New frontiers in extracellular proximity labeling: Novel matrisome targets, diverse cell types, and 3-D culture conditions

Authors

• JA Rich
• SI Coastes-Park
• S Gurung
• Y Liu
• A Jovial
• S Kaur
• D Roberts
• W Stetler-Stevenson
• D Peeney

Abstract

The matrisome encompasses the genes and proteins that embody extracellular matrix (ECM) and ECM-associated proteins. Investigation of protein-protein interactions (PPIs) between matrisome targets is a promising direction to improve understanding of ECM diseases and therapeutic opportunities. Proximity labeling, a powerful technique for investigating protein interactomes based on enzyme-fusion constructs and affinity chromatography, has considerable potential in detailing proximal interactors of the matrisome. We have successfully developed an extracellular proximity labeling (ePL) approach based on fusion constructs containing the biotinylation enzymes BioID2 and TurboID. We have combined this approach with mass spectrometry to gain new insights into the interactome of the matrisome protein tissue inhibitor of metalloproteinase 2 (TIMP2), an endogenous metalloproteinase inhibitor found to have therapeutic potential. We have now expanded this approach to other matrisome protein targets, including tissue inhibitor of metalloproteinase 3 (TIMP3) and thrombospondin-1 (THBS-1). TIMP3 has been shown to possess anti-tumor properties, with loss of TIMP3 function associated with poor prognosis in multiple cancers. THBS-1 is a larger (129 kDa) matrisome protein that is relatively promiscuous with diverse functions in the tumor microenvironment. The role of THBS-1 in cancer is nuanced and appears to manifest in context-dependent pro- and anti-tumorigenic effects. Experiments to expand ePL to novel gene transfer techniques (transient transfection), cell lines, and culture conditions (suspension cell models, spheroids, 3-D cocultures) are currently underway. We show that ePL is a suitable method for studying PPIs in new protein targets and experimental conditions and expect to continue applying the technique broadly to additional matrisome targets.

Scientific Focus Area: Cell Biology

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