Amyloids in Enamel Development

摘要 淀粉样蛋白是一类自发地自组装成跨β片状聚集体的蛋白质，这些聚集体具有 与淀粉样变性和神经退行性变有关。然而，最近，无毒和 相反，在哺乳动物的生物合成途径中扮演着重要的角色已经被发现挑战我们的 目前关于淀粉样蛋白在哺乳动物中的唯一作用的观点是细胞毒性的。在这里，我们建议釉原蛋白，即 发育中的釉质基质的主要蛋白质，适应交叉β片状结构，发育成纤维状 淀粉样蛋白实现结构支撑，引导体内和体外矿化。牙釉质，最坚硬和 人体内大多数矿化组织是由磷灰石纳米纤维组成的独特组织 直径约50纳米，长数百微米。人们一致认为，这种独特的 晶体形态和组织成棒状牙釉质和棒状牙釉质是蛋白质引导的单轴的结果 磷灰石的生长过程，但尚不清楚这种独特的微观和微观机制 纳米结构的发展。而釉质基质蛋白，特别是釉原蛋白的自组装作用， 被广泛认为是控制牙釉质结构发展的关键因素， 有机超分子聚集体和釉质结构之间令人信服的关系直到最近 当我们发现重组人全长釉原蛋白(RH174)形成时， 17纳米宽的丝带，长到几微米长。这样的丝带具有自我调节的能力 排列并形成类似于釉质棒中排列的磷灰石微晶外观的束状。分析 釉原蛋白的一级结构揭示了几个容易形成β-Sheet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) 变成纳米带(6.7 nm宽)，可能有淀粉样结构。 在缺乏釉质特异酶激肽释放酶4(KLK4)的小鼠牙釉质中，淀粉样染色呈阳性。两者都有 牙釉质组织和重组釉原蛋白纳米带的X射线衍射间距为4.7？ 具有β片状和淀粉样蛋白的特征。促进自组装釉原蛋白的酶促加工 精确地切割成23 kDa和20 kDa的片段，阻止了基质金属蛋白酶-20的进一步降解，因此 可能为分泌期成釉作用的矿化提供稳定的有机模板，而 第二种釉质蛋白水解酶能够分解和降解淀粉样釉原蛋白。在这里，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起关键作用 牙釉质的有机基质的发展和组织，以及对这种结构的探索 对于我们理解釉质的形成、其疾病和修复是必不可少的。

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.