New protein-engineered tools and technologies for identifying, deorphanizing, and targeting shed proteins in cancer

University Of California, San Francisco, San Francisco CA

Investigators

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Abstract

Project Summary Extracellular proteolysis is a hallmark of cancer and has major functional consequences for remodeling cell surface proteins including receptor activation and shedding of functional extracellular domains. In our first RO1 grant, we successfully developed mass spectrometry-based proteomics technologies using subtilgase conjugation to identify more than 2000 neo-proteolytic events on the cell surface; more than 90% of these were new to the literature. We developed recombinant antibody selection strategies that can generate antibody tools that selectively bind these neo-epitopes, or block them, and killed cancer cells. In this R01 renewal, we propose to extend the technology from interrogating membrane bound events to shed events that exert autocrine effects on cancer cells. We recently identified >200 shed candidates affected by oncogene transformation using chemical proximity labeling techniques, and here we plan to expand identification and de-orphanization of the binding functions for shed proteins. First, we will develop technologies to profile cell surface proteolysis and secretome in cancer cells and human plasma (SA1). While it is well-understood that cancer genes aberrantly regulate proteases, the substrates and molecular consequences of proteolytic events are poorly characterized. We propose to characterize extracellular cleavage events that generate neo-N-termini on the cancer cell lines using a mass spectrometry approach, and ultimately developing a publicly available extracellular proteomic atlas. We will identify shed fragments in conditioned media from cancer cells and search for them in plasma of cancer patients as potential biomarkers. Next, we will develop and utilize Multi-map proximity labeling techniques to identify partners that shed proteins bind and interact with on the cell membrane (SA2). Initially, we will identify autocrine partner proteins for two shed proteins vasorin and PTK7 we discovered previously as a first step in de- orphanizing potential functions. Applying our new multi-range proximity labeling proteomics technology (Multi- map) that can identify cell surface interactomes, we will globally probe for any autocrine binders in the sheddome of cancer cells. Lastly, we will develop antibody-based selective protease inhibitors by Epitope-Directed- Selections to determine the substrates of candidate dysregulated proteases (SA3). We want to address what are the cell surface proteases that are doing the cleavage and what are their substrates? We will start by employing recombinant antibodies we designed recently in our current R01 that prevent activation of matrix metallo-proteases that are key enzymes in remodeling cell surface proteins for invasion and metastasis. In sum, we believe our work will significantly impact the functional understanding of shedding events in cancer, and identify new biomarkers and immunotherapeutic targets.