Mechanisms of desmosome regulation and disassembly in the skin disease Pemphigus

描述(申请人提供)：天疱疮是一种破坏性的表皮起泡性疾病，在这种疾病中，针对存在于皮肤和粘膜中的细胞-细胞黏附分子产生自身抗体。天疱疮免疫球蛋白以桥粒为靶标，桥粒是一种将角蛋白中间细丝网络连接到细胞间粘附性强的区域的结构。在寻常型天疱疮(PV)中，自身抗体的主要靶点是桥粒钙粘附素亚家族成员桥粒钙粘附素-3(Dsg3)。这项研究概述了在人类角质形成细胞中桥粒组装和分解的基本机制，以及这些过程是如何被PV患者的免疫球蛋白干扰的。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结合如何调节桥粒的形成和分解，以响应天疱疮免疫球蛋白。我们假设，针对脂筏的桥粒蛋白对于桥粒组装过程中的桥粒蛋白聚集以及对PV-IgG反应的解体过程中桥粒钙粘附素的内吞是必不可少的。我们将利用PV患者的样本，包括皮肤活检和纯化的PV Ig G，确定PV Ig G如何影响人体皮肤中的桥粒，并使用细胞生物学和单分子生物物理方法确定PV Ig G如何影响Dsg3黏附功能。此外，我们将定义Dsg3中将蛋白质靶向脂筏的氨基酸决定因素，并使用缺乏脂筏靶向的突变体，确定与脂筏的关联如何影响桥粒桥粒蛋白的功能。类似的研究将对桥粒斑块蛋白进行，如桥粒亲和素和桥粒蛋白，以确定桥粒蛋白与脂筏膜结构域的联系层次。这些实验方法将被结合起来，以促进我们对角质形成细胞黏附的基本细胞机制的理解，并揭示这些机制如何在严重的表皮起泡疾病中受到损害。

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.