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脏疾病（ADPKD）是由编码多囊这1（PC1）和Polycystin 2（PC2）的基因突变引起的。由于肾脏替代治疗所需的必要，该疾病对患者的生活质量造成了非凡的伯恩（Burnen）的生活质量，并导致估计的年度医疗保健费用为57亿美元。尽管知道严重基因的身份，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突变的分子缺陷的了解不足，无法开发量身定制的治疗ADPDK。 PC1和PC2中引起疾病​​的突变导致大型充满流体的肾脏囊肿的积累，随后是肾功能的事件丧失。据信PC1和PC2可以组装，流动到并允许上皮细胞膜和主要顶端纤毛的离子通过和信号传导。因此，在这些位置缺乏功能多囊素主要负责ADPKD。 为了到达细胞表面和功能，必须合成PC1和PC2并在内质网（ER）中折叠。接下来，蛋白质必须流动到细胞表面和功能。尽管引起ADPKD的突变可能会影响这些步骤中的任何一项，但该建议将开发，优化，然后部署一种工具来分类哪种引起疾病的突变阻碍了功能性PC1在细胞表面上的出现。基于对PC2中某些突变体，我们的初步数据以及来自其他实验室的新兴工作的先前分析，这种方法将为科学和PKD社区提供新的试剂和研究，以研究PC1突变体的分子缺陷。我们的新型研究工具采用了酵母，这将为PC1对渠道功能的影响提供快速和定量的读数。酵母是一个理想的模型系统，因为它们的迅速产生，遗传性可延展性，以高通量格式使用，并且在许多例子中将有关疾病引起突变的研究转化为人类细胞和体内。 为了提供本工具的持续开发的原则证明，我们将快速和定量地对〜70识别PC1中的突变进行分类，以破坏PC1：PC2通道活动。本应用程序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活动。应用程序中的初步数据还表明，PC1可以与酵母中的PC2共表达并形成功能通道。该项目将帮助PKD社区测试与新和新兴的PC1突变体相关的缺陷，以帮助Springboard开发ADPKD的个性化疗法。