PROJECT SUMMARY/ABSTRACT The mammalian cell surface proteome (surfaceome) is a hub of communication that transmits information from the extracellular space to the intracellular machinery, controlling proliferation and cell fate. A major regulatory endpoint of the surfaceome and extracellular proteome is the lysosome. As the primary organelle for breaking down various macromolecules, lysosomes are central to nutrient sensing, storage and distribution, as well as modulating cell surface protein abundance. The transport of membrane and secreted proteins from the extracellular space to lysosomes occurs through many discrete processes, including autophagy, endocytosis, and pinocytosis. Given the critical roles of the surfaceome and lysosomes in integrating signals from the extracellular space, the trafficking between these two compartments is highly regulated across different cell types and in response to specific cues. Dysregulation of this trafficking is a hallmark of cancer and neurodegenerative diseases that disrupts the homeostatic balance between cell surface protein function, recycling, and lysosomal degradation. While an understanding of all proteins which move between the extracellular space and lysosomes remains incomplete, this pathway has proven transformative for targeted drug delivery and new therapeutic modalities like targeted protein degradation. An atlas of all potential proteins which can be used as lysosome targeting moieties in specific contexts would substantially expand therapeutic options for numerous diseases. However, a lack of methods for studying surface-to-lysosome trafficking in organ and cell-type specific ways has limited our ability to rationally hijack this pathway for translational applications. What is needed is a functional map across tissues, time, and disease states for identifying potential receptors that could be used to drive targeted protein degradation, as well as more broadly enable widespread utilization of selective trafficking pathways. In this proposal, I describe an innovative approach which unites whole lysosome isolation from distinct mouse tissues with chemoproteomics to identify proteins that traffic to lysosomes from the plasma membrane and extracellular space. We propose to map across tissues and time the transport of extracellular proteins to lysosomes both in healthy tissues and in murine tumor models. By correlating these in vivo discovery efforts with a novel high-throughput method for in vitro analysis of lysosome translocation kinetics, we will facilitate the rational utilization of new trafficking proteins. We go on to propose conceptual advances in designing ligands to harness lysosomal transport for targeted degradation of disease-causing extracellular proteins using de novo protein design. Finally, we propose a new approach to immunotherapy that directly translates our in vivo, in vitro, and ligand development efforts via lysosome targeting chimeras (LYTACs) that selectively degrade immune checkpoint proteins from cancer cells. The outcomes of this proposal will help revolutionize therapeutic design and drug delivery by mapping the scope and function of extracellular-to-lysosome communication.

PROJECT NARRATIVE Macromolecular trafficking between the cell surface and lysosomes is an essential cellular process with proven therapeutic promise, yet the identities and functions of proteins which transit between these two spaces have only been profiled on a limited basis. Here, we develop an innovative, tissue-specific approach to discover the identities of proteins which traffic to lysosomes in vivo, characterize these proteins’ trafficking ability, and to harness this lysosomal transport for targeted protein degradation of extracellular proteins and therapeutic delivery. Our work will directly provide translational avenues for context-specific drug delivery and establish an approach to remove deleterious proteins from the extracellular space.