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

常染色体显性遗传性多囊肾病(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。由于需要肾脏替代疗法，这种疾病给患者的生活质量带来了极大的负担，并导致美国估计每年的医疗费用为57亿美元。尽管我们知道致病基因的身份，但我们缺乏直接针对多囊藻毒素的治疗方法。到目前为止，对PC1和PC2突变背后的分子缺陷的了解不足，阻碍了针对ADPDK的量身定制疗法的开发。 PC1和PC2的致病突变会导致大量充满液体的肾囊肿积聚，继而最终导致肾功能丧失。PC1和PC2被认为是组装、运输到上皮细胞膜和初级顶端纤毛的离子通道和信号通路。因此，这些部位缺乏功能性多囊蛋白是ADPKD的主要原因。 为了达到细胞表面和功能，PC1和PC2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。虽然导致ADPKD的突变可能会影响这些步骤中的任何一个，但该提案将开发、优化并部署一个工具来分类哪些致病突变阻碍了功能性PC1在细胞表面的出现。基于先前对PC2突变体的分析，我们的初步数据，以及其他实验室的新工作，该方法将为科学界和PKD界提供新的试剂和分析方法，以研究PC1突变体的分子缺陷。我们的新型研究工具使用酵母，这将提供一个快速和定量的读数PC1‘S对通道功能的影响。酵母是一种理想的模式系统，因为它们的快速生成、遗传延展性、高通量形式的使用，以及许多关于致病突变的研究被翻译到人类细胞和体内的例子。 为了为这一工具的继续开发提供原则证明，我们将快速和定量地对PC1中已发现的70个突变如何破坏PC1：PC2通道活动进行分类。本申请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，PC1和PC2可以在酵母中共表达并形成功能通道。该项目将帮助PKD社区测试与新的和新兴的PC1突变相关的缺陷，以帮助跳板开发ADPKD的个性化治疗方法。

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.