Structural Biology of Keratin Filaments and Cornified Cell Envelopes

角蛋白丝和角化细胞包膜的结构生物学

• 批准号: 8344718
• 负责人: ALASDAIR C. STEVEN
• 金额: $ 0.66万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8344718
• 关键词: Amino Acids; Antibodies; Biocompatible Materials; Biology; Biomechanics; Cell membrane; Cells; Ceramides; Complex; Cytokeratin filaments; Cytoskeleton; Data; Deposition; Epidermis; Epithelium; Eukaryotic Cell; Genes; Goals; Gold; Intermediate Filaments; Keratin; Knock-out; Laboratories; Life; Lipids; Messenger RNA; Mouse Protein; Mus; Paper; Phenotype; Production; Property; Protein Family; Proteins; Publications; Reporting; Skin; Structural Protein; Structure; Surface; Thick; Transglutaminases; cell envelope; cell type; crosslink; env Gene Products; interest; keratinocyte; loricrin; member; structural biology

Terminally differentiated epidermal keratinocytes form the body's outer protective barrier that undergoes constant renewal as cornified squames slough off the exterior surface and are replaced by material migrating outwards from the inner living layers of the epidermis. Cornified squames are composite biomaterials with two components: a matrix of keratin filaments encased within the cornified cell envelope (CE). The CE is a multi-component 10nm-thick layer of insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the epidermis, a 5nm-thick layer of ceramide lipids (lipid envelope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the cross-linking of several structural proteins by transglutaminases. Intermediate filaments (IF) are ubiquitous constituents of the cytoskeletons of eukaryotic cells. They consist of five different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested also in the related IF of other cell types and in the interactions beetween keratin IF and the CE. The long-term goal of this project, previously pursued in both the Laboratory of Skin Biology (P. M. Steinert, P.I.) and the LSBR is to elucidate the production, assembly, structure and biomechanical properties of these cells and their components in the context of both normal and diseased skin. This project is now largely in abeyance but we did succeed in FY10 completing and submitting for publication a paper reporting the following results. In normal skin, some 75% of the protein mass of the CE is contributed by a single protein called loricrin. Despite the functional importance that this number would suggest, knocking out the loricrin gene in mice results in only a mild transient phenotype and the loricrin knockout CE maintains a normal thickness (15nm). However, an examination of the loricrin KO CE amino acid composition revealed changes in the amino acid content, suggesting that other proteins were incorporated into the CE to compensate for its loss. Recently our collaborator has determined that several members of the late cornified envelope (lce) protein family are significantly upregulated at the mRNA and protein level in the KO mouse, and these proteins are incorporated into the CE to functionally compensate for the loss of loricrin. We have generated immuno-gold EM data with anti-lce antibodies to confirm and extend these observations. This paper is currently in revision.

末端分化的表皮角质形成细胞形成人体的外部保护屏障，该保护性屏障会经历恒定的更新，因为玉米式蹲下了外表面，并被从表皮内部居住层向外迁移的材料所取代。玉米质蹲是具有两个组成部分的复合生物材料：角蛋白丝的基质，该基质包裹在蜂蜜细胞包膜内（CE）。 CE是在末端分化过程中沉积在细胞质膜内表面上的不溶性蛋白的多组分厚层。在表皮中，将5nm厚的神经酰胺脂质（脂质包膜）连接到外表面。蛋白质包膜的不溶性在很大程度上归因于跨谷氨酰胺酶的几种结构蛋白的交联。中间丝（如果）是真核细胞细胞骨架的普遍成分。它们由五种不同类型组成，其中最多和复杂的是I型和II型角蛋白，它们在上皮中广泛表达。我们还对其他细胞类型以及角蛋白IF与CE之间的相互作用感兴趣。该项目的长期目标，以前在皮肤生物学实验室（P. M. Steinert，P.I。）和LSBR中均在正常皮肤和患病的皮肤的背景下阐明这些细胞及其成分的生产，组装，结构和生物力学特性。 该项目现在主要是暂停的，但是我们确实成功完成了第10财年完成并提交出版的论文，报道了以下结果。在正常皮肤中，CE的蛋白质质量的约75％由一种称为Loricrin的蛋白质贡献。尽管该数字表明的功能很重要，但敲除小鼠的洛里克林基因只会导致轻度的瞬时表型，而洛里克林敲除CE保持正常的厚度（15nm）。 然而，对洛里克林KO CE氨基酸组成的检查显示氨基酸含量的变化，表明将其他蛋白质纳入了CE中，以补偿其损失。 最近，我们的合作者确定，在KO小鼠中的mRNA和蛋白质水平上，晚期晶状膜（LCE）蛋白质家族的几个成员显着上调，并且这些蛋白质被纳入CE中，以在功能上补偿Loricrin的损失。 我们已经产生了具有抗LECE抗体的免疫金EM数据，以确认和扩展这些观察结果。本文目前正在修订中。