Mapping Extracellular Protein–Protein Interactions Using Extracellular Proximity Labeling (ePL)

Proximity labeling (PL) has given researchers the tools
to explore
protein–protein interactions (PPIs) in living systems; however,
most PL studies are performed on intracellular targets. We have adapted
the original PL method to investigate PPIs within the extracellular
compartment, which we term extracellular PL (ePL). To demonstrate
the utility of this modified technique, we investigated the interactome
of the matrisome protein TIMP2. TIMPs are a family of multifunctional
proteins that were initially defined by their ability to inhibit metalloproteinases,
the major mediators of extracellular matrix (ECM) turnover. TIMP2
exhibits broad expression and is often abundant in both normal and
diseased tissues. Understanding the functional transformation of matrisome
regulators, such as TIMP2, during disease progression is essential
for the development of ECM-targeted therapeutics. Using dual orientation
fusion proteins of TIMP2 with BioID2/TurboID, we describe the TIMP2
proximal interactome (MassIVE MSV000095637). We also illustrate how
the TIMP2 interactome changes in the presence of different stimuli,
in different cell types, in unique culture conditions (2D vs 3D),
and with different reaction kinetics, demonstrating the power of this
technique versus classical PPI methods. We propose that screening
of matrisome targets in disease models using ePL will reveal new therapeutic
targets for further comprehensive studies.