Friday, May 9, 2025
5/9/2025
Molecular Mechanisms of Cellular Signaling by the Proton Pumping Vacuolar ATPase - PROJECT SUMMARY Altered pH, metabolic homeostasis and autophagy are hallmarks of cancer, neurodegenerative and infectious diseases. These fundamental processes are linked by a ubiquitous and essential ATP-dependent proton pump called the vacuolar ATPase (V-ATPase). The V-ATPase is a large, multisubunit, membrane-integral molecular machine whose canonical function is to acidify intracellular organelles in all eukaryotic cells according to their functional requirements. More recently, the V-ATPase has emerged in crucial non-canonical signaling roles including, but not limited to metabolic homeostasis and vesicular trafficking. This research program aims to discover the molecular mechanisms that drive V-ATPase’s ability to both acidify organelles and interact with cellular factors to mediate signaling pathways. The current hypothesis is that the ability of V- ATPase to multitask in these interdependent functions involves changes in V-ATPase subunit composition and conformations that are linked to the enzyme’s reaction cycle. To address these questions, (1) The complete reaction cycle of the V-ATPase will be delineated using cryo-electron microscopy (cryo-EM) coupled with in vitro biochemical assays (e.g., ATPase and proton pumping activity assays) and established yeast phenotypes for V-ATPase activity. (2) On early endosomes, the V-ATPase acts as a pH-sensor to recruit a GTPase (Arf-6) and its GEF (Cytohesin-2) for vesicular trafficking of substances to the lysosome. The molecular mechanism of this process will be dissected using protein-protein interaction assays, cryo-EM, and endocytosis/trafficking of albumin in kidney proximal tubule cells. (3) On lysosomes, the V-ATPase mediates metabolic signaling by forming distinct supercomplexes with members of the mechanistic target of rapamycin complex 1 (mTORC1) or AMP-dependent kinase (AMPK) pathways. It is hypothesized that the V-ATPase, bound to a GEF called Ragulator acts as a common molecular switch to promote either mTORC1 or AMPK signaling. The mechanism of V-ATPase-mediated metabolic signaling will be elucidated using protein-protein interaction studies, in vitro reconstitutions, affinity purification mass spectrometry, cryo-EM, and in-cell phosphorylation assays for mTORC1/AMPK activity. The feasibility of this work is supported by the candidate’s expertise in the field as well as robust preliminary experiments showing cryo-EM of V-ATPase (pertinent to goals 1, 2 and 3) and interaction of V-ATPase with Cytohesin-2 (goal 2) and Ragulator (goal 3). Collaborations with leaders in molecular dynamics and mass spectrometry further support these studies. Overall, this research program will not only improve our understanding of V-ATPase mechanics but also reveal key details of cellular pathways implicated in disease conditions.
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