The Molecular Mechanisms of Polycystin-1 Proteolytic Cleavage in Kidney Health and Polycystic Kidney Disease

项目总结/摘要 我们实验室的长期目标是了解由多囊泡编码的蛋白质的生物学功能。 肾脏疾病（PKD）基因，如PKD 1/多囊蛋白-1（PC 1），并确定PKD致病途径 当它们变异的时候这个建议是建立在我们以前发现的顺式自我蛋白水解切割的PC 1 GPS基序及其在调节PC 1的生物发生，运输和功能中的核心作用。我们的中央 假设GPS基序和相邻的接头形成一个二分力-转导模块， 介导PC 1的关键功能。这个项目的目标是使用分子，生化， 细胞和生物物理方法以及小鼠模型来剖析GPS连接模块在PC 1中的作用 贩运和功能。我们的目的是：1）测试假设，即β1链内的紧密联系， GPS是PC 1纤毛运输和功能所必需的，并被PKD 1突变破坏。我们预测 GPS基序内的该β-链的紧密结合是使PC 1能够运输到纤毛并诱导 MDCK细胞中的体外小管发生，并被PKD 1突变破坏; 2）检验高表达的假设， 刚性和短长度的接头是PC 1纤毛运输和功能所需的，并被破坏 PKD 1相关突变。我们预测，连接器的刚性是使PC 1能够运输到纤毛所必需的 并在MDCK细胞中诱导体外小管形成，并被PKD 1突变破坏;和3）确定 GPS-Linker模块在胚胎期小鼠肾脏中对PC 1运输和功能的体内作用 和产后发育。我们将生成两个新的Pkd 1敲入鼠标模型，影响GPS或 连接器分别。我们将比较它们的表型，并评估它们的表达，切割和运输。 不同发育阶段和肾单位节段中的突变PC 1蛋白。这些分析应 提供关于模块的两个组件如何一起操作以调节两者的机械见解。 PC 1在胚胎和出生后发育阶段的形式，以及它们的功能障碍如何导致 PKD。总的来说，我们预计，拟议的研究应导致更好地了解基本的 在肾脏健康和PKD中调节PC 1功能的机制，并可能为新的 通过操纵多囊蛋白-1中的力转导过程来靶向疾病的策略。

Project Summary/Abstract The long-term goals of our laboratory are to understand biological functions of proteins encoded by polycystic kidney disease (PKD) genes such as PKD1/polycystin-1 (PC1) and to determine PKD pathogenic pathways when they are mutated. This proposal is build on our previous discovery of cis-autoproteolytic cleavage of PC1 at the GPS motif and its central role in regulating PC1's biogenesis, trafficking and function. Our central hypothesis is that the GPS motif and the adjacent linker form a bipartite force-transduction module that mediates key functions of PC1. The goal of this project is to use a combination of molecular, biochemical, cellular, and biophysical methods as well as mouse models to dissect the role of the GPS-linker module in PC1 trafficking and function. Our Aims are: 1) Test the hypothesis that a tight association of the β1-strand within the GPS is required for PC1 ciliary trafficking and function, and is disrupted by PKD1 mutations. We predict that tight association of this β-strand within the GPS motif is required to enable PC1 to traffic to cilia and to induce in vitro tubulogenesis in MDCK cells, and is disrupted by PKD1 mutations; 2) Test the hypothesis that high rigidity and short length in the linker is required for PC1 ciliary trafficking and function, and is disrupted by PKD1-associated mutations. We predict that the rigidity of the linker is required to enable PC1 to traffic to cilia and to induce in vitro tubulogenesis in MDCK cells, and is disrupted by PKD1 mutations; and 3) Determine the in vivo role of the GPS-Linker module for trafficking and function of PC1 in the mouse kidney during embryonic and postnatal development. We will generate two new Pkd1 knockin mouse models affecting the GPS or the linker respectively. We will compare their phenotypes and assess the expression, cleavage, and trafficking of the mutant PC1 proteins in various developmental stages and nephron segments. These analyses should provide mechanistic insights into how the two components of the module operate together to regulate both forms of PC1 during embryonic and postnatal stages of development, and how their dysfunction might lead to PKD. Overall, we anticipate that the proposed studies should lead to a better understanding of the fundamental mechanisms that regulate PC1 function in kidney health and PKD, and might provide rationales for novel strategies to target the disease by manipulating the force-transduction process in polycystin-1.