Extracellular Proximity Labeling Reveals an Expanded Interactome for the Matrisome Protein TIMP2.

Classical methods of investigating 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 proximal 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 the cell or on the cell membrane. We have adapted the original PL 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 inhibitor 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 metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. 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 (BioID2 vs. TurboID); demonstrating the power of this technique versus 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.

Tissue inhibitors of metalloproteinases are proteolytic targets of matrix metalloproteinase 9.

Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.