ENAMEL MATRIX PROTEIN INTERACTION

淀粉原蛋白自组装成纳米球，并在氨基端包含一个自组装结构域。我们将追求假设驱动的实验策略，以解决影响牙釉质有机基质组装成能够指导生物矿化的结构的关键问题。我们将测试一个假设，即淀粉原蛋白自组装成纳米球需要一个不变的氨基末端自组装结构域。我们预测，与对照组相比，组装域的改变将改变纳米球的大小。为了验证这一假设，我们提出了两个具体目标：1)与对照相比，设计转基因动物。为了验证这一假设，我们提出了两个具体目标：1)设计转基因动物，表达含有自组装氨基末端“A”结构域改变或缺失的淀粉原蛋白，并表征所产生的牙釉质；2)直接测量淀粉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淀粉原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淀粉原蛋白或凝乳蛋白相互作用的蛋白质伴侣。我们鉴定了一个淀粉原蛋白相互作用蛋白（AIP）和几个簇绒蛋白相互作用蛋白（TIPs）。我们将验证AIP或TIP的生理功能是参与能够指导生物矿化的牙釉质有机基质的组装的假设。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。我们预测每一种减少的靶蛋白导致的牙釉质特异性的病理改变。最后，我们预测在釉质有机细胞外基质的组装过程中，其他蛋白质将发挥重要作用。我们将重点关注牙本质与牙釉质的关键接合处（DEJ），它将脆性牙釉质生物陶瓷固定在较软的牙本质上。我们假设DEJ形式的独特物理性质是由成釉细胞和成牙本质细胞合成的蛋白质混合物的结果。我们将寻找在DEJ形成过程中表达的关键蛋白伴侣。我们将以牙本质唾液磷蛋白（DSPP）为诱饵，筛选与DSPP相互作用的牙齿特异性转录物cDNA文库。在具体目标4中，我们建议寻找与牙本质基质蛋白DSPP相互作用的新蛋白。具体目标是从我们自己的成就和该领域其他人的成就中获得的数据的逻辑延伸。每一个具体目标都是相互补充和建立的。完成这些实验将扩展我们对正常和异常牙釉质和DEJ形成的理解。这些信息将使我们能够追求牙釉质和DEJ仿生的创造。

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.