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

• 批准号: 9348875
• 负责人: Feng Qian
• 金额: $ 11.56万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-21 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9348875
• 关键词: Adhesions; Affect; Atomic Force Microscopy; Autosomal Dominant Polycystic Kidney; Back; Biochemical; Biogenesis; Biophysical Process; Birth; CRISPR/Cas technology; Cells; Cilia; Cleaved cell; Computer Simulation; Cyst; Defect; Development; Disease; Distal; Embryo; Figs - dietary; Goals; Health; Homeostasis; Human; Image; In Vitro; Individual; Kidney; Kidney Failure; Knock-in Mouse; Length; Link; Liquid substance; Measures; Mediating; Mendelian disorder; Missense Mutation; Molecular; Mus; Mutant Strains Mice; Mutation; N-terminal; Nephrons; PKD1 gene; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Polycystic Kidney Diseases; Process; Property; Proteins; Recruitment Activity; Renal function; Renal tubule structure; Role; Series; Signal Transduction; Staging; Structure; Testing; Therapeutic; Transmembrane Domain; Tubular formation; Uncertainty; Work; abstracting; base; biophysical techniques; ciliopathy; falls; flexibility; functional restoration; in vivo; insight; molecular dynamics; mouse model; mutant; nephrogenesis; novel; novel strategies; polycystic kidney disease 1 protein; postnatal; success; trafficking; transmission process

Project Summary/Abstract Our long-term goal is to understand specific functions of the heterodimeric (PC1cFL) and uncleaved (PC1U) states of Polycystin-1 (PC1) in kidney development, homeostasis, and PKD pathogenesis. This project builds on our previous key findings involving cleavage of PC1 at the GPS motif and investigates molecular and biophysical mechanisms by which cleavage regulates the trafficking and function of PC1. The central hypothesis is that the GPS motif and the adjacent linker form a bipartite force-transduction module that mediates critical functions of PC1 in both the heterodimeric (PC1cFL) and uncleaved (PC1U) states. In PC1cFL, GPS cleavage generates a conformational change of the last β-strand (β-1) within the GPS motif, which enables ciliary trafficking and function via signal transmittance from the rigid linker to the N-terminal transmembrane domain. In PC1U, the linker transduces cleavage-independent forces. This proposal will use a multi-disciplinary approach to test the functional role of the GPS-linker module. 1) We will 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-associated mutations. We predict that tight association of this β-strand within the GPS motif is required to enable PC1 to recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 2) We will 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 recruit Rabep1/GGA1/Arl3, traffic to cilia, and induce in vitro tubulogenesis. 3) We will determine the in vivo role and mechanism of the GPS-linker module by generating two Pkd1 knockin mouse models, one with impaired GPS association and the other with a flexible linker. We will examine their kidney phenotypes at development and after birth, and analyze the biogenesis and trafficking of mutant PC1 in proximal and distal nephron cells. The proposed studies will provide novel mechanistic insights into the GPS-linker module and dissect their functional role for the two PC1 forms at kidney development and in different nephron-segments in postnatal periods. These insights should result in therapeutic strategies that restore normal PC1 function in a significant proportion of ADPKD patients by manipulating the GPS-linker module force-transduction process.

项目摘要/摘要 我们的长期目标是了解异二聚体（PC1CFL）和未开采（PC1U）的特定功能 肾脏发育，体内平衡和PKD发病机理中Polycystin-1（PC1）的状态。这个项目建立 关于我们以前的主要发现，涉及在GPS基序上切割PC1，并研究了分子和 裂解调节PC1的运输和功能的生物物理机制。中央 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在异二聚体（PC1CFL）和未溶解（PC1U）状态中的关键功能。在PC1CFL中， GPS裂解会在GPS基序内产生最后一个β链（β-1）的构象变化，该基序中 通过信号传输从刚性接头到N末端，启用睫状运输和功能 跨膜结构域。在PC1U中，接头传递了裂解无关的力。该建议将使用 测试GPS链链模块功能作用的多学科方法。 1）我们将检验假设 PC1睫状运输和功能需要β1链在GPS中的紧密关联，并且是 受PKD1相关突变的破坏。我们预测该β链在GPS基序中的紧密关联 必须使PC1能够募集Rabep1/gga1/arl3，流向纤毛，并诱导体外微管发生。 2）我们 将检验以下假设：PC1睫毛贩运和 功能，并被PKD1相关的突变所禁用。我们预测需要接头的刚度 使PC1能够募集RABEP1/GGA1/ARL3，流向纤毛的流量，并诱导体外肾变发生。 3）我们将确定 GPS链链模块的体内角色和机制，通过生成两个PKD1敲蛋白小鼠模型，一个 GPS关联受损，另一个具有灵活的接头。我们将检查他们的肾脏表型 发育和出生后，并分析突变体PC1在近端和不同的生物发生和运输 肾单位细胞。拟议的研究将为GPS链链链模块和 在肾脏发育和不同的肾分离中剖析其功能作用 产后期。这些见解应导致治疗策略，以恢复正常的PC1在A中的功能 通过操纵GPS链模块的力转导过程，ADPKD患者很大一部分。