Expression/Structure,Function Of Cornified Cell Envelope

角质化细胞膜的表达/结构、功能

基本信息

批准号：
6968014
负责人：
ALASDAIR C. STEVEN
金额：
--
依托单位：
NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

项目摘要

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. During FY04, several projects addressing CE structure and assembly were completed (see below). Further research in this area will be continued by Dr L Marekov in association with the LSBR, NIAMS. (1) Co-assembly of Envoplakin and Periplakin into Oligomers and Ca++ dependent Vesicle Binding: Plakin family members envoplakin and periplakin are part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In FY04, we completed an investigation of the properties and interactionsof purified recombinant human envoplakin and periplakin. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility. Envoplakin and periplakin formed soluble complexes with equimolar stoichiometry. Chemical cross-linking revealed that the major soluble form of all periplakin constructs and of envoplakin/periplakin rod domains was a dimer, while co-assembly of the full-length proteins resulted in formation of higher order oligomers. Electron microscopy of rotary-shadowed periplakin demonstrated thin flexible molecules with an average contour length of 88 nm for the rod-plus-tail fragment, and immunolabeling EM confirmed the molecule as a parallel, in-register, dimer. Both periplakin and envoplakin/periplakin oligomers were able to bind synthetic lipid vesicles whose composition mimicked the cytoplasmic side of the plasma membrane of eukaryotic cells. This binding was dependent on anionic phospholipids and Ca++. These findings raise the possibility that envoplakin and periplakin bind to the plasma membrane upon elevation of intracellular [Ca++] in differentiating keratinocytes, where they serve as a scaffold for cornified cell envelope assembly. (2) Effects of knocking-out trichohyalin. This project was initiated in LSB two years ago. During FY04, we designed and constructed the knock-out vector and contarcted to have the ES cells screened and injected into mice. The chimera mice were born and transferred to our collaborator, Dr. Allen Li at Oregon Health & Science University for further characterization. A total of 7 homogyzous mice were generated. Their first coat of hair showed a wavy phenotype initially which fits our prior hypothesis that trichohyalin is needed to strengthen hair shafts. These hairs, however, developed normally after two weeks. Over a longer timescale, the KO mice started to develop hair loss at about 6 months. Also their hair shaft appeared less strong as compared to wildtype shaft. More detailed biochemistry and EM analyses will be conducted to study the effects of trichohyalin loss in hair and other tissues such as skin and tongue. 3)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. 4) Role of epiplakin as a cytolinker. Epiplakin is a member of the plakin family with multiple copies of the plakin repeat domain (PRD). We studied the subcellular distribution and interactions of human epiplakin by immunostaining, overlay assays, and RNAi knockdown. Epiplakin decorated the keratin IF network and partially that of vimentin. In the binding assays, the repeat unit (PRD plus linker) showed strong binding and preferentially associated with assembled IF over keratin monomers. Epiplakin knock-down revealed disruption of IF networks in simple epithelial but not in epidermal cells. In rescue experiments, the repeat unit was necessary to prevent the collapse of IF networks in transient knock-down; however, it could only partially restore the keratin but not the vimentin IF network in stably knocked-down HeLa cells. We infer that epiplakin is a versatile cytolinker with functions involved in maintaining the integrity of IF networks in simple epithelial cells. Furthermore, we observed an increase of epiplakin expression in keratinocytes after the calcium switch suggesting the involvement of epiplakin in the process of keratinocyte differentiation. (5) 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. This project was completed in FY04.

