Function of Keratin 75 in enamel

项目摘要。 牙釉质由牙冠的外层组成，是人体最坚硬的组织。釉质 具有高硬度和极高机械韧性的独特组合，这是由于其复杂的 结构组织和化学成分分级。牙釉质容易患龋齿，这是最常见的 传染性慢性病，影响高达90%的学童和绝大多数成年人。龋齿是 由产生乳酸的细菌引起，乳酸能溶解牙釉质矿物。龋齿是一个复杂的问题 多因素疾病；具有显著的遗传成分。最近几个密切相关的上皮角蛋白， 在成釉细胞和成釉细胞中发现K75、K6a、K6b和K6c。重要的是，中的几个多态性 这些角蛋白与头发和皮肤疾病有关，会导致牙釉质的结构和机械缺陷， 并与更高的龋齿易感性有关。值得注意的是，所有这些多态都包含在编码区， 这指向了一种直接的结构效应。我们鉴定了K75和K6b在形成小鼠和猪牙釉质中的作用 质谱仪(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MS分析 确定了K6c，这强烈表明K75存在于这个高度交联的基质中。此外，a 先天性厚甲Krt75tm1Der敲入(Ki)159Asn缺失小鼠模型主要显示 牙釉质中的皮肤缺陷和微结构变化。根据我们最近的发现，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病易感性之间的联系。 我们推测，釉质中的角蛋白形成了高度交联的细丝网络，从而稳定了釉质。 结构和防止积累的微小损伤，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/或组成的变化可以导致增加 牙釉质对酸侵蚀的敏感性直接或通过积累机械损伤，即微裂纹， 它可以作为酸扩散的管道。三个目标被用来检验这一假说。目标1是 评价不同年龄Krt75tm1Der Ki小鼠釉质的结构和力学性能。目标2是为了 用公认的小鼠致龋性模型评估Krt75tm1Der Ki小鼠牙釉质的龋易感性 质询协议。目的3评估不溶性细胞外釉质基质的力学性能。 人牙釉质和Krt75tm1Der Ki和WT小鼠。在拟议研究中获得的数据将用作 R01应用的基础，最终目标是全面了解基因是如何 角蛋白和不溶牙釉质基质的其他成分的变化会影响龋齿的易感性， 牙釉质的结构和力学性能。这些研究将潜在地导致我们的 不溶性牙釉质基质在牙釉质机械增强中的作用及其机制 对龋病的复原力和新的龋病预防和治疗方法的开发，并将启发 用于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.