Function of Keratin 75 in enamel

角蛋白 75 在牙釉质中的功能

• 批准号: 10227364
• 负责人: ELIA BENIASH
• 金额: $ 6.78万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-19 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227364
• 关键词: Accounting; Acids; Adult; Affect; Age; Ameloblasts; Amino Acid Substitution; Amino Acids; Atomic Force Microscopy; Bacteria; Biocompatible Materials; Caries prevention; Chemicals; Child; Chronic Disease; Code; Complex; Crystallization; Data; Defect; Dental Caries Susceptibility; Dental Enamel; Dental caries; Dentists; Development; Diffusion; Disease; Electron Microscopy; Epithelial; Epithelium; Exposure to; Extracellular Matrix; Family suidae; Filament; Fluorescent Dyes; Fluorides; Foundations; Future; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Goals; Hair Diseases; Hardness; High Prevalence; Human; Human body; Immunofluorescence Immunologic; Keratin; Knock-in; Knock-in Mouse; Lactic acid; Laser Scanning Confocal Microscopy; Lead; Lesion; Link; Location; Mass Spectrum Analysis; Mechanics; Minerals; Mouth Diseases; Mus; Mutation; Peptide Hydrolases; Play; Porosity; Predisposition; Property; Proteins; Protocols documentation; Psychological reinforcement; Research; Resistance; Rod; Role; Scanning Electron Microscopy; Schools; Shapes; Site; Skin; Structure; Surface; Testing; Tissues; Tooth Crowns; Tooth structure; Transmission Electron Microscopy; base; comparative; contrast imaging; crosslink; demineralization; enamel matrix proteins; extracellular; human tissue; insight; link protein; mechanical properties; microCT; mouse model; nano; novel; novel strategies; organizational structure; pachyonychia congenita; photoemission; prevent; public health relevance; repaired; resilience; skin disorder; viscoelasticity

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.

项目摘要。 牙釉质包括牙冠的外层，是人体最坚硬的组织。搪瓷 由于其复杂的结构，具有高硬度和极高机械弹性的独特组合 结构组织和分级化学成分。牙釉质容易发生龋齿，这是最常见的 传染性慢性疾病，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，乳酸会溶解牙釉质矿物质。龋齿是一个复杂的 多因素疾病；具有重要的遗传成分。最近发现几种密切相关的上皮角蛋白， K75、K6a、K6b 和 K6c 是在成釉细胞和牙釉质中发现的。重要的是，其中的几个多态性 这些与头发和皮肤疾病相关的角蛋白会导致牙釉质的结构和机械缺陷， 并与较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。我们通过以下方法鉴定了 K75 和 K6b 在形成鼠和猪牙釉质中的作用： 质谱（MS）。我们进一步在人牙釉质和质谱分析中分离出不溶性有机部分 鉴定出 K6c，强烈表明在这种高度交联的基质中存在 K75。此外，一个 先天性厚甲症小鼠模型 Krt75tm1Der 敲入 (KI) 并删除 159Asn 显示主要 皮肤缺陷和牙釉质微观结构变化。根据我们最近的发现，我们建议确定 K75 在牙釉质中的功能作用，并阐明角蛋白突变与龋齿易感性之间的联系。 我们假设牙釉质中的角蛋白形成高度交联的细丝网络，从而稳定牙釉质 结构并防止微损伤的累积，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增加 牙釉质对酸的敏感性直接或通过机械损伤的积累，即微裂纹， 它可以作为酸扩散的导管。制定了三个目标来检验这一假设。目标 1 是 评估不同年龄 Krt75tm1Der KI 小鼠牙釉质的结构和机械特性。目标 2 是 使用成熟的小鼠致龋材料评估 Krt75tm1Der KI 小鼠牙釉质的龋齿易感性 挑战协议。目标 3 是评估不溶性细胞外牙釉质基质的机械性能 人类牙釉质和 Krt75tm1Der KI 和 WT 小鼠。在拟议的研究中获得的数据将作为 R01应用的基础，最终目标是全面了解遗传如何 角蛋白和不溶性牙釉质基质的其他成分的变化会影响龋齿的易感性， 釉质的结构和机械性能。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及其 抗龋能力和开发预防和治疗龋齿的新方法将激励 用于牙釉质修复的新型生物材料。