Examining the role of PC2 ciliary interactors and folding therapies for PC1 missense mutants in the context of ADPKD

常染色体显性遗传性多囊肾病(ADPKD)是最常见的潜在致死性单基因疾病，每600至1000名活产儿中就有一人受到影响。ADPKD的特点是肾脏内广泛的囊性增大，导致功能性肾组织进行性丧失。人类基于疾病的基因发现发现了多囊蛋白-1(PC1)和多囊蛋白-2(PC2)的突变，这是ADPKD的原因。ADPKD是遗传性囊性疾病的一部分，该疾病还包括常染色体显性遗传性多囊肝病(ADPLD)。ADPLD的主要特征是肝脏内的囊性病变，由PRKCSH和SEC63基因突变引起，这两个基因会影响PC1蛋白在内质网中的转位和成熟。最近，Somlo教授的实验室(美国耶鲁大学肾脏病研究所)发现，转录因子XBP1s的异源上调可能通过增强Prkcsh基因突变背景上的PC1功能，改善与ADPLD相关的细胞和体内表型。最近的研究表明，PC1错义突变的ADPKD患者的病程通常比PC1截短突变的患者病程要轻。因此，该项目基于这样的假设，即ADPLD和ADPKD具有共同的潜在分子遗传学机制，即PC1错义突变导致PC1活性降低。因此，我们假设由于PC1的错义突变，ADPLD和ADPKD具有共同的基于PC1活性降低的潜在分子遗传机制。)由于通过XBP1s增强的蛋白质折叠可能促进ADPLD中PC1的生物发生，我们希望研究这种效应是否也适用于由于PC1错义突变而导致的ADPKD。我们使用CRISPR/Cas9-系统在上皮细胞系和小鼠模型中选择性地将错义突变引入PC1。我们将确定在ADPLD突变中有效的转录因子XBP1s上调是否会影响ADPKD错义突变的细胞和体内表型。基于这些结果，我们将研究XBP1的上游诱导剂IRE1α的药物激活是否可以减少ADPKD模型中未折叠PC1的负担，从而影响由于PC1错义突变而导致的ADPKD表型。第二个目标是在酵母双杂交筛选中验证与PC2相互作用的纤毛蛋白列表。我们将首先在免疫共沉淀实验中确认结果，然后研究候选蛋白对易位/折叠同源稳定的影响。综上所述，该项目的结果将有助于更好地理解伴侣疗法改善PKD基因错义突变患者疾病的治疗潜力。

Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal monogenic disorder, affecting one in 600 to 1000 live births. ADPKD is characterized by extensive cystic enlargement in the kidney that results in progressive loss of functional renal tissue. Human disease-based gene discovery has identified mutations in polycystin-1 (PC1) and polycystin-2 (PC2) accounting for ADPKD. ADPKD is part of a spectrum of inherited cystic diseases that also includes autosomal dominant polycystic liver disease (ADPLD). ADPLD is mainly characterized by cysts in the liver and is caused by mutations in the genes PRKCSH and SEC63, which affect PC1 protein translocation and maturation in the endoplasmic reticulum. Recently, Professor Somlo’s laboratory (Nephrology, Yale University, USA) has shown that heterologous upregulation of the transcription factor XBP1s may improve the cellular and in vivo phenotype associated with ADPLD by augmenting functional PC1 activity on a Prkcsh gene mutation background. The effects of PC1 dosage in ADPKD have been implied by recent findings that ADPKD patients with missense mutations in PC1 typically have a milder course than patients with truncating mutations in PC1. This project is thus based on the hypothesis that ADPLD and ADPKD share the common underlying molecular genetic mechanism of reduced PC1 activity resulting from missense mutations in PC1. We therefore hypothesize that due to missense mutations in PC1, ADPLD and ADPKD share a common underlying molecular genetic mechanism based on reduced activity of PC1.) As enhanced protein folding via XBP1s may improve PC1 biogenesis in ADPLD, we wish to investigate whether that effect is also applicable in ADPKD due to PC1 missense mutations. We used the CRISPR/Cas9-system to selectively introduce missense mutations into PC1 in epithelial cell lines and mouse models. We will determine whether upregulation of transcription factor XBP1s that was effective in ADPLD mutations can impact the cellular and in vivo phenotypes of ADPKD missense mutations. Based on those results, we will then investigate whether pharmaceutical activation of Ire1α, the upstream inducer of XBP1, can decrease the burden of unfolded PC1 in ADPKD models and hence impact the ADPKD phenotype resulting from missense PC1 mutations.A second aim is to validate a list of ciliary proteins that have been proposed to interact with PC2 in a Yeast Two-Hybrid Screen. We will first confirm the result in a co-immunoprecipitation experiment and subsequently investigate the effect of the candidate protein on translocation/folding homeostasis.In summary, the results of this project should contribute to a better understanding of the therapeutic potential of chaperone therapies to ameliorate disease in patients with missense mutations in PKD genes.