Administrative Supplement to Purchase CLARIOstar Plus Platereader

购买 CLARIOstar Plus 读板器的行政补充文件

• 批准号: 10788703
• 负责人: Nathan Thompson Wright
• 金额: $ 6.26万
• 依托单位: JAMES MADISON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-20 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10788703
• 关键词: Adherence; Adhesives; Administrative Supplement; Antibodies; Calpain; Cardiomyopathies; Cells; Clinical; Cytoskeleton; Data; Desmosomes; Dilated Cardiomyopathy; Disease; Genes; Goals; Hair; Hypersensitivity; Incubated; Intermediate Filaments; Keratoderma; Link; Mechanical Stress; Methods; Molecular; Mutation; Pathogenicity; Proteins; Proteolysis; Rare Diseases; Site; Skin; Structural Protein; Structure; Syndrome; Testing; Tissues; Variant; Work; arrhythmogenic cardiomyopathy; desmoplakin; novel; novel therapeutics; pharmacologic; skin disorder; small molecule; tool

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.

项目概要 桥粒是一种蛋白质细胞-细胞粘附结构，仅 存在于承受显着机械应力的组织中。它的主要功能之一是 连接相邻细胞的中间丝网格。桥粒斑蛋白 (DSP) 是 桥粒内的中心结构蛋白之一。至少80% 致心律失常性心肌病 (AC) 病例与编码基因的变异有关 桥粒蛋白和大约 5-15% 的 AC 病例与 编码桥粒斑蛋白 (dsp) 的基因。 dsp 变体也与皮肤密切相关 脆性毛茸茸综合症 (SFWHS) 和毛茸茸的扩张型心肌病 和角皮病（DCWHK），两种罕见疾病，其特征是皮肤脆弱或脆弱 皮肤加心肌病。我们的实验室（PI Wright 和联合 PI Borzok）之前曾 共同努力确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，我们发现一个子集 dsp 变体导致 DSP 中通常闭塞的钙蛋白酶切割位点暴露， 使变体对蛋白水解过敏。这会导致蜂窝 DSP 丢失 水平，产生无功能的桥粒，并在临床上表现为薄弱的组织 稳定。我们之前还发现了多种可以逆转这种钙蛋白酶的策略 超敏反应：引入覆盖钙蛋白酶的二次突变 裂解位点，将 DSP 与封闭钙蛋白酶裂解位点的抗体一起孵育， 将 DSP 与阻止钙蛋白酶裂解的小分子一起孵育 地点。 虽然我们有数据表明这些操纵 DSP 的策略 降解是有效的，但该策略的分子基础尚未确定 进行了彻底的分析。在这里，我们建议检查其分子机制 这些策略如何在蛋白质水平上发挥作用（目标 1）。我们还将测试这些是否 策略在细胞环境中发挥作用（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附的参数 不足。