Enamelysin Processing Mechanisms in Amelogenesis

釉质生成中的釉质加工机制

• 批准号: 7873019
• 负责人: JOHN D BARTLETT
• 金额: $ 52.19万
• 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/7873019
• 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溶釉素（MMP-20）如何在体外和体内加工釉质蛋白，并确定这种加工如何允许适当的釉质发育。拟议的研究利用了釉质领域的一个独特发展：产生釉质溶解素敲除小鼠。釉质溶解素（-/-）小鼠表现出严重的釉质表型，包括发育不全的釉质和一个闭塞的杆模式，没有其他可检测到的组织畸形。基因敲除小鼠证明，釉质基质蛋白的蛋白水解是牙釉质形成的关键部分，并允许我们测试特定的假设，在体内的功能性影响的釉质溶解素裂解。这些假设是：1）溶釉素是在小鼠成釉的分泌阶段期间切割釉质蛋白的唯一蛋白酶，2）以溶釉素敲除小鼠为特征的釉质基质蛋白将不被切割，因此仍处于其分泌形式，3）成釉蛋白和/或成釉蛋白切割产物是釉棒组织所必需的，和4）全长釉质蛋白的组装催化晶体伸长的增加，一旦完成，接着是蛋白质水解，以分解结构，并为另一轮晶体延伸让路。通过对从釉质溶素敲除小鼠和野生型小鼠获得的发育中牙齿的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选择性剪接产物的相对丰度的重要新信息。通过确定溶釉素的底物特异性（目的2），我们将理解溶釉素切割位点偏好的相对贡献和优选切割位点的可及性（即，蛋白质折叠是否起作用？）以确定釉质基质的组成。为了验证我们的假设的有效性，我们建议利用基因敲除小鼠进行转基因实验，以确定釉原蛋白和/或成釉蛋白裂解产物是否是釉质棒组织所必需的（目标3），并确定全长釉质蛋白的蛋白水解是否是釉质微晶伸长所必需的（目标4）。该应用程序利用独特的工具（釉质溶解素敲除小鼠）和最近的高度先进的技术，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釉质发育。