LAMININ-5 AND HEMIDESMOSOMES IN ORAL EPITHELIAL CELLS

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部位。 细胞表面。在牙周病病程中，有 半桥粒完整性作为上皮的必要性和调节 细胞沿着牙齿迁移，形成“长连接” 在我们能够研究半桥粒在细胞中的作用之前。 对牙周病的发病机制我们必须有一个基本的了解 对这两个过程中涉及的分子成分的理解 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，我们计划将重点放在功能上 一种粘附性胞外糖蛋白组分的分析 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Aim I中，我们解决了对 层粘连蛋白-5异源三聚体参与半桥粒成核 集合。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 重组蛋白质和多肽将被检测其能力 A)诱导口腔上皮细胞快速黏附和b)与 半桥粒的核组装。作为替代战略 为了确定层粘连蛋白-5的重要功能结构域，在 目的2我们将使用多肽来竞争粘附性 完整的层粘连蛋白-5。在目标3中，我们希望确定底物 层粘连蛋白-5的结合部位(S)。为此，蛋白水解物片段 将对层粘连蛋白-5进行检测：a)它们在表面上的黏附能力 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被表征为 微测序。在目标4中，细胞与层粘连蛋白结合的影响- 5对半桥粒组分进行生化检测。 我们特别感兴趣的是层粘连蛋白-5 通过磷酸化事件在基质细胞信号转导中发挥作用。 最后，在目标5中，我们希望确定膜相互作用 层粘连蛋白5。这种联系对于稳定经济是必要的 牙齿表面的口腔上皮细胞。

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.