Abstract 3836: Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL)

Abstract

Tissue inhibitors of metalloproteinases (TIMPs) are a family of endogenous 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. TIMPs are important regulators of ECM structure, function and homeostasis. Since their original discovery, additional MMP-independent functions have been attributed to TIMP family members leading to their designation as multifunctional proteins with discrete functional domains. 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 TIMP-2, during the dynamic 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. Interactome studies are predominantly performed in non-living systems, and very rarely performed in complex culture systems such as 3D spheroids/co-cultures. To investigate the TIMP-2 interactome in complex biological systems we recently developed carboxyl- and amino-terminal fusion genes of TIMP-2 with the promiscuous biotinylating peptides BioID2 and TurboID. Packaged for retroviral delivery, this system gives us great flexibility in assessing the TIMP-2 interactome in simple versus complex 3D co-culture systems. We show, for the first time, that conditions can be optimized to support proximity labeling reactions in the extracellular milieu, representing an important technical resource for extracellular matrix biologists. Using this method, we identify novel interacting partners for TIMP-2 that provide insight into the tissue-specific actions of this protein in both healthy and diseased tissues. Citation Format: David Peeney, Sadeechya Gurung, Yueqin Liu, Carolyn Lazaroff, Christopher Richie, William G. Stetler-Stevenson. Mapping the interactome of the endogenous metalloproteinase inhibitor TIMP2 using extracellular proximity labeling (ePL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3836.