Administrative Supplement to Purchase CLARIOstar Plus Platereader

项目摘要 桥粒是一种蛋白质细胞-细胞黏附结构，只有 在承受巨大机械应力的组织中发现。它的主要功能之一是 以连接相邻细胞的中间纤维网格。Desmoplakin(DSP)是 桥粒桥粒内的中心结构蛋白之一。至少80%的 致心律失常的心肌病(AC)病例与编码基因变异有关 桥粒蛋白和大约5%-15%的AC病例与 编码桥粒蛋白(Dsp)的基因。DSP变体也与皮肤有很强的关联 脆性毛发综合征与扩张型心肌病伴毛发 和角皮病(DCWHK)，这两种罕见疾病的特征是皮肤脆弱或脆弱 皮肤加心肌病。我们的实验室(Pi Wright和co-Pi Borzok)之前 共同鉴定和表征了一种新的分子机制 这三种疾病都有共同的致病性。也就是说，我们发现 DSP变异体导致在DSP中露出通常闭塞的钙蛋白裂解位点， 使该变异体对蛋白质分解反应过敏。这导致了蜂窝dsp的损失。 水平，产生不起作用的桥粒，并在弱化的组织中临床表现 稳定性。我们之前还发现了多种策略可以逆转这种钙蛋白酶 超敏反应：引入覆盖在钙蛋白上的次级突变 裂解部位，将DSP与封闭Calain裂解部位的抗体孵育， 以及将DSP与小分子孵育，这些小分子可以阻止钙蛋白裂解 地点。 虽然我们有数据表明这些操纵数字信号处理器的策略 降解是有效的，但这些策略的分子基础还没有 被彻底分析过了。在此，我们建议研究其分子机制。 这些策略如何在蛋白质水平发挥作用(目标1)。我们还将测试这些 战略在一个单元的背景下是起作用的(目标2)。这两个目标将定义 我们如何使用药理学方法来纠正细胞粘连的参数 缺乏症。

Project Summary The desmosome is a proteinaceous cell-cell adherence structure that is only found in tissues that undergo significant mechanical stress. One of its main functions is to connect the intermediate filament latticework of adjacent cells. Desmoplakin (DSP) is one of the central structural proteins within the desmosome. At least 80% of Arrhythmogenic Cardiomyopathy (AC) cases are linked to variants in genes that encode desmosomal proteins and about 5-15% of AC cases are specifically linked to variants in the gene encoding desmoplakin (dsp). dsp variants are also strongly linked to Skin Fragility Wooly Hair Syndrome (SFWHS) and Dilated Cardiomyopathy with Wooly Hair and Keratoderma (DCWHK), two rare diseases characterized by fragile skin or fragile skin plus cardiomyopathy. Our labs (PI Wright and co-PI Borzok) have previously worked together to identify and characterize a novel molecular mechanism of pathogenicity common to all three of these diseases. Namely, we found that a subset of dsp variants result in the exposure of a usually occluded calpain cleavage site in DSP, rendering the variant hypersensitive to proteolysis. This results in a loss of cellular DSP levels, creates nonfunctioning desmosomes, and manifests clinically in weakened tissue stability. We also previously discovered multiple strategies that can reverse this calpain hypersensitivity: the introduction of secondary mutations that overlay the calpain cleavage site, incubation of DSP with antibodies that occlude the calpain cleavage site, and incubation of DSP with small molecules that block access to the calpain cleavage site. While we have data showing that these strategies to manipulate DSP degradation are effective, the molecular underpinnings of the strategies have not yet been thoroughly analyzed. Here, we propose to examine the molecular mechanism of how these strategies work on the protein level (aim 1). We will also test whether these strategies are functional within the context of a cell (aim 2). These two aims will define the parameters of how we can use pharmacological methods to correct a cell adhesive deficiency.