Molecular modulators of polycystin signaling

多囊蛋白信号传导的分子调节剂

基本信息

批准号：
10561693
负责人：
STEFAN SOMLO
金额：
$ 42.4万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-03-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10561693
关键词：
ATAC-seq Ablation Address Adult Affect Affinity Chromatography Antisense Oligonucleotides Apoptosis Attenuated Autosomal Dominant Polycystic Kidney Biological Assay Biology Cell Culture System Cell Culture Techniques Cell Cycle Regulation Cell Line Cell Proliferation Cells ChIP-seq Chromatin Chromatin Structure Cilia Complex Consensus Cyst Data Dialysis procedure Disease Dissociation Down-Regulation GLIS2 gene Gene Dosage Genes Genetic Genetic Diseases Genetic Transcription Genotype Growth In Vitro Inherited Investigation Kidney Kidney Diseases Knowledge Maps Modeling Molecular Mutation Oxidative Phosphorylation PKD2 protein Pathogenesis Pathway interactions Patients Polycystic Kidney Diseases Post-Translational Protein Processing Pre-Clinical Model Proliferating Property Proteins Proteolytic Processing Renal function Research Resolution Ribosomes Role Signal Pathway Signal Transduction Time Tissues Transgenic Organisms Translating Transplantation Transposase Up-Regulation differential expression dosage early onset expectation field study human disease improved in vitro Model in vivo in vivo Model innovation mouse model mutant novel novel therapeutics optimal treatments overexpression polycystic liver disease programs targeted treatment therapeutic target trafficking transcription factor transcriptome transcriptome sequencing transcriptomics

项目摘要

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that results from mutations in either of two proteins, polcysytin-1 (PC1) or polycystin-2 (PC2). More than two decades have passed since the genes encoding these proteins were discovered and there has been significant progress in understanding the functions of polycystins and their associated disease. Nonetheless, there remain substantial gaps in knowledge and lack of consensus about the precise functions of PC1 or PC2 and the mechanisms of ADPKD. Resolution of these gaps is of great significance given our expectation that optimal therapies for ADPKD are best developed based on the fundamental understanding of polycystin function in the mammalian kidney. Much of the current mechanistic understanding of polycystin function is based on studies of candidate pathways drawn from amongst known cellular mechanisms associated with functions such as differentiation, proliferation, transport and signaling. The lack of coalescence toward an interrelated unifying functional pathway in polycystin biology and the persistence of gaps in understanding of in vivo polycystin function despite extensive investigation suggests that the critical components of the most proximal polycystin signaling cascade have yet to be identified. Indeed, the polycystins were discovered as complex, entirely novel proteins and it stands to reason that they may function in a signaling pathway that is not among those that are currently well understood or studied. We made use of this concept in by applying an unbiased in vivo transcriptomic study using Translating Ribosome Affinity Purification (TRAP) RNASeq. From this, we identified upregulation of cell cycle and down regulation of oxidative phosphorylation as key pathways alterations in vivo. Among these, we found genotype dependent upregulation of a cilia associated transcription factor, Glis2, not previously considered to function in polycystin signaling or ADPKD pathogenesis. We made double mutants of Pkd1 with Glis2 in early onset and adult models and found Glis2 dosage-dependent rescue of cyst formation in both. Based on these findings we propose that Glis2 is a candidate for a downstream effector of PC1 function that is critical for cyst progression in ADPKD. In this study, we will determine the in vivo mechanism of action of Glis2 and establish its effects on cyst cell proliferation, apoptosis and ADPKD due to Pkd2. We will determine whether Glis2 is a target for therapy through both genetic and pharmacotherapeutic studies. We will assess whether in vivo genotype dependent transcriptional changes we have identified are similarly extended to cell culture systems with Pkd mutant genotypes. We will also evaluate the functional properties of Glis2 protein in Pkd mutant cell lines. In aggregate, these studies open a new direction of investigation for polycystin signaling and ADPKD pathogenesis.

常染色体显性多囊肾病（ADPKD）是一种常见的遗传性疾病，由以下原因引起：多囊蛋白-1 (PC1) 或多囊蛋白-2 (PC2) 两种蛋白中的任何一个发生突变。二十多年过去了自从编码这些蛋白质的基因被发现并且在这方面取得了重大进展了解多囊蛋白的功能及其相关疾病。尽管如此，仍然存在大量对于 PC1 或 PC2 的确切功能及其机制存在知识差距和缺乏共识 ADPKD。鉴于我们期望 ADPKD 的最佳治疗方法，解决这些差距具有重要意义最好是基于对哺乳动物肾脏中多囊蛋白功能的基本了解而开发。目前对多囊蛋白功能的大部分机制理解都是基于对候选途径的研究取自与分化、增殖等功能相关的已知细胞机制，传输和信号传输。多囊蛋白中缺乏相互关联的统一功能途径的聚结尽管进行了广泛的研究，但对体内多囊蛋白功能的理解仍然存在生物学上的差距表明最近的多囊蛋白信号级联的关键成分尚未确定。事实上，多囊蛋白被发现是一种复杂的、全新的蛋白质，并且理所当然地，它们可能在目前尚未充分理解或研究的信号通路中发挥作用。我们通过使用翻译核糖体进行无偏见的体内转录组学研究，利用了这一概念亲和纯化 (TRAP) RNASeq。由此，我们确定了细胞周期的上调和细胞周期的下调氧化磷酸化作为体内关键途径的改变。其中，我们发现基因型依赖性纤毛相关转录因子 Glis2 的上调，此前不认为该因子在多囊蛋白中发挥作用信号传导或 ADPKD 发病机制。我们在早发型和成人模型中制作了 Pkd1 和 Glis2 的双突变体并发现 Glis2 对囊肿形成的拯救作用呈剂量依赖性。根据这些发现，我们建议 Glis2 是 PC1 功能下游效应子的候选者，对 ADPKD 囊肿进展至关重要。在在这项研究中，我们将确定 Glis2 的体内作用机制并确定其对囊肿细胞的影响 Pkd2 导致的增殖、凋亡和 ADPKD。我们将通过以下方式确定 Glis2 是否是治疗靶点：遗传和药物治疗研究。我们将评估体内基因型是否依赖我们发现的转录变化同样扩展到具有 Pkd 突变体的细胞培养系统基因型。我们还将评估 Pkd 突变细胞系中 Glis2 蛋白的功能特性。总的来说，这些研究为多囊蛋白信号传导和 ADPKD 发病机制的研究开辟了新的方向。