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.

终末分化的表皮角质形成细胞形成身体的外部保护屏障，随着角质层从外表面脱落斯劳并被从表皮的内部活层向外迁移的物质所取代，该保护屏障经历不断的更新。角膜鳞状细胞是一种复合生物材料，由两种成分组成：包裹在角膜细胞包膜（CE）内的角蛋白细丝基质。CE是在终末分化期间沉积在细胞质膜内表面上的10nm厚的不溶性蛋白质的多组分层。在表皮中，5 nm厚的神经酰胺脂质层（脂质包膜）附着在外表面。蛋白质包膜的不溶性在很大程度上是由于几种结构蛋白质被转氨酶交联。中间丝是真核细胞骨架中普遍存在的组成部分。它们由五种不同的类型组成，其中数量最多和最复杂的是广泛表达于上皮中的I型和II型角蛋白。我们也有兴趣在其他类型的细胞相关的IF和角蛋白IF和CE之间的相互作用。该项目的长期目标，以前在皮肤生物学实验室（P。M。Steinert，P.I.）LSBR是为了阐明这些细胞及其组分在正常和患病皮肤中的产生、组装、结构和生物力学特性。 该项目目前基本上处于搁置状态，但我们在2010财年成功完成并提交了一篇报告以下结果的论文。在正常皮肤中，CE的蛋白质质量的约75%由称为兜甲蛋白的单一蛋白质贡献。尽管这个数字表明了功能的重要性，敲除小鼠中的兜甲蛋白基因仅导致轻微的瞬时表型，并且兜甲蛋白敲除CE保持正常的厚度（15nm）。 然而，对兜甲蛋白KO CE氨基酸组成的检查揭示了氨基酸含量的变化，表明其他蛋白质被掺入CE中以补偿其损失。 最近，我们的合作者已经确定，在基因敲除小鼠中，晚期皮质包膜（lce）蛋白家族的几个成员在mRNA和蛋白质水平上显著上调，这些蛋白质被整合到CE中，以在功能上补偿兜甲蛋白的损失。 我们已经用抗冰抗体产生了免疫金EM数据，以证实和扩展这些观察结果。该文件目前正在修订中。