ENAMEL MATRIX PROTEIN INTERACTION

釉原蛋白自组装成纳米球，并在氨基末端含有一个自组装结构域。我们将采用假设驱动的实验策略，研究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。我们将检验这一假设，即釉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，与对照相比，组装结构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)将转基因动物与对照组进行比较。为了验证这一假设，我们提出了两个具体的目标：1)设计表达釉原蛋白的转基因动物，该转基因动物含有自组装氨基末端A结构域的改变或缺失，并对所产生的釉质进行表征；2)直接测量釉原蛋白相互作用的强度。接下来，我们筛选了与釉原蛋白相互作用的未知蛋白伙伴(S)。接下来，我们筛选了以前未被识别的蛋白伴侣(S)，它们与釉原蛋白或端粒蛋白相互作用。我们鉴定了一个釉原蛋白相互作用蛋白(AIP)和几个端粒蛋白相互作用蛋白(TIPS)。我们将检验这一假设，即AIP或TIP的生理功能是参与釉质有机基质的组装，该基质有能力直接进行生物矿化。在具体目标3中，我们建议使用锤头状核酶来减少AIP或每个TIP靶蛋白的可用性。我们预测每一种减少的目标蛋白所产生的牙釉质中的致病基因组变化。最后，我们预测，在釉质有机细胞外基质的组装过程中，额外的蛋白质将发挥重要作用。我们将把注意力集中在牙本质到釉质交界处(DEJ)，它用于将脆性的釉质生物陶瓷固定在下面较软的牙本质上。我们假设DEJ的独特物理性质是成釉细胞和成牙本质细胞合成的蛋白质的混合物的结果。我们将寻找在DEJ形成过程中表达的关键蛋白质伙伴。我们将使用牙本质涎磷蛋白(DSPP)作为诱饵来筛选与DSPP相互作用的那些蛋白的牙齿特异转录的cDNA文库。在特定目标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获得的数据的合乎逻辑的延伸。每一个具体目标都与其他目标相辅相成，相辅相成。完成这些实验将扩大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继续创造牙釉质和DEJ仿生。

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.