Developing a new platform to characterize and treat disease-associated polycystin variants

开发一个新平台来表征和治疗与疾病相关的多囊蛋白变体

• 批准号: 10726754
• 负责人: Christopher James Guerriero
• 金额: $ 19.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10726754
• 关键词: Address; Affect; Alleles; Anabolism; Apical; Appearance; Autosomal Dominant Polycystic Kidney; Binding; Biological Assay; Biological Models; CCL13 gene; Cations; Cell membrane; Cell physiology; Cell surface; Cells; Cilia; Classification; Co-Immunoprecipitations; Communities; Cystic kidney; Data; Defect; Destinations; Development; Dialysis procedure; Disease; Disease Progression; Endoplasmic Reticulum; Epithelial Cells; FDA approved; Generations; Genes; Genetic; Genetic Diseases; Genetic Engineering; Genomics; Goals; Growth; Hand; Health Care Costs; Human; Ion Transport; Ions; KCNJ1 gene; Kidney Diseases; Length; Liquid substance; Location; Mammalian Cell; Measures; Membrane Proteins; Modeling; Molecular; Molecular Chaperones; Molecular Conformation; Mutation; N-terminal; PKD2 protein; Patients; Persons; Pharmaceutical Preparations; Positioning Attribute; Precision therapeutics; Proteins; Quality of life; Reagent; Renal Replacement Therapy; Renal function; Reporting; Research; Ribosomes; Signal Transduction; System; Testing; Therapeutic; Translating; Variant; Work; Yeast Model System; Yeasts; disease-causing mutation; epithelial Na+ channel; experience; high throughput screening; improved; in vivo; link protein; loss of function; mutant; novel; personalized medicine; polycystic kidney disease 1 protein; prevent; protein complex; response; success; targeted treatment; tolvaptan; tool; tool development

Autosomal dominant polycystic kidney disease (ADPKD) results from mutations in the genes encoding polycystin 1 (PC1) and polycystin 2 (PC2). The disease places an extraordinary burden on patient quality of life and results in estimated annual healthcare costs of $5.7 billion in the US due to the necessity for renal replacement therapy. Despite knowing the identity of the causative genes, we lack a treatment that directly targets the polycystins. A poor understanding of the molecular defects underlying PC1 and PC2 mutations has thus far prevented the development of tailored therapeutics to treat ADPDK. Disease-causing mutations in PC1 and PC2 lead to the accumulation of large fluid-filled renal cysts, which is followed by an eventual loss of kidney function. PC1 and PC2 are believed to assemble, traffic to, and permit ion passage and signaling from the epithelial cell membrane and the primary apical cilium. Therefore, the absence of functional polycystins at these locations is primarily responsible for ADPKD. To reach the cell surface and function, PC1 and PC2 must be synthesized and fold in the endoplasmic reticulum (ER). Next, the proteins must traffic to the cell surface and function. Although ADPKD-causing mutations can affect any of these steps, this proposal will develop, optimize, and then deploy a tool to classify which disease-causing mutations impede the appearance of functional PC1 at the cell surface. Based on previous analysis of select mutants in PC2, our preliminary data, and emerging work from other labs, this approach will provide new reagents and assays to the scientific and PKD community to study the molecular defects of PC1 mutants. Our novel research tool employs yeast, which will provide a rapid and quantitative read-out for PC1’s impact on channel function. Yeast are an ideal model system due to their rapid generation, genetic malleability, use in high-throughput formats, and the many examples in which studies on disease- causing mutations were translated into human cells and in vivo. To provide a proof-of-principle for the continued development of this tool, we will rapidly and quantitatively classify how ~70 identified mutations in PC1 disrupt PC1:PC2 channel activity. Background data provided in this application indicate that the yeast system faithfully reports on how mutations alter PC2 activity. Preliminary data in the application also show that PC1 can be co- expressed with PC2 in yeast and form a functional channel. This project will help the PKD community test the defects associated with new and emerging PC1 mutants to help springboard the development of personalized therapies for ADPKD.

常染色体显性多囊肾病（ADPKD）是由多囊蛋白1（PC1）和多囊蛋白2（PC2）编码基因突变引起的。该疾病给患者的生活质量带来了巨大的负担，由于需要肾脏替代治疗，美国每年的医疗费用估计为57亿美元。尽管知道致病基因的身份，但我们缺乏直接靶向多囊蛋白的治疗方法。迄今为止，对PC1和PC2突变背后的分子缺陷的认识不足阻碍了治疗ADPDK的定制疗法的开发。 PC1和PC2中的致病突变导致大量充满液体的肾囊肿的积累，随后最终丧失肾功能。PC1和PC2被认为是组装，交通，并允许离子通道和信号从上皮细胞膜和初级顶端纤毛。因此，在这些位置缺乏功能性多囊蛋白是ADPKD的主要原因。 为了到达细胞表面并发挥功能，PC 1和PC 2必须在内质网（ER）中合成和折叠。接下来，蛋白质必须运输到细胞表面并发挥作用。虽然ADPKD引起的突变可以影响这些步骤中的任何一个，但该提案将开发，优化，然后部署一种工具来分类哪些致病突变阻碍了细胞表面功能性PC1的出现。基于先前对PC2中选择突变体的分析，我们的初步数据以及其他实验室的新兴工作，这种方法将为科学和PKD社区提供新的试剂和检测方法，以研究PC1突变体的分子缺陷。我们的新研究工具采用酵母，这将提供一个快速和定量读出的PC1的通道功能的影响。酵母是一种理想的模型系统，这是由于它们的快速生成、遗传可塑性、以高通量形式使用，以及其中对致病突变的研究被转化到人细胞和体内的许多实例。 为了为该工具的持续开发提供原理证明，我们将快速定量地对PC1中约70个已鉴定的突变如何破坏PC1：PC2通道活性进行分类。本申请中提供的背景数据表明酵母系统忠实地报告了突变如何改变PC2活性。应用中的初步数据还表明，PC 1可以与PC 2在酵母中共表达，并形成功能通道。该项目将帮助PKD社区测试与新出现的PC1突变体相关的缺陷，以帮助开发ADPKD的个性化疗法。