Investigate kidney cyst formation and a cilia-mediated signaling network

研究肾囊肿的形成和纤毛介导的信号网络

• 批准号: 8685254
• 负责人: ZHAOXIA SUN
• 金额: $ 36.21万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8685254
• 关键词: Address; Affinity Chromatography; Autosomal Dominant Polycystic Kidney; Binding; Biochemical; Biology; C-terminal; Cell Nucleus; Cell physiology; Cell surface; Cells; Chromatin; Chromatin Remodeling Factor; Cilia; Coin; Couples; Coupling; Cyst; Cystic kidney; Cytoplasmic Protein; Data; Defect; Development; Disease; Disease Progression; Disease model; Epigenetic Process; Epithelial Cells; Etiology; Functional disorder; Future; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetic Screening; Genetic Transcription; Genus Hippocampus; Goals; Histone Deacetylase Inhibitor; Human; Infertility; Kidney; Kidney Diseases; Knock-out; Knowledge; Link; Mediating; Modeling; Molecular; Mus; Mutate; Nature; Obesity; Organelles; Pathogenesis; Pathway interactions; Phenotype; Play; Polycystic Kidney Diseases; Precipitation; Proteins; Proteomics; Research; Retinal Degeneration; Role; Sea; Signal Pathway; Signal Transduction; Signaling Protein; Structure; System; Tail; Testing; Transcriptional Regulation; Transducers; Yeasts; Zebrafish; base; cellular transduction; ciliopathy; cilium biogenesis; design; expectation; human disease; in vivo; insight; mutant; nephrogenesis; novel; prevent; response; therapy design; transcription factor; yeast two hybrid system

DESCRIPTION (provided by applicant): Our long-term goal is to understand the etiology of polycystic kidney disease (PKD) and to provide insight for better treatment of PKD and other ciliopathies. The main goal of this project is to dissect a newly identified Arl13b-Sea-Reptin network in cilia-mediated signaling and its role in kidney cyst formation. The cilium, a previously obscure cell surface structure, plays a critical role in PKD and an increasing number of other diseases. Although many cilia-associated proteins have been identified, little is known about the functional relationship between these proteins and how signals are transduced from this cell surface organelle into the cell and the nature of elicited cellular responses. Lack of such knowledge is hindering our understanding of the role of cilia in vertebrate development and human disease. To understand cilia-mediated signaling and its role in kidney cyst formation in an integrated fashion, we combine zebrafish genetics with proteomic approaches. In a large-scale genetic screen, we isolated 13 genes that can cause kidney cyst when mutated. In addition to multiple IFT genes that are important for cilia biogenesis, also isolated in this scree are novel genes arl13b, sea and transcriptional regulators reptin and pontin. Further studies revealed that Arl13b is a ciliary protein required for cilia biogenesis, while Sea is mainly a cytoplasmic protein required for cilia signaling. Unexpectedly, we found that Arl13b co-purifies with Sea and that Sea physically interacts with Reptin through tandem affinity purification and yeast two-hybrid screens. Considering that Reptin is a well-known epigenetic/transcriptional regulator, we hypothesize that Arl13b-Sea- Reptin links the cilium to transcriptional regulation and defects in this pathway contribute to cyst formation. We propose three specific aims to test our hypothesis. In aim 1, we will characterize the role of the Arl13b-Sea-Reptin connection in cilia-mediated signaling and PKD pathogenesis. In aim 2, we will expand our understanding of this network by identifying and characterizing additional binding partners of Arl13b and Sea and transcriptional targets of Reptin. Finally, we will start to address the functional conservation of this network by analyzing Arl13b function in the mouse kidney. Together, this study will validate a newly identified connection between cilia on the cell surface and transcriptional regulation in the nucleus. It will also identify new players in this signaling network and thus open up new directions for future studies. Knowledge gained from this study is an important step in obtaining a detailed understanding of how cilia normally function, what their downstream targets are, and how their dysfunction contribute to PKD, thus will provide insights for rational design of effectiv treatments against this disease and other ciliopathies.

描述（由申请人提供）：我们的长期目标是了解多囊肾病 (PKD) 的病因，并为更好地治疗 PKD 和其他纤毛病提供见​​解。该项目的主要目标是剖析新发现的 Arl13b-Sea-Reptin 网络在纤毛介导的信号传导中及其在肾囊肿形成中的作用。纤毛，以前 模糊的细胞表面结构，在 PKD 和越来越多的其他疾病中起着至关重要的作用。尽管已鉴定出许多纤毛相关蛋白，但人们对这些蛋白之间的功能关系、信号如何从细胞表面细胞器转导到细胞以及引发的细胞反应的性质知之甚少。缺乏这方面的知识阻碍了我们对纤毛在脊椎动物发育和人类疾病中的作用的理解。 为了以综合方式了解纤毛介导的信号传导及其在肾囊肿形成中的作用，我们将斑马鱼遗传学与蛋白质组学方法结合起来。在大规模的基因筛查中，我们分离出了 13 个突变时可导致肾囊肿的基因。除了对纤毛生物发生很重要的多个 IFT 基因外，该屏幕中还分离出新基因 arl13b、sea 和转录调节因子 reptin 和 pontin。进一步的研究表明，Arl13b是纤毛生物发生所需的纤毛蛋白，而Sea主要是纤毛信号传导所需的细胞质蛋白。出乎意料的是，我们通过串联亲和纯化和酵母双杂交筛选发现 Arl13b 与 Sea 共纯化，并且 Sea 与 Reptin 发生物理相互作用。考虑到 Reptin 是一种众所周知的表观遗传/转录调节因子，我们假设 Arl13b-Sea-Reptin 将纤毛与转录调节联系起来，并且该途径的缺陷导致囊肿形成。 我们提出三个具体目标来检验我们的假设。在目标 1 中，我们将描述 Arl13b-Sea-Reptin 连接在纤毛介导的信号传导和 PKD 发病机制中的作用。在目标 2 中，我们将通过识别和表征 Arl13b 和 Sea 的其他结合伙伴以及 Reptin 的转录靶点来扩展我们对该网络的理解。最后，我们将开始解决功能守恒问题 通过分析小鼠肾脏中的 Arl13b 功能来构建该网络。 总之，这项研究将验证细胞表面纤毛与细胞核转录调控之间新发现的联系。它还将识别该信号网络中的新参与者，从而为未来的研究开辟新的方向。从这项研究中获得的知识是详细了解纤毛如何正常发挥作用、其下游靶标是什么以及其功能障碍如何导致 PKD 的重要一步，因此将为合理设计针对该疾病和其他纤毛病的有效治疗方法提供见解。