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例活产中就有一例受影响。ADPKD的特征是肾脏广泛囊性增大，导致功能性肾组织进行性丧失。基于人类疾病的基因发现已经确定了多囊蛋白-1（PC 1）和多囊蛋白-2（PC 2）中的突变，这些突变导致ADPKD。ADPKD是一系列遗传性囊性疾病的一部分，还包括常染色体显性遗传性多囊肝病（ADPLD）。ADPLD的主要特征是肝脏中的囊肿，并且是由基因PRKCSH和SEC 63的突变引起的，其影响PC 1蛋白在内质网中的移位和成熟。最近，Somlo教授的实验室（美国耶鲁大学肾脏病学）已经表明，转录因子XBP 1 s的异源上调可以通过在Prkcsh基因突变背景下增强功能性PC 1活性来改善与ADPLD相关的细胞和体内表型。最近的研究结果表明，PC 1错义突变的ADPKD患者通常比PC 1截短突变的患者病程更轻，这暗示了PC 1剂量对ADPKD的影响。因此，该项目基于ADPLD和ADPKD共享由PC 1中的错义突变导致的PC 1活性降低的共同潜在分子遗传机制的假设。因此，我们假设由于PC 1的错义突变，ADPLD和ADPKD共享基于PC 1活性降低的共同潜在分子遗传机制。由于通过XBP 1 s增强的蛋白质折叠可以改善ADPLD中的PC 1生物合成，我们希望研究这种效应是否也适用于由于PC 1错义突变引起的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PC 1。我们将确定在ADPLD突变中有效的转录因子XBP 1 s的上调是否会影响ADPKD错义突变的细胞和体内表型。在此基础上，我们将进一步研究药物激活XBP 1的上游诱导物Ire 1 α是否能够降低ADPKD模型中未折叠PC 1的负荷，从而影响PC 1错义突变导致的ADPKD表型。第二个目的是验证酵母双杂交筛选中提出的与PC 2相互作用的纤毛蛋白列表。我们将首先确认结果的免疫共沉淀实验，随后调查的效果的候选蛋白易位/折叠homeostasis.In总结，该项目的结果应有助于更好地了解伴侣疗法的治疗潜力，以改善疾病的患者PKD基因的错义突变。

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.