ENAMEL PROTEIN IN TISSUE SPECIFIC BIOMINERALIZATION

组织特异性生物矿化中的牙釉质蛋白

• 批准号: 6104690
• 负责人: ALAN G FINCHAM
• 金额: $ 22.53万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-15 至 2001-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104690
• 关键词: ameloblasts; amelogenin; antisense nucleic acid; chemical structure function; crystallization; dental development; dental materials; endopeptidases; hydroxyapatites; laboratory mouse; odontoblasts; protein engineering; protein sequence; protein structure function; proteolysis; recombinant proteins; site directed mutagenesis; tissue /cell culture; tooth enamel

The overall objective of this proposal is to understand the function of enamel gene products (i.e., amelogenins, enamelins and enamel proteinases), to advance the understanding of the process of amelogenesis, to create a strategy to produce an enamel bioceramic as a novel dental restorative biomaterial, and to improve the diagnosis of the various types of amelogenesis imperfecta. We hypothesize the motifs within the anionic enamelin protein (e.g.tuftelin) structure serve to nucleate the initial crystalline phase, and that motifs within amelogenin protein aggregates regulate the size, orientation and patterns of the growing enamel crystals. We further predict that proteases (e.g. calcium-dependent metalloproteinases) degrade proteins and facilitate the removal of water and protein from the mineralizing extracellular matrix. To address this hypothesis, the following Specific Aims have been formulated: (1) To determine the function of amelogenins and enamelins during mouse amelogenesis in in vitro ameloblast cell culture, in transfected odontoblast cell cultures and mouse molar tooth organ culture systems using antisense inhibition strategies. (2) To determine the function of enamelins to nucleate octacalcium phosphate (OCP) or hydroxyapatite (HAP) in vitro. Recombinant enamelins will be evaluated as nucleators for OCP or HAP in vitro. (3) To determine the function of amelogenins to regulate the size and patterns of OCP or HAP crystal growth in vitro. Recombinant amelogenins will be evaluated as regulators for the crystal growth and patterns of growth in vitro. Subsequent protein engineering approaches using site-directed mutagenesis will enable the identification of the motifs required for OCP or HAP crystal growth along the c-axis as well as the patterns of crystal growth. (4) To determine the function of enamel proteases to regulate both structural properties and the stepwise removal of enamel proteins and subsequently to regulate crystal formation. We propose to isolate and characterize the enamel protease gene product. (5) To fabricate enamel HAP crystal formation in vitro. Enamel replacement therapy is a strategy to use recombinant enamel protein motifs to fabricate an enamel bioceramic as a possible dental enamel restorative biomaterial. These experiments are a logical extension of the proposed functional studies to define how enamel proteins regulate enamel OCP and HAP crystal formation.

该提案的总体目标是了解 牙釉质基因产物（即釉原蛋白、牙釉质和牙釉质） 蛋白酶），以促进对过程的理解 釉质形成，制定一种生产牙釉质生物陶瓷的策略 新型牙科修复生物材料，并提高诊断能力 各种类型的釉质发育不全。 我们假设主题 阴离子牙釉质蛋白（例如 tuftelin）结构内的作用是 使初始结晶相成核，并且牙釉蛋白内的基序 蛋白质聚集体调节蛋白质聚集体的大小、方向和模式 牙釉质晶体生长。 我们进一步预测蛋白酶（例如 钙依赖性金属蛋白酶）降解蛋白质并促进 从矿化细胞外基质中去除水和蛋白质。 为了解决这一假设，我们制定了以下具体目标 制定：（1）确定牙釉蛋白和牙釉蛋白的功能 在体外成釉细胞培养的小鼠成釉细胞过程中， 转染的成牙本质细胞培养物和小鼠磨牙器官培养物 使用反义抑制策略的系统。 (2) 确定 釉质使磷酸八钙 (OCP) 成核的功能或 羟基磷灰石（HAP）体外。 将评估重组牙釉质 作为 OCP 或 HAP 体外成核剂。 (3) 确定函数 釉原蛋白调节 OCP 或 HAP 晶体的大小和图案 体外生长。 重组牙釉蛋白将作为调节剂进行评估 用于体外晶体生长和生长模式。 随后的 使用定点突变的蛋白质工程方法将 能够识别 OCP 或 HAP 晶体所需的基序 沿c轴的生长以及晶体生长的模式。 (4) 确定釉质蛋白酶调节两种结构的功能 特性和逐步去除牙釉质蛋白，然后 调节晶体形成。 我们建议分离并表征 釉质蛋白酶基因产物。 (5) 搪瓷HAP晶体的制作 体外形成。 牙釉质替代疗法是一种使用策略 重组牙釉质蛋白基序来制造牙釉质生物陶瓷 一种可能的牙釉质修复生物材料。 这些实验是 所提议的功能研究的逻辑延伸，以定义如何 牙釉质蛋白调节牙釉质 OCP 和 HAP 晶体的形成。