Expression, Structure And Function Of The Cornified Cell

复层鳞状上皮中屏障功能的主要组成部分是皮质细胞被膜（CE）。这是在终末分化期间沉积在细胞质膜内表面上的10 nm厚的高度不溶性蛋白质的多组分层。在表皮的情况下，5 nm厚的神经酰胺脂质层（脂质包膜）附着于外表面。蛋白质包膜的不溶性在很大程度上是由于几种结构蛋白被转氨酶交联。蛋白质和脂质成分的生物学和组装研究是该实验室的主要工作。具体而言，我们正在研究：（i）从各种来源分离的CE中的蛋白质的交联，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，并提供关于结构和功能的信息;（ii）几种关键的结构蛋白，例如兜甲蛋白、富含脯氨酸的小蛋白（SPR）家族、外皮蛋白、envoplakin和periplakin;（iii）共价连接到CE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;和（v）使用体外合成脂质囊泡（svi）模型系统重建CE样结构的尝试。 CE蛋白包膜结构和组装。 (1)在这一年中，我们长期努力获得复层鳞状上皮中皮质细胞包膜屏障结构的组成部分之一，periplakin的结构信息，取得了成功。纯化蛋白的旋转阴影图像显示非常灵活的85-90 nm长的棒状分子。针对periplakin的C-末端的抗体仅标记分子的一端，首次为预测的双链卷曲螺旋中的两条链的平行排列提供了证据。 (2)细胞间的相互作用periplakin已通过免疫荧光分析在培养的人角质形成细胞和体外实验。其N端结构域与丝状肌动蛋白结合，而C端结构域与角蛋白8和角蛋白14结合，角蛋白8和角蛋白14分别是简单上皮和复层上皮的中间丝网络的组成部分。电子显微镜下观察到的周斑蛋白分子的细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)与杰斐逊医学院的研究人员合作进行的实验表明，在细胞凋亡期间被激活的半胱天冬酶6特异性地切割靠近C末端的周斑蛋白，有效地将其中间丝结合部分与负责肌动蛋白结合的结构域分离（A. E. Kalinin）。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periplakin寡聚体分子中单个链的比对，产生来自寡聚体的一系列交联肽并通过氨基酸测序进行分析。 (5)为了获得负责中间丝结合的periplakin结构域的3D结构信息，含有该C-末端区域的periplakin的一系列GST融合构建体已在细菌中表达并纯化。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.