EXPRESSION, STRUCTURE AND FUNCTION OF THE CORNIFIED CELL ENVELOPE

复层鳞状上皮屏障功能的主要成分 就是角化的细胞包膜。这是一种厚度为15 nm的多组分 一层高度不溶的蛋白质沉积在内表面 细胞的质膜。就表皮而言，5纳米厚 外层附着一层神经酰胺脂类(脂膜)。 浮出水面。蛋白质包膜的不溶性在很大程度上是由于 通过转谷氨酰胺酶使组成蛋白发生交联化。 我们已经详细研究了其中的两种蛋白质，氯化蛋白和小分子 富含脯氨酸(SPR)的家庭。我们已经表达了人的三氯氯丙烷 细菌，并用它来表征它的结构，生化 性质，以及表皮谷氨酰胺转氨酶的体外交联性。 同样，我们在细菌中表达了几种SPR蛋白和 在体外表征了它们的生物化学和交联性。 SPR蛋白质的溶液核磁共振结构研究正在进行中。 初步数据表明，中心肽重复结构域采用了新的 3-环状蛋白折叠。我们已经描述了这种差异 SPR1和SPR2蛋白在小鼠表皮中的表达特性 和上皮组织。我们的数据表明SPR蛋白似乎 在细胞包膜结构中作为交叉桥蛋白发挥作用。我们 使用受控蛋白分解法来解剖角化细胞 包皮表皮和活体包皮中形成的包膜 角质形成细胞的体外培养。蛋白质包膜的大部分 由氯氰菊酯(70-80%)(与少量(2-20%)混合)组成 SPRS。通过在甲醇中碱性水解来去除共价键 包皮细胞外膜的神经酰胺脂类，我们已经能够探索 此结构的最内部方面，对应于首字母 它的组装阶段。我们的数据表明细胞包膜组装是 在角蛋白细丝和许多相关的部位开始 蛋白质与桥粒相遇。我们发现，脂质中的神经酰胺 包膜由不同种类的分子组成 大小，并且共价连接到总蛋白以及可能的其他 蛋白质，在蛋白质包膜上。

A major component of barrier function in stratified squamous epithelia is the cornified cell envelope. This is a multi-component 15 nm thick layer of highly insoluble protein deposited on the inner surface of the plasma membrane of the cells. In the case of the epidermis, a 5 nm thick layer of ceramide lipids (lipid enevlope) is attached to the exterior surface. The insolubility of the protein envelope is due in large part to the crosslinking of the constituent proteins by transglutaminases. We have studied two of these proteins in detail, loricrin and the small proline rich (SPR) families. We have expressed human loricrin in bacteria and used it to characterize its structure, biochemical properties, and crosslinking by epidermal transglutaminases in vitro. Similarly, we have expressed in bacteria several SPR proteins and characterized their biochemical and crosslinking properties in vitro. Solution nmr structural studies on SPR proteins are in progress. Preliminary data suggest the central peptide repeat domains adopt novel 3-loop- like protein folds. We have characterized the differential expression properties of the SPR1 and SPR2 proteins in mouse epidermis and epithelial tissues. Our data suggest the SPR proteins seem to function as crossbridging proteins in the cell envelope structure. We have used controlled proteolysis to dissect apart the cornified cell envelope formed in foreskin epidermis in vivo and in epidermal keratinocyte cultures in vitro. The bulk of the protein envelope consists of loricrin (70-80%( admixed with smaller amounts (2-20%) of SPRs. By alkaline hydrolysis in methanol to remove covalently bound ceramide lipids of foreskin cell envelopes, we have been able to explore the innermost aspects of this structure, corresponding to the initial stages of its assembly. Our data suggest that cell envelope assembly is initiated at the site where keratin filaments and many associated proteins meet desmosomes. We have found that the ceramides of the lipid envelope consist of a heterogeneous population of molecules of varying size, and are covalently attached to involucrin, as well as perhaps other proteins, on the protein envelope.