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

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

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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.

项目摘要/摘要我们实验室的长期目标是了解由多囊编码的蛋白质的生物学功能肾脏疾病（PKD）基因，例如PKD1/Polycystin-1（PC1），并确定PKD致病途径当它们突变时。该提议建立在我们以前发现的pc1的顺式溶液裂解的基础上在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。我们的中心假设是GPS基序和相邻的接头形成了一个两部分力量转导模块，该模块是介导PC1的关键功能。该项目的目的是结合分子，生化，细胞和生物物理方法以及小鼠模型，以剖析PC1中GPS链接模块的作用贩运和功能。我们的目的是：1）检验以下假设：β1链紧密相关 GPS是PC1睫状运输和功能所必需的，并且通过PKD1突变被禁用。我们预测需要在GPS基序内进行β链的紧密关联才能使PC1能够访问纤毛并诱导 MDCK细胞中的体外微管发生，并被PKD1突变破坏。 2）检验高的假设 PC1睫毛运输和功能需要刚性和短长度，并被破坏 PKD1相关突变。我们预测，需要链接器的刚度才能使PC1访问纤毛并诱导MDCK细胞中的体外微管发生，并受到PKD1突变的破坏； 3）确定 GPS - 链链链模块在胚胎期间在小鼠肾脏中运输和功能的体内作用和产后发展。我们将生成两个影响GP或GPS的新PKD1敲击鼠模型链接器分别。我们将比较他们的表型并评估表达，乳沟和贩运在各个发育阶段和肾单位段中的突变PC1蛋白。这些分析应该提供有关模块运算符的两个组件如何调节两个组件的机械洞察力在发育的胚胎和产后阶段，PC1的形式，以及它们的功能障碍可能导致 PKD。总体而言，我们预计拟议的研究应该可以更好地了解基本调节PC1在肾脏健康和PKD中功能的机制，并可能为新颖的理由提供原理通过操纵polycystin-1中的力 - 变换过程来针对疾病的策略。