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

Authors

• B Chimukangara
• S Curran
• L Bishop
• X Li
• W Chang
• O Cisse
• L Ma
• J Kovacs

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 TIMP2 using carboxyl- and amino-terminal fusion peptides of TIMP2 with BioID2 and TurboID. Expanding on this pipeline, we have started to screen the interactomes of new matrisome targets in simple 2D and complex 3D culture conditions. 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 versus classical PPI methods. Furthermore, we expand this method to interrogate the interactomes of new matrisome targets such as TIMP3 and Thrombospondin-1. We propose that the screening of matrisome PPIs in disease models using ePL will reveal new therapeutic targets for further comprehensive studies. Knowledge of disease specific PPIs may also garner understanding of patient-specific therapeutic resistance to conventional and next-generation therapies.

Scientific Focus Area: Molecular Biology and Biochemistry

This page was last updated on Monday, September 25, 2023