Function of Keratin 75 in enamel

项目摘要。 牙釉质包括牙冠的外层，是人体最硬的组织。釉质 具有高硬度和极端的机械弹性的独特组合，由于其复杂的 结构组织和分级化学成分。牙釉质容易发生龋齿， 传染性慢性疾病，影响高达90%的学童和绝大多数成年人。龋 由产生乳酸的细菌引起，乳酸溶解釉质矿物质。 多因素疾病;有重要的遗传因素。最近几个密切相关的上皮角蛋白， K75、K6 a、K6 b和K6 c在成釉细胞和釉质中均有表达。重要的是，一些多态性， 这些与毛发和皮肤疾病相关的角蛋白，导致牙釉质的结构和机械缺陷， 并且与更高的龋齿易感性有关。值得注意的是，所有这些多态性都包含在编码区中， 这表明了直接的结构效应。我们鉴定了K75和K6 b在鼠和猪牙釉质形成中的作用， 质谱法（MS）。我们进一步从人牙釉质中分离出一种不溶性有机组分，并进行了质谱分析 鉴定了K6 c，并且它强烈表明在这种高度交联的基质中存在K75。而且有 先天性甲肥厚小鼠模型Krt 75 tm 1Der基因敲入（KI）并缺失159 Asn， 皮肤缺损和牙釉质的微观结构变化。根据我们最近的研究结果，我们建议确定 K75在釉质中的功能作用，并阐明角蛋白突变与龋齿易感性之间的联系。 我们推测，釉质中的角蛋白形成高度交联的细丝网络，从而稳定釉质 结构和防止微损伤的积累，有助于其机械弹性。我们进一步 假设蛋白质网络中的结构和/或组成变化可以导致蛋白质网络中的蛋白质含量增加。 牙釉质直接或通过机械损伤（即微裂纹）的累积对酸侵蚀的敏感性， 其可用作酸扩散的导管。有三个目标被开发来测试这个假设。目标1： 评价不同年龄Krt 75 tm 1Der KI小鼠牙釉质的结构和机械性能。目标2是 使用完善的鼠致龋模型评估Krt 75 tm 1Der KI小鼠牙釉质的龋齿易感性， 挑战协议。目的3是评估不溶性细胞外釉基质的机械性能， 人釉质和Krt 75 tm 1Der KI和WT小鼠。在拟议研究中获得的数据将作为 R 01应用的基础，最终目标是全面了解基因是如何 不溶性釉质基质的角蛋白和其它组分的变化影响龋齿易感性， 搪瓷的结构和机械性能。这些研究可能会导致我们的范式转变， 了解不溶性牙釉质基质在牙釉质机械强化中的作用， 对龋齿的恢复力和龋齿预防和治疗的新方法的开发，将激发 用于牙釉质修复的新型生物材料。

Project Summary. Dental enamel comprises the outer layer of tooth crowns and is the hardest tissue of the human body. Enamel possesses a unique combination of high hardness and extreme mechanical resilience due to its intricate structural organization and graded chemical composition. Enamel is prone to dental caries, the most prevalent infectious chronic disease, affecting up to 90% of school children and the vast majority of adults. Caries is caused by bacteria producing lactic acid, which dissolves the enamel mineral. Caries is a complex multifactorial disease; with a significant genetic component. Recently several closely related epithelial keratins, K75, K6a, K6b and K6c, were discovered in ameloblasts and enamel. Importantly, several polymorphisms in these keratins, associated with hair and skin disorders, lead to structural and mechanical defects of enamel, and are linked to higher caries susceptibility. Notably, all these polymorphisms are contained in coding regions, which points to a direct structural effect. We identified K75 and K6b in forming murine and porcine enamel by mass spectrometry (MS). We further isolated an insoluble organic fraction in human enamel and MS analysis identified K6c and it strongly suggests the presence of K75 in this highly cross-linked matrix. Furthermore, a mouse model of pachyonychia congenita Krt75tm1Der knock-in (KI) with deletion of 159Asn displays major skin defects and microstructural changes in enamel. Building on our recent findings, we propose to identify the functional role of K75 in enamel and elucidate the link between mutations in keratins and caries susceptibility. We hypothesize that keratins in enamel form heavily cross-linked filament networks, which stabilize enamel structure and prevent accumulation of microdamage, contributing to its mechanical resilience. We further hypothesize that either structural and/or compositional changes in the protein network can lead to an increased enamel susceptibility to acid attack directly or through accumulation of mechanical damage, i.e. microcracks, which can serve as conduits for acid diffusion. Three aims were developed to test this hypothesis. Aim 1 is to assess structural and mechanical properties of enamel in Krt75tm1Der KI mice at different ages. In Aim 2 is to assess caries susceptibility of enamel in Krt75tm1Der KI mice, using a well-established murine cariogenic challenge protocol. Aim 3 is to assess mechanical properties of the insoluble extracellular enamel matrix from human enamel and Krt75tm1Der KI and WT mice. The data acquired in the proposed studies will serve as a basis for R01 application with the ultimate goal to gain a comprehensive understanding of how the genetic variations in keratins and other components of the insoluble enamel matric affect caries susceptibility, structural and mechanical properties of enamel. These studies will potentially lead to a paradigm shift in our understanding of the roles of the insoluble enamel matrix in mechanical reinforcement of enamel and its resilience to caries and development of new approaches for caries prevention and treatment and will inspire novel biomaterials for enamel repair.