Regulation of WNK signaling by potassium and Mo25: structure, function and physiology

钾和 Mo25 对 WNK 信号传导的调节：结构、功能和生理学

基本信息

批准号：
10298458
负责人：
AYLIN RACHEL RODAN
金额：
$ 47.48万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-13 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10298458
关键词：
Alanine Animal Model Binding Binding Sites Biochemical Biological Assay Biophysics Blood Calorimetry Cerebrovascular Disorders Cesium Chloride Ion Chlorides Chronic Kidney Failure Crystallization Data Development Drosophila genus Drosophila melanogaster Epithelial Equilibrium Funding Future Goals Grant Hand Health Human Hypertension In Vitro Ion Transport Kidney Kidney Diseases Lead Lysine Malpighian Tubules Maps Measures Methods Modeling Molecular Molecular Conformation Molecular Genetics Morbidity - disease rate Mouse Protein Mutagenesis Mutation Organ Outcome Oxidative Stress Pathologic Pathway interactions Phosphotransferases Physiological Physiology Positioning Attribute Potassium Potassium Chloride Process Proline Protein Isoforms Publishing Regulation Renal function Research Research Personnel Roentgen Rays Role Scaffolding Protein Signal Pathway Signal Transduction Structure Testing Therapeutic Tissues Titrations adverse outcome base biological adaptation to stress collaborative approach conformer design dimer experience human model hyperkalemia in vitro activity in vivo inhibitor/antagonist innovation insight monomer mortality mutant new therapeutic target novel novel therapeutic intervention paralogous gene post stroke salt sensitive hypertension side effect tool translational impact

项目摘要

Mutations in human WNK (With No Lysine) kinases are associated with hyperkalemia, hypertension and chronic kidney disease. However, despite extensive characterization of (patho)physiological roles of WNKs in the kidney and extrarenal tissues, there is surprisingly little understanding of how WNKs themselves are regulated. WNKs regulate epithelial ion transport in the mammalian kidney. The applicants’ long-term goal is to achieve mechanistic understanding of epithelial ion transport mechanisms relevant to human kidney function. Chloride ion is known to regulate WNKs. The overall objective of this application is to define and understand new mechanisms of WNK regulation. This renewal application builds on three significant advances from the currently funded grant. First, potassium inhibits Drosophila and mammalian WNKs through chloride-independent mechanisms. Second, the scaffold protein Mo25 (Mouse protein 25/Cab39) is an important regulator of WNK signaling, and activates WNKs independent of its known effects on SPAK (Ste20-related proline alanine rich kinase) and OSR1 (oxidative stress response) kinases. Third, potassium and Mo25 have differential effects on the kidney-expressed mammalian WNKs 1, 3 and 4. The central hypothesis is that potassium and Mo25 directly regulate WNK kinase activity, with differential effects on mammalian WNK isoforms. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the mechanism and physiological consequences of WNK regulation by potassium; 2) Elucidate novel mechanisms of Mo25 regulation of WNK signaling; and 3) Determine the molecular basis for differential WNK isoform regulation by potassium and Mo25. The approach is innovative by leveraging insights from biophysical and structural studies to determine molecular mechanisms of epithelial ion transport regulation, using a unique platform, the Drosophila Malpighian tubule, that has powerful molecular genetic tools and tractable physiologic readouts. Assays have been established, and demonstrated feasible in the investigators’ hands, to: 1) identify WNK potassium binding sites, generate potassium-insensitive WNK mutants, and test their effects on transepithelial ion transport; 2) determine how Mo25 regulates WNK activity in vitro and in the tubule, and probe the interactions between Mo25, potassium and chloride in WNK regulation; and 3) test differences in potassium and Mo25 regulation of WNKs 1, 3 and 4. Successful completion of the proposed studies will elucidate the most comprehensive mechanistic understanding of WNK regulation achieved to date. This is significant, because delineation of the importance of these WNK regulatory mechanisms in various (patho)physiological contexts, together with the molecular insights gained from the studies proposed here, will allow the development of targeted approaches to therapeutically modulate WNK signaling. This has translational impact in a broad range of conditions, including the treatment of dyskalemias, salt-sensitive hypertension, cardio- and cerebrovascular disease, and kidney disease.

人WNK（无赖氨酸）激酶的突变与高钾血症，高血压和慢性有关肾脏疾病。但是，dospite在肾脏中的（病原）生理作用（病情）的广泛表征和外部组织，人们对WNK本身的调节几乎没有什么了解。 wnks 调节哺乳动物肾脏中的上皮离子运输。申请人的长期目标是实现与人肾功能相关的上皮离子转运机制的机械理解。氯化物已知离子可以调节WNK。该应用程序的总体目的是定义和了解新的 WNK调节的机制。此续签应用程序以目前的三个重大进展为基础资助赠款。首先，钾通过独立于氯化物抑制果蝇和哺乳动物的WNK 机制。第二，支架蛋白MO25（小鼠蛋白25/cab39）是WNK的重要调节剂信号传导，并激活WNK，独立于其对SPAK的已知影响（Ste20相关的脯氨酸丙氨酸富含）激酶）和OSR1（氧化应激反应）激酶。第三，钾和MO25对肾脏表达的哺乳动物WNK 1、3和4。中心假设是直接的钾和MO25 调节WNK激酶活性，对哺乳动物WNK同工型具有差异作用。在强大的初步指导下数据，中心假设将通过追求三个具体目标来检验：1）确定机制和 WNK调节钾的生理后果； 2）阐明MO25调节的新型机制 WNK信号传导； 3）确定钾差异WNK同工型调节的分子基础和MO25。通过利用生物物理和结构研究的见解来确定这种方法是创新的上皮离子转运调节的分子机制，使用唯一平台，果蝇马尔皮亚 Tubele，具有强大的分子遗传工具和可拖动的生理读数。测定法在研究人员的手中建立并证明了可行的，以：1）确定WNK钾结合位点，产生对钾不敏感的WNK突变体，并测试其对旋转离子转运的影响； 2）确定 MO25如何在体外和管中调节WNK活性，并探测MO25，钾之间的相互作用和WNK调节中的氯化物； 3）WNK 1、3和4的钾和MO25调节的测试差异。成功完成拟议的研究将阐明最全面的机械理解迄今为止已实现的WNK法规。这很重要，因为描述了这些WNK的重要性各种（病原）生理环境中的调节机制，以及从中获得的分子见解这里提出的研究将允许开发有针对性的方法来调节WNK 信号。这已经转化了各种条件的影响，包括治疗dyskalemias，盐敏感的高血压，心脏和脑血管疾病以及肾脏疾病。