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

项目总结 尽管它在肾脏和其他器官的细胞外酸化以及在关键的 在细胞内过程中，质子泵ATPase(V-ATPase)活性在分子水平上的调节很差 明白了。在之前的资助期间，两种与V-ATPase密切相关的蛋白质调节其 功能鉴定为Ncoa7和Dmxl1。这项提案的总体目标是确定机制 通过它们与V-ATPase相互作用来调节肾脏的质子分泌，从而维持全身 酸/碱平衡。长期目标是为调控制定策略(包括药物和多肽设计)。 酸化过程不仅在肾脏中不适当地上调或下调，在疾病中也是如此 影响其他细胞和器官的。Ncoa7基因缺失小鼠在采集过程中V-ATPase亚单位的表达显著降低 导管间质细胞(ICs)导致远端RTA，而体外肾上皮细胞Dmxl1基因敲除导致 细胞内囊泡酸化不足，与骨化V-ATPase亚基被击倒的程度相同。目标1 将决定Ncoa7调节V-ATPase亚单位表达的机制，侧重于翻译， WT和Ncoa7基因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突变研究及其在V-ATPase依赖的酸化中的作用来确定 事件将会被确定。目标2将解决新的假设，即V-ATPase在 与直觉相反，ICs的质膜对系统性酸碱信号的反应需要部分拆解 包裹在细胞内运输小泡上的大的、空间上阻碍V-ATPase的全酶。在工作中 假说是Rav1p酵母V-ATPase组装蛋白的同源物Dmxl1与V-ATPase配位 V-ATPase循环到质膜和从质膜循环的组装/拆卸，共同调节V-ATPase 肾脏ICs和其他细胞的活性和质子分泌。具有重要功能的V-ATPase结合序列 Dmxl1还将通过蛋白质相互作用和突变研究来鉴定。因此，我们的一个主要创新方面 建议的研究概念是两个新发现的V-ATPase相互作用蛋白参与了 V-ATPase功能在“上游”表达水平和“下游”组装水平的调节。两者都有 AIMS 1和AIMS 2利用集成的细胞和分子技术与转基因小鼠结合 模型、分离的ICs和体外培养的肾细胞，以及使用纯化的蛋白和特定的 子域。拟议的研究具有重要意义：a)因为它将允许该领域向前推进，而不是至多 基础细胞水平通过阐明新的V-ATPase依赖的酸化调节通路，以及b)因为 相互作用部位分析将为未来药物和/或细胞生物制剂的设计提供信息，以上调或下调调控 在不适当的过度激活状态(例如，在许多癌症中，病毒感染)或下调状态下的V-ATPase活性 (如dRTA)。

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).