Mechanisms of desmosome regulation and disassembly in the skin disease Pemphigus

描述（由申请人提供）：天疱疮是一类破坏性表皮起泡疾病，其中产生针对皮肤和粘膜中存在的细胞-细胞粘附分子的自身抗体。天疱疮IgG靶向桥粒，一种将角蛋白中间丝网络与强细胞-细胞粘附区域偶联的结构。在寻常型天疱疮（PV）中，自身抗体的主要靶点是桥粒芯糖蛋白-3（Dsg 3），它是粘附分子桥粒钙粘蛋白亚家族的成员。本提案中概述的工作研究了人类角质形成细胞中桥粒组装和拆卸的基本机制以及PV患者IgG如何破坏这些过程。该提案的一个主要焦点是桥粒蛋白如何靶向脂筏膜微区，以及桥粒蛋白与脂筏的关联如何响应天疱疮IgG调节桥粒形成和分解。我们推测，桥粒蛋白靶向脂筏是必不可少的桥粒组装过程中的桥粒蛋白聚类，并在响应PV IgG的拆卸过程中的桥粒钙粘蛋白的内吞作用。我们将利用PV患者样本，包括皮肤活检和纯化的PV IgG，以确定PV IgG如何影响人类皮肤中的桥粒，并使用细胞生物学和单分子生物物理学方法确定PV IgG如何影响Dsg 3粘附功能。此外，我们将定义Dsg 3中的氨基酸决定簇，其将蛋白质靶向脂筏，并使用筏靶向缺陷的突变体，确定与脂筏的关联如何影响桥粒芯蛋白功能。将用桥粒斑蛋白如斑嗜蛋白和桥粒斑蛋白进行类似的研究，以确定桥粒蛋白与脂筏膜结构域缔合的层次结构。这些实验方法将被整合，以促进我们的角质形成细胞粘附的基本细胞机制的理解，并揭示这些机制是如何在一个严重的表皮起泡疾病妥协。

DESCRIPTION (provided by applicant): Pemphigus is a class of devastating epidermal blistering diseases in which autoantibodies are generated against cell-cell adhesion molecules present in the skin and mucous membranes. Pemphigus IgG target desmosomes, a structure that couples the keratin intermediate filament network to regions of strong cell-cell adhesion. In pemphigus vulgaris (PV), the primary target of the autoantibodies is desmoglein-3 (Dsg3), a member of the desmosomal cadherin subfamily of adhesion molecules. The work outlined in this proposal investigates the fundamental mechanisms of desmosome assembly and disassembly in human keratinocytes and how these processes are disrupted by PV patient IgG. A major focus of the proposal is how desmosomal proteins are targeted to lipid raft membrane microdomains, and how the association of desmosomal proteins with lipid rafts regulates desmosome formation and disassembly in response to pemphigus IgG. We hypothesize that desmosomal protein targeting to lipid rafts is essential for desmosomal protein clustering during desmosome assembly, and for endocytosis of desmosomal cadherins during disassembly in response to PV IgG. We will utilize PV patient samples, including skin biopsies and purified PV IgG, to determine how PV IgG affect desmosomes in human skin and to determine how PV IgG impact Dsg3 adhesive function using cell biological and single molecular biophysical approaches. Additionally, we will define the amino-acid determinants in Dsg3 that target the protein to lipid rafts and, using mutants deficient in raft targeting, determine how association with lipid rafts impacts desmoglein function. Similar studies will be performed with desmosomal plaque proteins such as the plakophilins and desmoplakin to determine the hierarchy of desmosome protein association with lipid raft membrane domains. These experimental approaches will be integrated to advance our understanding of basic cellular mechanisms of keratinocyte adhesion and to reveal how these mechanism are compromised in a serious epidermal blistering disorder.