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

项目概要 尽管它在肾脏和其他器官的细胞外酸化以及关键的细胞外酸化中发挥着核心作用 细胞内过程中，分子水平上质子泵浦 ATP 酶 (V-ATP 酶) 活性的调节很差 明白了。在之前的资助期间，两种与 V-ATPase 密切相关的蛋白质来调节其 已确定功能 - Ncoa7 和 Dmxl1。该提案的总体目标是确定机制 通过它们与 V-ATP 酶相互作用来调节肾脏的质子分泌，从而维持全身性 酸/碱平衡。长期目标是制定监管策略（包括药物和肽设计） 酸化过程不仅在肾脏中而且在疾病中被不当上调或下调 影响其他细胞和器官。 Ncoa7缺失小鼠在收集细胞中V-ATP酶亚基表达显着降低 导管嵌入细胞 (IC) 导致远端 RTA，而体外肾上皮细胞中 Dmxl1 敲低则导致 细胞内囊泡酸化缺陷与骨性 V-ATP 酶亚基的敲低程度相同。目标1 将确定 Ncoa7 调节 V-ATP 酶亚基表达的机制，重点关注翻译、 WT 和 Ncoa7 敲除小鼠的转录和降解途径。 Ncoa7 的 V-ATP 酶结合序列 将通过蛋白质相互作用和诱变研究及其在 V-ATP 酶依赖性酸化中的作用来鉴定 事件将被确定。目标 2 将解决 V-ATP 酶胞吐作用和积累的新假设 与直觉相反，IC 的质膜响应全身酸/碱信号需要部分分解 包裹细胞内转运囊泡的大空间位阻 V-ATP 酶全酶。工作中 假设 Dmxl1（Rav1p 酵母 V-ATPase 组装蛋白的同源物）协调 V-ATPase 组装/拆卸，V-ATP酶循环进出质膜，共同调节V-ATP酶 肾 IC 和其他细胞的活性和质子分泌。功能上重要的 V-ATP 酶结合序列 Dmxl1 还将通过蛋白质相互作用和诱变研究来鉴定。因此，我们的一个主要创新方面 拟议的研究的概念是两个新鉴定的 V-ATPase 相互作用蛋白参与了 在“上游”表达水平和“下游”装配水平调节V-ATP酶功能。两者都 目标 1 和 2 利用整合细胞和分子技术与转基因小鼠相结合 模型、分离的 IC 和体外肾细胞培养物，以及使用纯化的蛋白质和特定的相互作用域研究 子域。拟议的研究意义重大：a）因为它最多不会让该领域向前发展 通过阐明新的 V-ATP 酶依赖性酸化调节途径，b) 因为 相互作用位点分析将为药物和/或细胞渗透生物制剂的未来设计提供信息，以上调或下调 V-ATP酶活性在不适当的过度激活状态下（例如，在许多癌症、病毒感染中）或下调 （例如 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).