Expression, Structure And Function Of The Cornified Cell

层状鳞状上皮屏障功能的主要组成部分是角化细胞包膜（CE）。这是一种多组分10纳米厚的高度不溶性蛋白质层，在细胞终末分化过程中沉积在质膜的内表面。在表皮的情况下，一层5纳米厚的神经酰胺脂质（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转谷氨酰胺酶的几个结构交联。研究蛋白质和脂质组分的生物学和组装是本实验室的主要工作。具体而言，我们正在研究：(i)从各种来源分离的ce中蛋白质的交联，以探索哪些蛋白质通过哪些谷氨酰胺和赖氨酸交联在一起，并提供结构和功能信息；（ii）几种关键结构蛋白，如loricrin、小脯氨酸富蛋白（SPR）家族、involucrin、envoplakin和periplakin；（iii）与CE共价结合的神经酰胺脂质；（iv）在培养的角质形成细胞中产生的CE组装的最早阶段；(v)尝试使用体外合成脂质囊泡（svi）模型系统重建ce样结构。

A major component of barrier function in stratified squamous epithelia is the cornified cell envelope (CE). This is a multi-component 10 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells during terminal differentiation. In the case of the epidermis, a 5 nm 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 by transglutaminases. Studies on the biology and assembly of the protein and lipid components are a major effort of this laboratory. Specifically, we are studying: (i) the cross-linking of proteins in CEs isolated from a variety of sources to explore which proteins are cross-linked together through which glutamines and lysines, and to provide information on structure and function; (ii) several key structural proteins such as loricrin, the small proline rich protein (SPR) families, involucrin, envoplakin and periplakin; (iii) the ceramide lipids which become covalently attached to the CE; (iv) the earliest stages of CE assembly produced in cultured keratinocytes; and (v) an attempt to recreate a CE-like structure using an in vitro synthetic lipid vesicle (svi) model system. CE protein envelope structure and assembly. (1) During this year our long-time efforts toward obtaining structural information on one of the components of cornified cell envelope barrier structure in stratified squamous epithelia, periplakin, met with success. Rotary-shadowed images of purified protein revealed very flexible 85-90 nm-long rod-like molecules. Antibodies against C-terminus of periplakin labeled only one end of the molecule, providing evidence for parallel alignment of two chains in predicted double stranded coil-coil for the first time. (2) Cellular interactions of periplakin have been characterized by immunofluorescent analysis in cultured human keratinocytes and by in vitro experiments. Its N-terminal domain turned out to be responsible for binding to filamentous actin, while C-terminal domain showed selective binding to keratins 8 and 14, components of intermediate filaments network in simple and stratified epithelia correspondingly. Elongated shape of periplakin molecule revealed by electron microscopy should allow it to efficiently interconnect actin microfilament and the intermediate filament networks, playing role in cell network integration. (3) Experiments done in collaboration with an investigator at Jefferson Medical College, showed that caspase 6, which is activated during apoptosis, specifically cleaves periplakin close to C-terminus, effectively separating its intermediate filaments binding part from domain which is responsible for actin binding (A. E. Kalinin). It remains to be investigated whether this event plays a role in terminal differentiation of cells in stratified epithelia. (4) To further characterize alignment of individual chains in periplakin oligomeric molecule, series of cross-linked peptides from the oligomer were generated and analyzed by amino acid sequencing. (5) To obtain 3D structural information on periplakin domain responsible for intermediate filaments binding, series of GST-fusion constructs of periplakin, containing this C-terminal region, have been expressed in bacteria and purified. Crystallization trials are ongoing.