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).
The research proposed is relevant to public health because it seeks to discover news ways of regulating the function of specialized cells in the kidney that control the amount of acid in the blood, thus maintaining its pH within levels compatible with life. Sometimes this process does not work correctly, leading to diseases known together as ?acidosis?. Our work is relevant to NIH's mission, which is aimed at finding mechanisms that cause these diseases in order to develop drugs and other strategies to cure them.
