Abstract 2348: Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL)

Abstract

Abstract Classical methods to investigate protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within or on the cell membrane. We describe a method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein Tissue inhibitors of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of matrix metalloproteinases (MMPs), the major mediators of ECM breakdown and turnover. TIMP2 is a unique family member, with a broad expression profile that is expressed in both normal and diseased tissues, even in those with minimal metalloproteinase activity. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential to the development of ECM targeted therapeutics. This knowledge may also garner understanding of therapeutic resistance and the failure of conventional and next-generation cancer therapies. Using carboxyl- and amino-terminal fusion peptides of TIMP2 with BioID2 and TurboID, we describe the TIMP2 interactome in unique tissue compartments. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell lines, and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique and comparing our findings with classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies. Citation Format: David Peeney, Sadeechya Gurung, Josh Rich, Sasha Coates-Park, Yueqin Liu, William G. Stetler-Stevenson. Mapping the interactome of matrisome targets using extracellular proximity labeling (ePL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2348.