Regulation of SNARE-mediated autophagosome-lysosome fusion by accessory proteins

PROJECT SUMMARY: The efficient management of sub-cellular injury, protein misfolding and organelle damage through the process of autophagy is critical for maintaining cell health and preventing the development of disease including neurodegeneration and cancer. One of the first steps in autophagy involves sequestering sub-cellular material within a vesicular structure called an autophagosome. Autophagosomes are then brought into proximity with lysosomes, the degrative organelle of the cell, to enable trans-SNARE assembly and subsequent merger of the two compartments; this delivers the autophagosome-encapsulated material to the acidic and proteolytic environment of the lysosomal lumen for degradation. Without efficient autophagosome-lysosome (Auto-Lyso) fusion, sub-cellular material accumulates, severely impacting cellular health and function. It is critical to elucidate how Auto-Lyso fusion is regulated to understand the molecular determinants of autophagy-associated disease. Primary objectives of my lab are to describe how a SNARE-binding protein, sec1-family domain containing protein 1 (SCFD1), and a Ca2+ sensor, synaptotagmin 7 (SYT7) regulate Auto-Lyso fusion. SCFD1 was recently reported to be a regulator of Auto-Lyso fusion. Interestingly, mutations or aberrant expression of SCFD1 has also recently been implicated in the development of neurodegenerative disease. This project will characterize SCFD1 function at the molecular level and describe the mechanisms that underlie how dysregulation of SCFD1 leads to disease. The second main project outlined in this proposal examines the Ca2+ regulation of Auto-Lyso fusion through the action of a lysosome-resident Ca2+-binding protein, SYT7. It has been shown that cancer cells upregulate both SYT7 expression and autophagy to facilitate proliferation, but a connection between SYT7 and autophagy has not been reported. The lysosomal lumen is enriched in Ca2+ and it has been demonstrated that Ca2+ is released from the lysosome during autophagy. Although Ca2+ is a tightly regulated and ubiquitous trigger of myriad membrane fusion reactions, a bone fide Ca2+ sensing protein for Auto-Lyso fusion has not been established. We hypothesize that SYT7 is activated by Ca2+ release from the lysosome to trigger Auto-Lyso fusion. Our lab will address these basic questions through a combination of in vitro biochemistry and advanced fluorescence imaging of mammalian cells to describe how Auto-Lyso fusion is regulated at the molecular level. Of importance, part of our in vitro biochemical approach includes a unique single-molecule technique with microsecond time resolution, known as nanodisc-black lipid membrane electrophysiology. This proposal marks the first use of this technique in the context of autophagy. Together, this work will provide novel insights into the regulation of Auto-Lyso fusion and describe the detailed molecular mechanisms that implicate both SCFD1 and SYT7 in the development of autophagy-associated disorders.