LAMININ-5 AND HEMIDESMOSOMES IN ORAL EPITHELIAL CELLS

口腔上皮细胞中的层粘连蛋白-5 和半桥粒

• 批准号: 6324662
• 负责人: JONATHAN C. JONES
• 金额: $ 22.13万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6324662
• 关键词: cell adhesion; cytoskeleton; epithelium; extracellular matrix; gingiva; integrins; intercellular connection; intermolecular interaction; laminin; molecular cloning; oral mucosa; protein isoforms; recombinant proteins; tissue /cell culture

Hemidesmosomes, cell-matrix connector devices, are used by oral epithelial to attach to the connective tissue of the gingiva as well as the tooth surface. In the cytoplasm of an oral epithelial cell hemidesmosomes are also sites of cytoskeleton anchorage to the cell surface. During the course of periodontal disease, there is, of necessity, modulation in hemidesmosome integrity as epithelial cells migrate along the tooth to form the "long junctional epithelium". Before we can study the role of hemidesmosomes in the pathogenesis of periodontal disease we must have a basic understanding of the molecular components involved in both the assembly and structural maintenance of this complex morphological entity. To this end, in Project 1 of this program project application, we plan to focus our efforts on functional analyses of an adhesive extracellular glycoprotein component of hemidesmosomes called laminin-5 and its impact on oral epithelial cells. In Aim I we address the identification of domains of the laminin-5 heterotrimer involved in nucleation of hemidesmosome assembly. This will involve preparation of recombinant laminin-5 proteins as well as laminin-5 peptides. In Aim 2, these recombinant proteins and peptides will be assayed for their ability a) to induce rapid adhesion of oral epithelial cells and b) to nucleate assembly of hemidesmosomes. As alternative strategies for identification of functional important domains of laminin-5, in Aim 2 we will use peptides to compete the adhesive properties of intact laminin-5. In Aim 3, we wish to identify the substrate binding site(s) of laminin-5. To this end, proteolytic fragments of laminin-5 will be assayed for a) their ability to adhere to surfaces and/or b) to compete with native laminin-5 for substrate binding. Functionally active fragments will be further characterized by microsequencing. In Aim 4, the impact of cell binding to laminin- 5 on hemidesmosome components will be assayed biochemically. We are particularly interested in the possibility that laminin-5 plays a role in matrix-cell signaling via phosphorylation events. Finally, in Aim 5, we wish to identify membrane interactions of laminin-5. This linkage is necessary for the stabilization of the oral epithelium on the tooth surface.

半桥粒，细胞基质连接装置，用于口腔 上皮细胞也附着在牙龈的结缔组织上 作为牙齿表面。 在口腔上皮细胞的细胞质中 半桥粒也是细胞骨架锚定的位点 细胞表面。在牙周病的病程中，有 必要性，半桥粒完整性的调节作为上皮 细胞沿着牙齿迁移形成“长连接” 在我们研究半桥粒在上皮细胞中的作用之前 对于牙周病的发病机制我们必须有一个基本的了解 了解参与这两个过程的分子成分 该综合体的组装和结构维护 形态实体。 为此，在本方案的项目1中 项目申请方面，我们计划将重点放在功能上 粘附细胞外糖蛋白成分的分析 称为层粘连蛋白-5的半桥粒及其对口腔上皮的影响 细胞。 在目标 I 中，我们解决了域的识别问题 层粘连蛋白 5 异源三聚体参与半桥粒成核 集会。 这将涉及重组层粘连蛋白 5 的制备 蛋白质以及层粘连蛋白 5 肽。 在目标 2 中，这些 将检测重组蛋白和肽的能力 a) 诱导口腔上皮细胞快速粘附，b) 半桥粒的核组装。 作为替代策略 用于鉴定层粘连蛋白 5 的重要功能域， 目标 2 我们将使用肽来竞争粘合性能 完整的层粘连蛋白-5。 在目标 3 中，我们希望识别底物 层粘连蛋白 5 的结合位点。 为此，蛋白水解片段 将检测层粘连蛋白 5 a) 其粘附表面的能力 和/或b)与天然层粘连蛋白5竞争底物结合。 功能活性片段将进一步表征为 微测序。 在目标 4 中，细胞与层粘连蛋白结合的影响 将对半桥粒成分的5进行生化分析。 我们对 laminin-5 的可能性特别感兴趣 通过磷酸化事件在基质细胞信号传导中发挥作用。 最后，在目标 5 中，我们希望确定膜相互作用 层粘连蛋白-5。这种联系对于稳定系统是必要的 牙齿表面的口腔上皮。