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 由于其错综复杂，具有高硬度和极端机械弹性的独特组合 结构组织和分级化学成分。搪瓷容易龋齿，最普遍 传染性慢性病，影响多达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。最近几个密切相关的上皮角蛋白， K75，K6A，K6B和K6C被发现在木板和搪瓷中。重要的是，有几种多态性 这些角质素与头发和皮肤疾病有关，导致牙釉质的结构和机械缺陷， 并与更高的龋齿敏感性有关。值得注意的是，所有这些多态性都包含在编码区域中， 这指出了直接的结构效应。我们通过 质谱法（MS）。我们进一步隔离了人类搪瓷和MS分析中的不溶性有机部分 确定的K6C，强烈建议在此高度交联的基质中存在K75。此外， Pachyonychia congenita krt75tm1der敲门（Ki）的鼠标模型，删除159ASN显示主要 牙釉质的皮肤缺陷和微观结构变化。在我们最近的发现的基础上，我们建议确定 K75在搪瓷中的功能作用，并阐明了角蛋白和性能敏感性之间的突变之间的联系。 我们假设牙釉质中的角蛋白形成了密集的交联丝网，从而稳定牙釉质 结构并防止微宏的积累，从而有助于其机械弹性。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 牙釉质对酸攻击的敏感性直接或通过机械损伤的积累，即微裂纹， 可以用作酸扩散的导管。开发了三个目标来检验这一假设。目标1是 评估不同年龄段KRT75TM1DER KI小鼠中搪瓷的结构和机械性能。在AIM 2中是 使用公认的鼠类致富性，评估Krt75TM1Der Ki小鼠中搪瓷的Caries易感性 挑战协议。 AIM 3是评估不溶性细胞外搪瓷基质的机械性能 人搪瓷和krt75tm1der ki和wt小鼠。拟议的研究中获得的数据将作为 R01应用的基础具有最终目标，以全面了解遗传 角蛋白和其他成分的变化不溶性搪瓷矩阵影响龋齿敏感性， 牙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 理解不溶性搪瓷基质在搪瓷及其机械增强中的作用 对龋齿的韧性和开发用于预防和治疗的新方法，并将激发 用于搪瓷修复的新型生物材料。