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

多囊蛋白-1 蛋白水解切割在肾脏健康和多囊肾病中的分子机制

基本信息

批准号：
9383569
负责人：
Feng Qian
金额：
$ 47.03万
依托单位：
UNIVERSITY OF MARYLAND BALTIMORE
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-15 至 2022-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9383569
关键词：
ARL3 gene Adhesions Affect Atomic Force Microscopy Autosomal Dominant Polycystic Kidney Back Biochemical Biogenesis Biological Process CRISPR/Cas technology Cilia Cleaved cell Computer Simulation Cyst Defect Development Disease Embryo Functional disorder GTP-Binding Protein alpha Subunits, Gs Genes Goals Health Human Hydrophobicity Image Impairment In Vitro Individual Kidney Kidney Failure Knock-in Mouse Laboratories Lead Length Link Liquid substance MDCK cell Measures Mediating Mendelian disorder Missense Mutation Modeling Molecular Molecular Conformation Mus Mutate Mutation N-terminal Nephrons PKD2 protein Pathogenicity Pathway interactions Patients Phenotype Play Polycystic Kidney Diseases Process Property Proteins Proteolysis Renal function Role Series Structure Testing Transmembrane Domain Uncertainty Work base biophysical techniques falls functional restoration improved in vivo insight molecular dynamics mouse model mutant nanomechanical nanomechanics novel strategies polycystic kidney disease 1 protein postnatal protein function success trafficking transmission process

项目摘要

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.

项目概要/摘要我们实验室的长期目标是了解多囊蛋白编码的蛋白质的生物学功能肾脏疾病 (PKD) 基因，例如 PKD1/多囊蛋白-1 (PC1) 并确定 PKD 致病途径当它们发生突变时，该提议是建立在我们之前发现的 PC1 顺式自蛋白水解裂解的基础上的。 GPS 基序及其在调节 PC1 的生物发生、运输和功能中的核心作用。假设 GPS 基序和相邻的连接子形成一个二分力传导模块，介导 PC1 的关键功能该项目的目标是结合使用分子、生物化学、细胞和生物物理方法以及小鼠模型来剖析 PC1 中 GPS 链接器模块的作用我们的目标是：1) 检验β1 链在β1 链内紧密结合的假设。 GPS 是 PC1 纤毛运输和功能所必需的，并且会被 PKD1 突变破坏。 GPS 基序内的该 β 链的紧密结合是使 PC1 能够运输到纤毛并诱导 MDCK 细胞中的体外肾小管发生，并被 PKD1 突变破坏；2) 检验高的假设；连接子的刚性和短长度是 PC1 纤毛运输和功能所必需的，并且被破坏我们预测连接子的刚性是 PC1 能够运输到纤毛所必需的。并在 MDCK 细胞中诱导体外肾小管形成，并被 PKD1 突变破坏；3) 确定 GPS-Linker 模块对胚胎期小鼠肾脏中 PC1 运输和功能的体内作用我们将生成两个影响 GPS 或产后发育的新 Pkd1 敲入小鼠模型。我们将分别比较它们的表型并评估它们的表达、切割和运输。这些分析应该对不同发育阶段和肾单位片段中的突变 PC1 蛋白进行分析。提供关于模块的两个组件如何一起运行以调节两者的机制见解 PC1 在胚胎和产后发育阶段的形式，以及它们的功能障碍如何导致总的来说，我们预计所提出的研究应该能够更好地理解基本原理。调节 PC1 在肾脏健康和 PKD 中的功能的机制，并可能为新的通过操纵多囊蛋白-1 中的力传导过程来靶向疾病的策略。