CELL AND GENETICS APPROACHES TO ENAMEL BIOMIMETICS

描述（由申请人提供）：破译结构/功能关系是获得生物医学知识的基础，同时为创造新材料和药物提供基础。一种破译结构/功能结果的有力工具是对小鼠进行种系遗传操作。在这个应用中，我们应用种系操作来连接釉质基质成釉蛋白的结构和功能。牙釉质是一种复合生物陶瓷组织，其独特的材料性能归功于其生物制造模式。成釉细胞产生细胞外釉质蛋白基质，用于控制晶体习性和晶体束的组织，使数千个纳米级晶体在单个细胞的控制下组织和组合在一起。我们假设单个成釉细胞的纳米晶体的分组和组织是由成釉蛋白内关键结构域的功能实现的结果。成釉蛋白被描述成至少有两个结构域，一个N‘端和一个C’端结构域[Iwata等人]。[07]，基于以下发现：在切割后，N'-和C'-成釉蛋白结构域不再位于釉质棒的同一物理位置。N‘端在杆的边界周围富集，就像护套覆盖在刀刃上一样，而C’端则表现得完全不同。我们假设成釉酶的N端可能负责细胞与基质的相互作用，维持在牙釉质中观察到的高度图案化的棒与棒之间的边界。我们假设C'端负责产生牙釉质生物陶瓷组织的蛋白质与矿物质相互作用。结果将通过牙釉质结构的改变和牙釉质在敲入条件下与野生型动物的材料特性分析来衡量。该实验策略的结果将有助于我们对功能基因组学和蛋白质组学的理解，同时进一步了解脊椎动物体内唯一的外胚层来源的生物矿化组织的形成。我们研究小组的初步数据表明，这种敲入方法将对成釉细胞蛋白的结构/功能关系产生新的见解，成釉细胞蛋白是釉质有机基质组装和生物矿化的第二大丰富蛋白。

DESCRIPTION (provided by applicant): Deciphering structure/function relationships underpins the acquisition of biomedical knowledge while providing the basis to create novel materials and drugs. A powerful tool for deciphering structure/function outcomes has been germline genetic manipulation in mice. In this application, we apply germline manipulation to link structure to function for the enamel matrix protein ameloblastin. Enamel is a composite bioceramic tissue with unique material properties that are owed to its mode of biological fabrication. Ameloblast cells create an extracellular enamel protein matrix that serves to control both crystallite habit and the organization of crystallite bundles, allowing thousands of nanoscale crystallites to be organized and grouped together under the control of a single cell. We hypothesize that the grouping and organization of the nanocrystallites by a single ameloblast cell is the outcome achieved by the function(s) of critical domain(s) within the ameloblastin protein. The ameloblastin protein has been described as being organized into at least two domains, an N'- and a C'- terminal domain [Iwata et al. '07], based on the finding that following cleavage the N'- and C'- ameloblastin domains are no longer found in the same physical site in the enamel rod. The N'-terminus is enriched around the rod boundaries, much as a sheath covers a knife blade, while the C'-terminal domain behaves quite differently. We hypothesize that the N'-terminus of ameloblastin is likely responsible for the cell-to-matrix interactions that maintain the highly patterned rod-to-interrod boundaries observed in enamel. We hypothesize that the C'-terminus is responsible for the protein-to-mineral interactions producing the enamel bioceramic tissue. Outcomes will be measured by changes to stereotypic enamel architecture and by analysis of the material properties of the enamel in the knockin condition compared to wildtype animals. The outcomes from this experimental strategy will contribute to our understanding of functional genomics and proteomics while furthering our understanding of the formation of the only ectoderm-derived biomineralized tissue in the vertebrate body. Preliminary data from our research team suggest that this knockin approach will yield novel insights into the structure/function relationship for the ameloblastin protein, the second most abundant protein contributing to enamel organic matrix assembly and biomineralization. PUBLIC HEALTH RELEVANCE: The function(s) for the second most abundant protein of the forming mammalian enamel matrix is not known. Here, we map the function(s) of ameloblastin protein domains to the production of the enamel matrix required to control enamel biomineralization. In performing this project we will create an animal model useful to study caries, the most prevalent-, infectious-disease of humankind and provide one more design specification for creating an enamel biomimetic, useful for restoring enamel lost to trauma and disease.