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

项目摘要 人皮肤屏障并不总是如预期的那样起作用;事实上，人皮肤屏障的外层中的缺陷可能导致皮肤损伤。 皮肤是生活在美国的人们的大量健康问题。作为一个 例如，据估计，多达9000万美国人患有某种形式的特应性皮炎。异位性 皮炎和其它形式的严重干燥皮肤，如寻常鱼鳞病，与缺陷或 聚丝蛋白原及其加工片段聚丝蛋白的突变。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，表现为红色，干燥，鳞状皮肤。在一起， 聚丝蛋白原和角蛋白1和10是参与人皮肤角质层的关键蛋白质， 阐明其功能背后的生物化学对于提高我们对这些蛋白质是如何在细胞中发挥作用的理解是重要的。 人体皮肤屏障有效。我们希望，推进我们对蛋白质结构如何决定 角质层中的功能将产生治疗人类皮肤病的新方法。 该建议旨在解决我们对密钥的原子分辨率结构的理解中的不足之处 表皮蛋白，即原丝聚蛋白和角蛋白1和10。特别是，目前还不清楚（1） 前丝聚蛋白与靶蛋白结合的精确机制;（2）结构之间是否存在相关性 前丝聚集蛋白B结构域的功能;（3）前丝聚集蛋白与靶蛋白的相互作用是否直接特异性地 终末表皮分化的事件;（4）角蛋白背后的分子机制是什么 中间丝聚集;和（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。考虑到聚丝蛋白原与 角蛋白1和10与多种人类疾病，我们相信集中我们的研究在这些医学 重要的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，我们研究了参与功能性蛋白质的生物化学和结构特性。 人类皮肤屏障。我们的第一个目标是使用X射线晶体学和生化分析技术， 确定前丝聚蛋白与其靶点之一膜联蛋白II结合的结构基础。第二 aim在没有结合分子的情况下检查了聚丝蛋白原的x射线晶体结构， 对关键的EF-手间接头残基进行突变分析，以了解这种突变的功能作用。 蛋白质区第三个目的是研究角蛋白中间丝与蛋白质相互作用的结构基础。 使用X射线晶体学和生物化学技术对原丝聚蛋白分子的不同部分进行了分析。 实现这些目标将为生物化学和原子分辨率的原理提供新的见解 关键表皮蛋白的结构参与维持人体皮肤屏障的完整性。

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.