复层鳞状上皮屏障功能的主要组成部分是角化细胞包膜（CE）。这是一种多成分的 10 nm 厚的高度不溶性蛋白质层，在终末分化过程中沉积在细胞质膜的内表面上。对于表皮，外表面附着一层 5 nm 厚的神经酰胺脂质（脂质包膜）。蛋白质包膜的不溶性很大程度上是由于转谷氨酰胺酶的几种结构的交联。对蛋白质和脂质成分的生物学和组装的研究是该实验室的主要工作。具体来说，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) 共价连接至 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； (v) 尝试使用体外合成脂质囊泡 (Svi) 模型系统重建类似 CE 的结构。 2004 财年期间，完成了几个涉及 CE 结构和组装的项目（见下文）。 L Marekov 博士将与 LSBR、NIAMS 合作继续开展该领域的进一步研究。 (1) Envoplakin 和 Periplakin 共组装成寡聚物和 Ca++ 依赖性囊泡结合：Plakin 家族成员 envoplakin 和 Periplakin 是终末分化的复层鳞状上皮中角化细胞包膜的一部分。 2004 财年，我们完成了对纯化的重组人 envoplakin 和 periplakin 的特性和相互作用的研究。我们发现envoplakin在体外生理条件下不溶，需要与periplakin共组装才能溶解。 Envoplakin 和 periplakin 形成等摩尔化学计量的可溶性复合物。化学交联揭示所有periplakin构建体和envoplakin/periplakin杆结构域的主要可溶形式是二聚体，而全长蛋白质的共组装导致形成更高级的寡聚物。旋转阴影周斑蛋白的电子显微镜显示出薄的柔性分子，杆加尾片段的平均轮廓长度为 88 nm，免疫标记 EM 证实该分子是平行的、对准的二聚体。 periplakin 和 envoplakin/periplakin 寡聚物都能够结合合成脂质囊泡，其组成模仿真核细胞质膜的细胞质侧。这种结合依赖于阴离子磷脂和 Ca++。这些发现提出了一种可能性，即在分化的角质形成细胞中细胞内[Ca++]升高时，envoplakin和periplakin与质膜结合，它们充当角化细胞包膜组装的支架。 (2)敲除毛玻璃蛋白的效果。该项目于两年前在LSB启动。 2004财年期间，我们设计并构建了敲除载体，并签约筛选ES细胞并注射到小鼠体内。嵌合体小鼠出生并转移给我们的合作者俄勒冈健康与科学大学的 Allen Li 博士进行进一步表征。总共产生7只纯合小鼠。他们的第一层毛发最初表现出波浪状的表型，这符合我们之前的假设，即需要毛透明蛋白来强化毛干。然而，两周后这些毛发就正常发育了。在更长的时间范围内，KO 小鼠在大约 6 个月时开始出现脱发。而且，与野生型毛干相比，它们的毛干显得不太坚固。将进行更详细的生物化学和电镜分析，以研究毛透明蛋白损失对头发和其他组织（例如皮肤和舌头）的影响。 3)通过培养的人角质形成细胞中的免疫荧光分析和体外实验对periplakin的细胞相互作用进行了表征。其 N 端结构域负责与丝状肌动蛋白结合，而 C 端结构域则选择性结合角蛋白 8 和 14，即简单上皮和复层上皮中相应的中间丝网络的组成部分。电子显微镜显示的周斑蛋白分子的细长形状应使其能够有效地互连肌动蛋白微丝和中间丝网络，在细胞网络整合中发挥作用。 4) Epiplakin 作为细胞链接剂的作用。 Epiplakin 是 plakin 家族的成员，具有多个 plakin 重复结构域 (PRD) 副本。我们通过免疫染色、叠加分析和 RNAi 敲低研究了人表皮蛋白的亚细胞分布和相互作用。 Epiplakin 装饰角蛋白 IF 网络，部分装饰波形蛋白。在结合测定中，重复单元（PRD 加接头）显示出很强的结合力，并且优先与组装的 IF 相关而不是角蛋白单体。 Epiplakin 敲除揭示了简单上皮细胞中 IF 网络的破坏，但表皮细胞中没有。在救援实验中，重复单元对于防止中频网络在瞬时击倒中崩溃是必要的。然而，它只能部分恢复稳定敲低的 HeLa 细胞中的角蛋白，而不能恢复波形蛋白 IF 网络。我们推断 Epiplakin 是一种多功能细胞接头，其功能涉及维持简单上皮细胞中 IF 网络的完整性。此外，我们观察到钙转换后角质形成细胞中表斑蛋白表达增加，表明表斑蛋白参与角质形成细胞分化过程。 (5) 与杰斐逊医学院的研究人员合作进行的实验表明，在细胞凋亡过程中被激活的 caspase 6 特异性切割靠近 C 末端的 periplakin，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。该项目于 2004 财年完成。