Amyloids in Enamel Development

淀粉样蛋白在牙釉质发育中的作用

• 批准号: 9177618
• 负责人: Stefan Friedrich Habelitz
• 金额: $ 67.97万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9177618
• 关键词: Agreement; Amelogenesis; Amelogenesis Imperfecta; Amyloid; Amyloid fibers; Amyloidosis; Apatites; Appearance; Architecture; Biological Process; Biology; Caliber; Characteristics; Dental Enamel; Development; Disease; Enamel Formation; Enzymes; Epithelial; Fiber; Fracture; Genetic; Growth; Human; Human body; In Vitro; Ions; Kinetics; Knockout Mice; Length; MMP-20; Mammals; Measures; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; N-terminal; Nanostructures; Nerve Degeneration; Pathology; Pathway interactions; Peptide Hydrolases; Peptides; Play; Point Mutation; Probability; Process; Proteins; Proteolysis; Recombinants; Resistance; Role; Series; Site; Staging; Staining method; Stains; Structure; Testing; Tissues; Transgenic Mice; Width; amelogenin; amyloid formation; amyloid structure; base; beta pleated sheet; calcium phosphate; cytotoxic; enamel matrix proteins; in vivo; kallikrein 4; mineralization; mouse model; mutant; nanofiber; nanostructured; protein aminoacid sequence; proteinase In; repaired; retinal rods; self assembly

ABSTRACT Amyloids are a class of proteins that spontaneously self-assemble into cross β-sheet aggregates which have been associated with amyloidosis and neurodegeneration. However, recently, amyloids that are non-toxic and rather play a functional role in a mammalian biosynthetic pathway have been discovered challenging our current views on the sole role of amyloids in mammals to be cytotoxic. Here we propose that amelogenin, the main protein of the developing enamel matrix, adapts cross-β sheet configuration and develops into fibrillar amyloids to achieve structural support to guide mineralization in vivo and in vitro. Enamel, the hardest and most mineralized tissue in the human body, is comprised of a unique organization of apatite nanofibers of about 50 nm in diameter and several hundreds of micrometers in length. There is agreement that the unique crystal morphology and organization into rod and interrod enamel is the result of a protein-guided uniaxial growth process of apatite, but it is unclear by which molecular mechanisms this unique micro- and nanostructure develops. While the role of self-assembly of enamel matrix proteins, in particular amelogenin, has widely been recognized as a crucial factor in controlling the structural development of enamel, a convincing relationship between organic supramolecular aggregates and enamel structure has only recently been observed, when we discovered that the recombinant human full-length amelogenin protein (rH174) forms ribbons of 17 nm in width, which grow to several micrometers in length. Such ribbons have the ability to self- align and form bundles resembling the appearance of aligned apatite crystallites in an enamel rod. Analysis of the primary structure of amelogenin revealed several domains with high propensity to form β-sheets, including the possibilty to form amyloids, and produced a 14 residue N-terminal peptide (14P2) that readily assembled into nanoribbons (6.7nm wide), with possible amyloid structure. Amyloid stains were positive in enamel from mice lacking the enamel-specific enzyme kallikrein 4 (KLK4). Both enamel tissue and recombinant amelogenin nanoribbons showed x-ray diffraction spacings at 4.7Å characteristic of β sheets and amyloids. Enzymatic processing of self-assembled amelogenin promoted precise cleavage into the 23 kDa and 20 kDa fragments which resisted further degradation by MMP-20, thus possibly providing a stable organic template for mineralization during secretory stage amelogenesis, whereas the second enamel protease is able to disassemble and to degrade amelogenin amyloids. Herein we will further investigate the presence of amyloids in enamel and propose that amyloids play a key role in the development and organization of the organic matrix of enamel and that an exploration of such structures is essential to our understanding of enamel formation, its diseases and repair.

抽象的 淀粉样蛋白是一类自发自组装成交叉 β 片层聚集体的蛋白质，其具有 与淀粉样变性和神经变性有关。然而，最近，淀粉样蛋白无毒且 相反，在哺乳动物生物合成途径中发挥功能性作用已被发现，这对我们的研究提出了挑战 目前认为淀粉样蛋白在哺乳动物中的唯一作用是细胞毒性。在这里，我们提出牙釉蛋白， 发育中的牙釉质基质的主要蛋白质，适应交叉β片层构型并发育成纤维状 淀粉样蛋白获得结构支持以指导体内和体外矿化。珐琅质是最坚硬的 人体中矿化程度最高的组织，由磷灰石纳米纤维的独特组织组成 直径约50纳米，长度数百微米。大家一致认为，独特的 晶体形态和组织成棒状和棒间釉质是蛋白质引导单轴的结果 磷灰石的生长过程，但尚不清楚这种独特的微观和 纳米结构发展。虽然牙釉质基质蛋白（特别是釉原蛋白）自组装的作用， 已被广泛认为是控制牙釉质结构发育的关键因素， 有机超分子聚集体与牙釉质结构之间令人信服的关系直到最近才被证实 观察到，当我们发现重组人全长牙釉蛋白（rH174）形成 宽度为 17 纳米的带状物，长度可增长至几微米。此类丝带具有自 排列并形成类似于牙釉质棒中排列的磷灰石微晶外观的束。分析 釉原蛋白的一级结构揭示了几个具有高度形成β-折叠倾向的结构域，包括 形成淀粉样蛋白的可能性，并产生易于组装的 14 个残基 N 端肽 (14P2) 纳米带（6.7纳米宽），可能具有淀粉样结构。 缺乏牙釉质特异性酶激肽释放酶 4 (KLK4) 的小鼠牙釉质中淀粉样蛋白染色呈阳性。两个都 牙釉质组织和重组牙釉蛋白纳米带显示 X 射线衍射间距为 4.7Å β折叠和淀粉样蛋白的特征。促进自组装牙釉蛋白的酶促加工 精确切割成 23 kDa 和 20 kDa 片段，从而抵抗 MMP-20 的进一步降解，从而 可能为分泌阶段釉质形成过程中的矿化提供稳定的有机模板，而 第二种牙釉质蛋白酶能够分解和降解牙釉质淀粉样蛋白。在此我们将 进一步研究牙釉质中淀粉样蛋白的存在，并提出淀粉样蛋白在牙釉质中发挥关键作用 牙釉质有机基质的发展和组织，以及对这种结构的探索 对于我们了解牙釉质的形成、疾病和修复至关重要。