Defining protein:protein interactions for the regulation of renal V-ATPase function: role in expression, assembly and trafficking.

定义蛋白质：调节肾 V-ATP 酶功能的蛋白质相互作用：在表达、组装和运输中的作用。

• 批准号: 10207619
• 负责人: Dennis Brown
• 金额: $ 54.17万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207619
• 关键词: ATP phosphohydrolase; Acid-Base Equilibrium; Acidosis; Acids; Address; Affect; Amino Acids; Binding; Biological; Blood; Cell Culture Techniques; Cell membrane; Cell surface; Cells; Complex; Cues; Cyclic AMP; Data; Degradation Pathway; Disease; Distal; Down-Regulation; Drug Design; Epithelial Cells; Event; Exocytosis; Family; Funding; Future; Gene Expression; Genetic Transcription; Goals; Holoenzymes; Homologous Gene; In Vitro; Incentives; Intercalated Cell; Intercalated Duct; Intracellular Transport; Intuition; Kidney; Knock-out; Knockout Mice; Knowledge; Life; Link; Malignant Neoplasms; Maps; Mission; Molecular; Mus; Mutagenesis; Mutation; Organ; Peptides; Pharmaceutical Preparations; Play; Process; Property; Proteins; Proton Pump; Protons; Public Health; Pump; Recycling; Regulation; Regulatory Pathway; Renal tubular acidosis; Research; Role; Site; Techniques; Translations; United States National Institutes of Health; Vesicle; Virus Diseases; Work; Yeasts; base; bone; cell type; design; disease-causing mutation; experimental study; extracellular; genetic regulatory protein; human disease; innovation; insight; kidney cell; knock-down; mouse model; news; novel; protein protein interaction; renal epithelium; response; spatiotemporal; trafficking; transcriptome sequencing; vacuolar H+-ATPase; vesicle transport

PROJECT SUMMARY Despite its central role in extracellular acidification in the kidney and other organs, as well as in critical intracellular processes, the regulation of proton-pumping ATPase (V-ATPase) activity at the molecular level is poorly understood. During the prior funding period, two proteins that associate strongly with the V-ATPase to regulate its function were identified – Ncoa7 and Dmxl1. The overall objective of this proposal is to determine the mechanisms by which they interact with the V-ATPase to regulate proton secretion by the kidney, thereby maintaining systemic acid/base balance. The long-term goal is to develop strategies (including drug and peptide design) for the regulation of acidification processes that are inappropriately up- or downregulated not only in the kidney, but also in diseases affecting other cells and organs. Ncoa7 null mice have markedly decreased V-ATPase subunit expression in collecting duct intercalated cells (ICs) resulting in distal RTA, while Dmxl1 knockdown in renal epithelial cells in vitro causes deficient intracellular vesicle acidification to the same degree as knockdown of bone-fide V-ATPase subunits. Aim 1 will determine the mechanism by which Ncoa7 regulates V-ATPase subunit expression, focusing on translational, transcriptional and degradation pathways in WT and Ncoa7 knockout mice. The V-ATPase-binding sequence of Ncoa7 will be identified by protein interaction and mutagenesis studies, and its role in V-ATPase-dependent acidification events will be determined. Aim 2 will address the novel hypothesis that V-ATPase exocytosis and accumulation on the plasma membrane of ICs in response to systemic acid/base cues requires, counter-intuitively, a partial disassembly of the large, sterically hindering V-ATPase holoenzymes that coat intracellular transport vesicles. The working hypothesis is that Dmxl1, a homolog of the Rav1p yeast V-ATPase assembly protein, coordinates V-ATPase assembly/disassembly with V-ATPase recycling to and from the plasma membrane, which together regulate V-ATPase activity and proton secretion in kidney ICs and other cells. The functionally important V-ATPase-binding sequence of Dmxl1 will also be identified by protein interaction and mutagenesis studies. Thus, a major innovative aspect of our proposed studies is the concept that two newly-identified V-ATPase interacting proteins are involved in the regulation of V-ATPase function at the “upstream” expression level and the “downstream” assembly level. Both Aims 1 and 2 make use of integrated cell and molecular techniques in conjunction with genetically modified mouse models, isolated ICs and renal cell cultures in vitro, and interaction domain studies using purified proteins and specific subdomains. The proposed research is significant: a) because it will allow the field to move forward not at the most basic cellular level by elucidating new V-ATPase dependent acidification regulatory pathways, and b) because the interaction site analysis will inform the future design of drugs and/or cell permeant biologics to up- or down-regulate V-ATPase activity in states of inappropriate hyperactivation (e. g., in many cancers, viral infection) or downregulation (such as dRTA).

项目摘要 尽管在肾脏和其他器官中以及临界中的细胞外酸化中具有核心作用 细胞内过程，质子 - 泵化ATPase（V-ATPase）在分子水平上的调节很差 理解齿。在以前的资金期间，两种与V-ATPase强烈关联以调节其的蛋白质 识别功能 - NCOA7和DMXL1。该提案的总体目的是确定机制 通过它们与V-ATPase相互作用以调节肾脏的质子分泌，从而保持全身性 酸/基础平衡。长期目标是制定法规的策略（包括药物和肽设计） 不仅在肾脏中，而且在疾病中，不仅上调或下调的酸化过程 影响其他细胞和器官。 NCOA7无效小鼠在收集中显着降低了V-ATPase亚基表达 导致远端RTA的导管插入细胞（ICS），而肾上皮细胞的DMXL1在体外原因敲低 明确的细胞内囊泡酸化与骨纤维V-ATPase亚基的敲低相同。目标1 将确定NCOA7调节V-ATPase亚基表达的机制，重点是翻译， WT和NCOA7敲除小鼠的转录和降解途径。 NCOA7的V-ATPase结合序列 将通过蛋白质相互作用和诱变研究及其在V-ATPase依赖性酸化中的作用来鉴定 将确定事件。 AIM 2将解决一个新的假设，即V-ATPase胞吐作用和积累 响应系统性酸/碱提示的IC的质膜需要违反局部拆卸 大型，在空间上阻碍的V-ATPase全酶，涂层细胞内运输蔬菜。工作 假设是DMXL1是RAV1P酵母V-ATPase组装蛋白的同源物，坐标为V-ATPase 带有V-ATPase回收的组装/拆卸质膜，该质膜一起调节V-ATPase 肾脏IC和其他细胞中的活性和质子分泌。功能上重要的V-ATPase结合序列 DMXL1也将通过蛋白质相互作用和诱变研究来鉴定。那是我们的主要创新方面 拟议的研究是一个概念，即两个新识别的V-ATPase相互作用蛋白参与 在“上游”表达水平和“下游”组装水平上对V-ATPase函数的调节。两个都 目标1和2使用集成细胞和分子技术与转基因小鼠结合 在体外模型，孤立的IC和肾细胞培养物以及使用纯化蛋白和特定的相互作用结构域研究 子域。拟议的研究很重要：a），因为它将允许该领域前进 通过阐明新的V-ATPase依赖性酸化调节途径，而b），因为 相互作用的分析将为未来的药物和/或细胞Pereant Biologics的设计提供上调或下调 不适当过度激活状态（例如，在许多癌症，病毒感染）或下调状态下的V-ATPase活性 （例如DRTA）。