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

基本信息

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
项目类别：
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

项目摘要

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) 是由编码多囊蛋白 1 (PC1) 和多囊蛋白 2 (PC2) 的基因突变引起的。该疾病给患者的生活质量带来了巨大的负担，预计每年的医疗费用高达 57 亿美元。由于需要进行肾脏替代治疗，尽管我们知道致病基因的身份，但我们缺乏直接针对多囊蛋白的治疗方法，对 PC1 和潜在的分子缺陷了解甚少。迄今为止，PC2 突变阻碍了治疗 ADPDK 的定制疗法的开发。 PC1 和 PC2 的致病突变会导致大量充满液体的肾囊肿积聚，最终导致肾功能丧失，PC1 和 PC2 被认为是组装、运输并允许离子通过和发出信号的。因此，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作用，PC1 和 PC2 必须在内质网 (ER) 中合成并折叠，接下来，蛋白质必须运输到细胞表面并发挥作用，尽管引起 ADPKD 的突变可能会影响这些步骤中的任何一个。该提案将开发、优化并部署一种工具，根据之前对 PC2 中选定突变体的分析、我们的初步数据以及其他方面的新工作，对哪些致病突变阻碍功能性 PC1 的出现进行分类。在实验室中，这种方法将为科学界和 PKD 界提供新的试剂和检测方法，以研究 PC1 突变体的分子缺陷。我们的新型研究工具采用酵母，这将为 PC1 对通道功能的影响提供快速、定量的读数。由于其快速生成、遗传可塑性、高通量形式的使用以及将致病突变研究转化为人体细胞和体内的许多例子，它们是理想的模型系统。为了为该工具的持续开发提供原理验证，我们将快速定量地对 PC1 中约 70 个已识别的突变如何破坏 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如实报告了这一情况。该应用中的初步数据还表明，PC1 可以与 PC2 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。帮助开发针对 ADPKD 的个性化疗法。