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

项目摘要 尽管它在肾脏和其他器官的细胞外酸化中起着核心作用， 在细胞内过程中，质子泵ATP酶（V-ATP酶）活性在分子水平上的调节很差 明白在之前的资助期间，两种与V-ATP酶密切相关的蛋白质调节其功能。 鉴定了功能-Ncoa 7和Dmxl 1。本提案的总体目标是确定 它们通过与V-ATP酶相互作用来调节肾脏的质子分泌，从而维持系统性 酸碱平衡长期的目标是发展策略（包括药物和肽的设计）的监管 不仅在肾脏，而且在疾病中， 影响其他细胞和器官。Ncoa 7基因敲除小鼠在收集细胞中V-ATP酶亚基表达显著降低， 导管插入细胞（IC）导致远端RTA，而体外肾上皮细胞中的Dmxl 1敲低导致 缺乏胞内囊泡酸化到与真正的V-ATP酶亚基敲低相同的程度。要求1 将确定Ncoa 7调节V-ATP酶亚基表达的机制，重点是翻译， 在WT和Ncoa 7敲除小鼠中的转录和降解途径。Ncoa 7的V-ATPase结合序列 将通过蛋白质相互作用和诱变研究确定，及其在V-ATP酶依赖性酸化中的作用 事件将被确定。目的2将阐述新的假设，即V-ATP酶的胞吐和积累， 与直觉相反，IC的质膜对系统性酸/碱提示的反应需要部分分解 大的，空间阻碍的V-ATP酶全酶，包被细胞内运输囊泡。工作 假设是Rav 1 p酵母V-ATP酶装配蛋白同源物Dmxl 1与V-ATP酶协调 与V-ATP酶的组装/拆卸循环到质膜和从质膜，它们一起调节V-ATP酶 肾IC和其他细胞中的活性和质子分泌。V-ATP酶结合序列的功能重要性 还将通过蛋白质相互作用和诱变研究鉴定dmxl 1。因此，我们的一个主要创新方面 提出的研究是这样一个概念，即两个新鉴定的V-ATP酶相互作用蛋白参与了 在“上游”表达水平和“下游”组装水平调节V-ATP酶功能。两 目的1和2利用整合的细胞和分子技术结合基因修饰小鼠， 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异性 子域。拟议的研究是重要的：a）因为它将使该领域向前发展，而不是最多 通过阐明新的V-ATP酶依赖性酸化调节途径，在基本细胞水平，和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， V-ATP酶活性在不适当的超活化状态下（e.例如，在一个实施例中，在许多癌症中，病毒感染）或下调 (such作为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).