Exploration of Novel Strategies to Preserve Desmoplakin Variant Function

探索保留桥粒斑蛋白变异功能的新策略

• 批准号: 10576702
• 负责人: Nathan Thompson Wright
• 金额: $ 44.01万
• 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/10576702
• 关键词: Adherence; Adhesives; Affinity; Antibodies; Binding; Biochemical; Biological Assay; Calcium; Calpain; Cardiomyopathies; Cell Adhesion; Cell Line; Cell-Cell Adhesion; Cells; Clinical; Complement; Cytoskeleton; Data; Desmosomes; Diagnosis; Dilated Cardiomyopathy; Disease; Epithelial Cells; Exhibits; Fluorescence Microscopy; Gene Mutation; Genes; Goals; Hair; Heart Diseases; Hypersensitivity; In Vitro; Infiltration; Intermediate Filaments; Keratoderma; Link; Location; Mechanical Stress; Methods; Molecular; Molecular Probes; Mutagenesis; Mutation; Paper; Pathogenicity; Peptide Hydrolases; Pharmaceutical Preparations; Pharmacology; Proteins; Proteolysis; Rare Diseases; Site; Skin; Solvents; Stretching; Structural Protein; Structure; Surface; Syndrome; Testing; Tissues; Translating; Variant; Western Blotting; Work; X-Ray Crystallography; arrhythmogenic cardiomyopathy; desmoplakin; efficacy testing; experimental study; mutant; novel; novel strategies; novel therapeutics; preservation; prevent; protein degradation; reconstitution; response; screening; 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.

项目摘要 脱粒体是一种蛋白质细胞 - 细胞粘附结构，仅是 在经历重大机械应力的组织中发现。它的主要功能之一是 连接相邻细胞的中间丝晶格。 Desmoplakin（DSP）是 脱骨体内的中央结构蛋白之一。至少80％ 心律失常性心肌病（AC）病例与编码的基因中的变体有关 脱发蛋白和约5-15％的AC病例特别关联 编码Desmoplakin（DSP）的基因。 DSP变体也与皮肤密切相关 脆弱的羊毛头发综合征（SFWH）和羊毛头发的扩张心肌病 和角膜皮（DCWHK），两种罕见疾病，具有脆弱的皮肤或脆弱的疾病 皮肤和心肌病。我们的实验室（Pi Wright和Co-Pi Borzok）以前有 共同努力识别和表征一种新颖的分子机制 这三种疾病共有的致病性。即，我们发现 DSP变体导致在DSP中暴露于通常遮挡的钙蛋白酶裂解位点， 对蛋白水解的过度敏感。这导致细胞DSP的损失 水平，产生无功能的脱骨小体，并在临床上表现在弱组织中 稳定。我们以前还发现了多种可以扭转这种钙蛋白酶的策略 高敏性：引入叠加钙蛋白酶的次级突变 切割部位，DSP与阻塞钙蛋白酶裂解位点的抗体孵育， DSP与小分子一起孵育，该分子阻止了进入钙蛋白酶的裂解 地点。 我们有数据表明这些操纵DSP的策略 降解是有效的，策略的分子基础尚未 已对其进行了彻底的分析。在这里，我们建议检查 这些策略如何在蛋白质水平上起作用（AIM 1）。我们还将测试是否 策略在单元格的背景下起作用（AIM 2）。这两个目标将定义 我们如何使用药理学方法校正细胞粘合剂的参数 不足。