Investigate kidney cyst formation and a cilia-mediated signaling network

描述(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和其他纤毛疾病提供见解。该项目的主要目的是剖析一个新发现的Ar13b-Sea-Reptin网络在纤毛介导的信号转导中的作用及其在肾囊肿形成中的作用。纤毛，以前的 细胞表面结构模糊，在PKD和越来越多的其他疾病中起着至关重要的作用。虽然已经鉴定了许多纤毛相关蛋白，但对这些蛋白之间的功能关系、信号如何从细胞表面细胞器传递到细胞以及引发细胞反应的性质知之甚少。缺乏这样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。为了了解纤毛介导的信号及其在肾囊肿形成中的作用，我们将斑马鱼遗传学和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个基因，这些基因在突变时会导致肾囊肿。除了对纤毛生物发生重要的多种IFT基因外，在本片段中分离到的还包括新基因Arl13b、SEA和转录调控因子eptin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主要是纤毛信号转导所需的细胞质蛋白。出乎意料的是，我们发现Arl13b与SEA共纯化，SEA通过串联亲和纯化和酵母双杂交筛选与Reptin物理相互作用。考虑到Reptin是一种众所周知的表观遗传/转录调控因子，我们假设Ar13b-Sea-Reptin将纤毛与转录调控联系起来，这一途径中的缺陷有助于包囊的形成。我们提出了三个具体目标来检验我们的假设。在目标1中，我们将表征Arl13b-Sea-Reptin连接在纤毛介导的信号转导和PKD发病中的作用。在目标2中，我们将通过识别和表征Arl13b和SEA的其他结合伙伴和Reptin的转录靶标来扩大我们对该网络的理解。最后，我们将开始解决功能守恒问题 该网络通过分析Arl13b在小鼠肾脏中的功能。总之，这项研究将验证新发现的细胞表面纤毛与细胞核转录调控之间的联系。它还将确定这一信令网络中的新参与者，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正常功能的重要一步，它们的下游靶点是什么，它们的功能障碍如何导致PKD，从而为合理设计针对该疾病和其他纤毛疾病的有效治疗方案提供见解。

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.