Structural Biology of Keratin Filaments and Cornified Cell Envelopes

终末分化的表皮角质形成细胞形成了人体的外部保护屏障，随着角化的鳞片从外表面脱落，并被从表皮内生活层向外迁移的物质所取代，该屏障不断更新。角化鳞片是由两种成分组成的复合生物材料：角化细胞包膜(CE)内包裹的角蛋白细丝基质。CE是在终末分化过程中沉积在细胞质膜内表面的一层10 nm厚的多组分不溶蛋白。在表皮中，一层5纳米厚的神经酰胺类脂(脂膜)附着在外表面。蛋白质包膜的不溶性在很大程度上是由于转谷氨酰胺酶使几种结构蛋白发生交联。中间丝是真核细胞细胞骨架中普遍存在的成分。它们由五种不同的类型组成，其中数量最多、结构最复杂的是在上皮细胞中广泛表达的I型和II型角蛋白。我们还对其他细胞类型的相关IF以及角蛋白IF和CE之间的相互作用感兴趣。这个项目的长期目标，以前在两个皮肤生物学实验室(P.M.Steinert，P.I.)LSBR将在正常皮肤和疾病皮肤的背景下阐明这些细胞及其成分的产生、组装、结构和生物力学特性。 这个项目现在基本上被搁置了，但我们确实在2010财年成功地完成了一篇论文，并提交了一篇报告以下成果的论文。在正常皮肤中，CE约75%的蛋白质质量是由一种名为氯蛋白的单一蛋白质贡献的。尽管这个数字表明了功能重要性，但在小鼠中敲除氯氯蛋白基因只会导致轻微的瞬时表型，并且氯氯蛋白基因敲除CE保持正常厚度(15 Nm)。然而，对Clicrin KO CE氨基酸组成的检查显示，氨基酸含量发生了变化，表明CE中加入了其他蛋白质以弥补其损失。最近，我们的合作者发现，在KO小鼠中，晚期角化包膜(Lce)蛋白家族的几个成员在mRNA和蛋白质水平上显著上调，这些蛋白被结合到CE中，以功能补偿Clicrin的丢失。我们已经用抗冰抗体生成了免疫金EM数据，以证实和扩展这些观察结果。这篇论文目前正在修订中。

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.