Expression, Structure And Function Of The Cornified Cell

复层鳞状上皮屏障功能的一个主要组成部分是角化细胞膜(CE)。这是在终末分化过程中，细胞质膜内表面沉积的一层10纳米厚的高度不溶的多组分蛋白质。在表皮的情况下，5纳米厚的神经酰胺脂(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对几种结构的交联性。对蛋白质和脂肪成分的生物学和组装的研究是这个实验室的主要工作。具体地说，我们正在研究：(I)从各种来源分离的CE中蛋白质的交联性，以探索哪些蛋白质通过哪些谷氨酸和赖氨酸交联在一起，并提供结构和功能方面的信息；(Ii)几个关键的结构蛋白质，如Clicrin，富含脯氨酸的小蛋白(SPR)家族，总蛋白，Envoplakin和Periplakin；(Iii)与CE共价连接的神经酰胺脂类；(Iv)在培养的角质形成细胞中产生的CE组装的早期阶段；以及(V)尝试使用体外合成脂泡(SVI)模型系统重建类似CE的结构。 CE蛋白的包膜结构和组装。 (1)在这一年里，我们为获得复层鳞状上皮角化细胞包膜屏障结构之一的结构信息所做的长期努力取得了成功。纯化蛋白的旋转阴影图像显示了非常灵活的85-90 nm长的棒状分子。针对周斑蛋白C末端的抗体只标记了分子的一端，首次为预测的双链线圈中的两条链平行排列提供了证据。 (2)用免疫荧光分析和体外实验研究了周斑蛋白与人角质形成细胞的相互作用。其N-末端结构域负责与丝状肌动蛋白结合，而C-末端结构域选择性地与角蛋白8和角蛋白14结合，角蛋白8和角蛋白14是单层和复层上皮细胞中中间丝网络的组成部分。电子显微镜显示的周膜蛋白分子的拉长形状使其能够有效地连接肌动蛋白微丝和中间丝网络，在细胞网络整合中发挥作用。 (3)与杰斐逊医学院的一名研究人员合作进行的实验表明，在细胞凋亡过程中被激活的caspase 6特异性地切割靠近C末端的周斑蛋白，有效地将其中间细丝结合部分与负责肌动蛋白结合的结构域(A.E.Kalinin)分开。这一事件是否在复层上皮细胞的终末分化中起作用还有待研究。 (4)为了进一步表征PERPARKIN低聚分子中各链的排列顺序，从低聚物中制备了一系列的交联肽，并进行了氨基酸序列分析。 (5)为了获得负责中间细丝结合的周斑蛋白结构域的三维结构信息，我们在细菌中表达并纯化了一系列包含该C-末端区域的周斑蛋白GST-融合载体。结晶试验正在进行中。

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.