后进行蛋白水解以分解结构并为另一轮晶体延伸让路。通过对从釉质素敲除和野生型小鼠中获得的发育中牙齿的牙釉质基质进行蛋白质组学表征（目标1），将获得关于各种牙釉质蛋白裂解和选择性剪接产物的相对丰度的重要新信息。通过确定釉质素的底物特异性（目标 2），我们将了解釉质素裂解位点偏好和首选裂解位点的可及性（即蛋白质折叠是否发挥作用？）在确定釉质基质的组成中的相对贡献。为了测试我们假设的有效性，我们建议利用基因敲除小鼠进行转基因实验，以确定牙釉质和/或成釉素裂解产物是否是牙釉质棒组织所必需的（目标3），并确定全长牙釉质蛋白的蛋白水解是否是牙釉质微晶伸长所必需的（目标4）。该应用利用独特的工具（釉质素敲除小鼠）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面向肽库，以帮助确定蛋白质加工如何实现正确的牙釉质发育。