Dissecting new mechanisms of lysosome quality control in health and disease

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PROJECT SUMMARY Lysosomes function as critical nodes for macromolecular recycling, metabolic rewiring, and pro-growth signaling in cells. Accordingly, defects in lysosome function underlie degenerative diseases and aging while hyperactivation of lysosomes are associated with cancer. Prior studies have shown that highly aggressive Pancreatic ductal adenocarcinoma (PDA) cells upregulate lysosome biogenesis and activity to facilitate degradation, clearance and recycling of incoming cargo material delivered by increased rates of autophagy and macropinocytosis. Whether qualitative differences endow PDA lysosomes with unique structural and functional properties to cope with a higher demand for substrate clearance remains unknown. To answer this question, we have conducted the first comparative proteomics analysis of lysosomes isolated from PDA versus normal cells and have identified members of the Ferlin family of membrane repair factors, Myoferlin and Dysferlin, as selectively enriched on the membrane of PDA lysosomes. We propose that Ferlin proteins confer increased protection against lysosomal membrane stress in PDA cells. Ferlin proteins are normally localized on the plasma membrane of cell types subjected to heightened mechanical stress, such as skeletal muscle, where they facilitate repair of the lipid bilayer. Accordingly, mutations in DYSF are associated with two forms of muscular dystrophy whereby impaired membrane resealing compromises myoblast maturation, fusion and plasma membrane repair. Therefore, we hypothesize that PDA cells hijack and repurpose Ferlin proteins at the lysosome membrane to protect the integrity of this organelle. In support of this hypothesis, our preliminary findings show that PDA lysosomes are more resistant to acute chemically induced membrane permeabilization relative to normal cells. Mechanistically, lysosome localization of MYOF is necessary and sufficient for maintenance of lysosome quality control and its suppression leads to profound defects in lysosome morphology, PDA cell proliferation and in vivo tumor growth. The goal of this study is to investigate how MYOF functions to protect the lysosome membrane in mechanistic detail and to determine the impact of blocking lysosome quality control on cellular metabolism, pro-growth signaling and disease progression. In summary, our discovery and proposed studies will be the first to determine a novel function for Ferlin proteins at the lysosome membrane and provide insight into how enhanced lysosome quality control regulates cellular homeostasis and disease pathogenesis.