Determining Structure and Function of Human Skin Barrier Proteins Using X-ray Crystallography

项目总结 人类的皮肤屏障并不总是像预期的那样起作用；事实上，人类外层的缺陷 对于生活在美国的人来说，皮肤是造成大量健康问题的原因。作为一个整体 例如，据估计，多达9000万美国人患有某种形式的特应性皮炎。特应性 皮炎和其他形式的严重干燥皮肤，如寻常型鱼鳞病，与皮肤缺陷或 Profilaggrin及其加工片段的突变。角蛋白1和角蛋白10蛋白的突变 也解释了各种遗传性皮肤病，表现为皮肤发红、干燥、鳞状。一起， Profilaggrin和Keratin1和10是涉及人类皮肤角质层的关键蛋白质，以及 阐明它们功能背后的生物化学对于提高我们对 人体皮肤屏障起作用了。希望通过提高我们对蛋白质结构决定的知识 角质层的功能将产生治疗人类皮肤病的新方法。 这一建议旨在解决我们对KEY原子解析结构的理解上的一个缺陷 表皮蛋白，即Profilaggrin和Keratin1和10。尤其是，仍不清楚(1)什么是 精确的机制是Profilaggrin与目标蛋白结合；(2)结构之间是否存在相关性 和profilaggrin B结构域的功能；(3)profilaggrin与靶蛋白的直接特异性相互作用 终末表皮分化事件；(4)角蛋白背后的分子机制是什么 中间丝聚集；以及(5)可以增强关键皮肤屏障蛋白的结构知识 为新设计的局部疗法铺平道路。鉴于Profilaggrin和Profilaggrin的直接关联 角蛋白1和角蛋白10与多种人类疾病有关，我们认为我们的研究集中在这些医学上 重要蛋白质将帮助我们解决这些悬而未决的问题。 在这个项目中，我们研究了与功能相关的关键蛋白质的生化和结构特性。 人体皮肤屏障。我们的第一个目标是使用x射线结晶学和生化分析技术 确定Profilaggrin与其靶标之一--膜联蛋白II之间结合的结构基础 目的研究在没有结合分子的情况下profilaggrin的x射线晶体结构，并进行 对关键的EF手间连接子残基进行突变分析，以了解其功能作用 蛋白质区域。第三个目的是研究角蛋白中间丝与 使用x射线结晶学和生化技术对Profilaggrin分子的不同部分进行分析。 实现这些目标将为生物化学和原子分辨的原理提供新的见解 与维持人体皮肤屏障完整性有关的关键表皮蛋白质的结构。

PROJECT SUMMARY The human skin barrier does not always function as intended; in fact, defects in the outer layer of human skin account for a significant number of health problems for people living in the United States. As one example, it is estimated that up to 90 million Americans suffer from some form of atopic dermatitis. Atopic dermatitis and other forms of severely dry skin, such as ichthyosis vulgaris, are associated with defects or mutations in profilaggrin and its processed fragment, filaggrin. Mutations in keratin 1 and keratin 10 proteins also account for a variety of inherited skin disorders manifested by red, dry, scaly skin. Together, profilaggrin and keratins 1 and 10 are critical proteins involved in the stratum corneum of human skin, and elucidating the biochemistry behind their function is important for improving our understanding of how the human skin barrier works. The hope is that advancing our knowledge of how protein structure dictates function in the stratum corneum will generate new methods for treatment of human skin diseases. This proposal aims to address a deficiency in our understanding of the atomic resolution structure of key epidermal proteins, namely profilaggrin and keratins 1 and 10. In particular, it is still unclear (1) what the precise mechanisms are for profilaggrin binding to target proteins; (2) is there correlation between structure and function of the profilaggrin B domain; (3) does profilaggrin interaction with target proteins direct specific events in terminal epidermal differentiation; (4) what are the molecular mechanisms behind keratin intermediate filament aggregation; and (5) can enhanced structural knowledge of key skin barrier proteins pave the way for newly designed topical therapeutics. Given the direct association of profilaggrin and keratins 1 and 10 with multiple human diseases, we believe focusing our studies on these medically important proteins will help us address these outstanding questions. In this project, we examine the biochemical and structural properties of key proteins involved in a functional human skin barrier. Our first aim uses x-ray crystallography and biochemical analysis techniques to determine the structural basis of binding between profilaggrin and one of its targets, annexin II. The second aim examines the x-ray crystal structure of profilaggrin in the absence of bound molecules and performs mutational analysis on key inter-EF-hand linker residues in order to understand the functional role of this protein region. The third aim examines the structural basis for keratin intermediate filament interaction with different parts of the profilaggrin molecule using x-ray crystallography and biochemical techniques. Accomplishing these aims will provide novel insight into the principle biochemistry and atomic resolution structure of key epidermal proteins involved in maintaining the integrity of the human skin barrier.