ENAMEL MATRIX PROTEIN INTERACTION

牙釉质基质蛋白相互作用

• 批准号: 6516577
• 负责人: Michael Lansdell Paine
• 金额: $ 31.16万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6516577
• 关键词: amelogenin; dental development; dentin; dentinogenesis; enamel organ; gene expression; gene targeting; genetic library; genetically modified animals; laboratory mouse; phosphoproteins; protein protein interaction; protein sequence; protein structure function; yeast two hybrid system

Amelogenin undergoes self-assembly into nanospheres and contains a self-assembly domain located at the amino-terminus. We will pursue hypothesis driven experimental strategy into critical issues impacting upon assembly of the enamel organic matrix into a structure competent to direct biomineralization. We will test the hypothesis that amelogenin self- assembly into nanospheres requires an unaltered amino-terminal self- assembly domain. We predict that alteration in the assembly domain will alter the size of the nanospheres compared to controls. To test this hypothesis we propose two specific aims: 1) To engineer transgenic animals compared to controls. To test this hypothesis we propose two specific aims: 1) To engineer transgenic animals expressing amelogenin that contains alterations or deletions to the self-assembly amino-terminal "A"-domain and to characterize the resulting enamel; 2) to directly measure the strength of amelogenin interactions. Next, we screened for previously unrecognized protein partner(s) that interact with amelogenin interactions. Next, we screened for previously unrecognized protein partner(s) that interact with amelogenin or with tuftelin. We identified an amelogenin interacting protein (AIP) and several tuftelin interacting proteins (TIPs). We will test the hypothesis that the physiologic function for an AIP or TIP is to participate in the assembly of an enamel organic matrix that is competent to direct biomineralization. In specific aim 3 we propose to reduce the availability of the AIP or each TIP target protein using hammerhead ribozymes. We predict pathogenomic alterations in the resulting enamel specific for each reduced target protein. Lastly, we predict that additional proteins will play essential roles during assembly of the enamel organic extracellular matrix. We will focus attention on the critical dentine to enamel junction (DEJ) which serves to anchor the brittle enamel bioceramic onto the underlying softer dentine. We hypothesize that the unique physical properties of the DEJ form as the result of an admixture of proteins synthesized by ameloblasts and dentinoblasts. We will search for critical protein partners expressed during the formation of the DEJ. We will use dentine sialophosphoprotein (DSPP) as bait to screen a cDNA library of tooth specific transcripts for those proteins that interact with DSPP. In specific aim 4, we propose to search for novel proteins that interact with the dentine matrix protein DSPP. The specific aims are a logical extension of data derived from our own accomplishments and from others in the field. Each specific aim complements and builds upon the others. Completing these experiments will extend our understanding of normal and abnormal enamel and DEJ formation. This information will allow us to pursue the creation of an enamel and DEJ biomimetic.

牙釉蛋白经过自组装成纳米球，并包含位于氨基末端的自组装结构域。我们将采用假设驱动的实验策略来解决影响牙釉质有机基质组装成能够直接生物矿化的结构的关键问题。我们将测试牙釉蛋白自组装成纳米球需要未改变的氨基末端自组装结构域的假设。我们预测，与对照相比，组装域的改变将改变纳米球的尺寸。为了检验这一假设，我们提出了两个具体目标：1）与对照动物相比，改造转基因动物。为了检验这一假设，我们提出了两个具体目标：1) 改造表达牙釉蛋白的转基因动物，其中牙釉蛋白含有自组装氨基末端“A”结构域的改变或缺失，并表征所得牙釉质； 2）直接测量釉原蛋白相互作用的强度。接下来，我们筛选了以前未被识别的与牙釉蛋白相互作用的蛋白质伴侣。接下来，我们筛选了以前未被识别的与牙釉蛋白或 tuftelin 相互作用的蛋白质伴侣。我们鉴定了一种牙釉蛋白相互作用蛋白 (AIP) 和几种 tuftelin 相互作用蛋白 (TIP)。我们将检验以下假设：AIP 或 TIP 的生理功能是参与能够直接生物矿化的牙釉质有机基质的组装。在具体目标 3 中，我们建议使用锤头核酶降低 AIP 或每个 TIP 靶蛋白的可用性。 我们预测每种减少的靶蛋白所产生的牙釉质会发生特异性的病原学改变。最后，我们预测其他蛋白质将在牙釉质有机细胞外基质的组装过程中发挥重要作用。我们将重点关注关键的牙本质与牙釉质连接处 (DEJ)，该连接处用于将脆性牙釉质生物陶瓷锚定到下面较软的牙本质上。我们假设 DEJ 的独特物理特性是成釉细胞和成牙质细胞合成的蛋白质混合的结果。我们将寻找 DEJ 形成过程中表达的关键蛋白质伙伴。我们将使用牙本质唾液酸磷蛋白 (DSPP) 作为诱饵，筛选牙齿特异性转录本的 cDNA 文库，以寻找与 DSPP 相互作用的蛋白质。在具体目标 4 中，我们建议寻找与牙本质基质蛋白 DSPP 相互作用的新型蛋白。具体目标是从我们自己的成就和该领域其他人获得的数据的逻辑扩展。每个具体目标都相互补充并建立在其他目标的基础上。完成这些实验将扩展我们对正常和异常牙釉质以及 DEJ 形成的理解。这些信息将使我们能够继续开发牙釉质和 DEJ 仿生材料。