CELL AND GENETICS APPROACHES TO ENAMEL BIOMIMETICS

描述（由申请人提供）：破译结构/功能关系是获取生物医学知识的基础，同时为创造新材料和药物提供基础。破译结构/功能结果的有力工具是小鼠种系遗传操作。在此应用中，我们应用种系操作将牙釉质基质蛋白成釉细胞的结构与功能连接起来。牙釉质是一种复合生物陶瓷组织，由于其生物制造方式而具有独特的材料特性。成釉细胞产生细胞外釉质蛋白基质，用于控制微晶习性和微晶束的组织，从而允许数千个纳米级微晶在单个细胞的控制下组织和分组在一起。我们假设单个成釉细胞对纳米微晶的分组和组织是由成釉细胞内关键结构域的功能实现的结果。 成釉细胞蛋白被描述为被组织成至少两个结构域，一个 N'- 和一个 C'- 末端结构域 [Iwata 等人，2015]。 '07]，基于以下发现：裂解后，在牙釉质棒的同一物理位点不再发现 N'- 和 C'- 成釉细胞蛋白结构域。 N' 末端在杆边界周围富集，就像刀鞘覆盖刀片一样，而 C' 末端结构域的行为则截然不同。我们假设成釉细胞的 N' 末端可能负责细胞与基质的相互作用，从而维持牙釉质中观察到的高度图案化的杆与杆间边界。我们假设 C' 末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。结果将通过刻板牙釉质结构的变化以及与野生型动物相比在敲入条件下牙釉质的材料特性分析来衡量。 这一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，同时进一步加深我们对脊椎动物体内唯一外胚层衍生的生物矿化组织的形成的理解。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，成釉细胞蛋白是第二丰富的蛋白质，有助于牙釉质有机基质组装和生物矿化。 公共健康相关性：形成哺乳动物牙釉质基质的第二丰富蛋白质的功能尚不清楚。在这里，我们将成釉素蛋白结构域的功能映射到控制牙釉质生物矿化所需的牙釉质基质的产生。在执行这个项目时，我们将创建一个可用于研究龋齿（人类最普遍的传染病）的动物模型，并为创建牙釉质仿生材料提供又一个设计规范，可用于恢复因创伤和疾病而损失的牙釉质。

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.