Toward Understanding the Functional Landscape of Proteolysis at the Cell Surface

Project Summary The proteome, or collection of proteoforms expressed in a biological system, is dynamic and heterogeneous. As our appreciation for the complexity of the proteome has evolved, so have the technologies we use to interrogate its composition. Cells often undergo significant remodeling of their surface protein and lipid compositions in disease, contributing to disease progression and therapeutic resistance. Even so, a lack of suitable analytical and chemical methods has meant that many surface proteins have been left understudied. Cell surface proteins account for only ~1% of all protein molecules in the cell, a prohibitive barrier of sensitivity and dynamic range. The dysregulation of protease activity in cancer cells prompts cleavage of proteins which may act as signaling molecules to neighboring cells or may circulate globally. Here, we hypothesize that proliferative oncogenes globally alter the surfaceome via protease activity by remodeling of extracellular proteome, shedding peptides as local and organism-wide signaling molecules, and inducing unique proteomic neighborhoods on the cell surface. To test this hypothesis, I will develop a streamlined surface protein profiling strategy using chemical biology tools, enabling unparalleled surveying of proteolysis on the cell surface. I will develop enrichment reagents to cleavage products on the cell surface and within the secreted space which are compatible with mass spectrometry workflows and will enable high-throughput characterization of proteins' abundance, modification states, conformation, and spatiotemporal relationships. I will utilize protein engineering approaches to uncover the activities and functional relevance of disease-identified cell surface proteoforms and their neighborhoods. By engineering heterobifunctional antibodies, where one arm binds a membrane target and the second arm recruits a protease of interest, I aim to dissect the functional roles of specific cell surface proteins, including cleaved forms, in cancer progression. The results of this research will produce, for the first time, a list of dysregulated human surface proteins in cancer with functional annotation of each protein isoform and proximal neighborhood. Moreover, development of novel surface protein enrichment and secretome strategies and protein engineering techniques will enable high-throughput profiling and functional annotation of cell surface proteoforms, paving the way for the identification of novel therapeutic targets and biomarkers. Furthermore, my research will extend beyond cancer biology to investigate cell surface remodeling in various other disease contexts, including neurodegenerative disorders. In summary, this research seeks to address fundamental questions regarding the role of cell surface remodeling in disease biology and to develop innovative methodologies for studying the cell surface proteome. By uniting highly- specific surfaceome mapping and protein engineering, this work will facilitate the development of precision medicine approaches that target the underlying molecular drivers of disease while minimizing side effects.