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Characterization and Regulation of MT1-MMP Cell Surface-Associated Activity

Characterization and Regulation of MT1-MMP Cell Surface-Associated Activity

Sonia Pahwa, Manishabrata Bhowmick, Sabrina Amar, Jian Cao, Alex Y Strongin, Rafael Fridman, Stephen J Weiss, Gregg B Fields

Chem Biol Drug Des. 2019 Jun;93(6):1251-1264.

PMID: 30480376

Abstract:

Quantitative assessment of MT1-MMP cell surface-associated proteolytic activity remains undefined. Presently, MT1-MMP was stably expressed and a cell-based FRET assay developed to quantify activity toward synthetic collagen-model triple-helices. To estimate the importance of cell surface localization and specific structural domains on MT1-MMP proteolysis, activity measurements were performed using a series of membrane-anchored MT1-MMP mutants and compared directly with those of soluble MT1-MMP. MT1-MMP activity (kcat /KM ) on the cell surface was 4.8-fold lower compared with soluble MT1-MMP, with the effect largely manifested in kcat . Deletion of the MT1-MMP cytoplasmic tail enhanced cell surface activity, with both kcat and KM values affected, while deletion of the hemopexin-like domain negatively impacted KM and increased kcat . Overall, cell surface localization of MT1-MMP restricts substrate binding and protein-coupled motions (based on changes in both kcat and KM ) for catalysis. Comparison of soluble and cell surface-bound MT2-MMP revealed 12.9-fold lower activity on the cell surface. The cell-based assay was utilized for small molecule and triple-helical transition state analog MMP inhibitors, which were found to function similarly in solution and at the cell surface. These studies provide the first quantitative assessments of MT1-MMP activity and inhibition in the native cellular environment of the enzyme.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4241292	MMP 14 (Stomelysin-3) Substrate			Price

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