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Chem Biol Drug Des. 2018 Nov 27. doi: 10.1111/cbdd.13450. [Epub ahead of print]

Characterization and regulation of MT1-MMP cell surface-associated activity.

Author information

1
Departments of Chemistry and Biology, Torrey Pines Institute for Molecular Studies, Port St. Lucie, FL, 34987, USA.
2
Department of Chemistry & Biochemistry, Florida Atlantic University, Jupiter, FL, 33458, USA.
3
Departments of Medicine/Cancer Prevention and Pathology, Stony Brook University, Stony Brook, NY, 11794, USA.
4
Cancer Research Center, Sanford Burnham Prebys Medical Research Institute, La Jolla, CA, 92037, USA.
5
Department of Pathology and the Karmanos Cancer Institute, Wayne State University, Detroit, MI, 48201, USA.
6
Division of Molecular Medicine & Genetics, Department of Internal Medicine, and the Life Sciences Institute, University of Michigan, Ann Arbor, MI, 48109, USA.
7
The Scripps Research Institute/Scripps Florida, Jupiter, FL, 33458, USA.

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 towards 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. This article is protected by copyright. All rights reserved.

KEYWORDS:

Cell Surface Proteolysis; Cell-based Assay; Collagenolysis; Matrix Metalloproteinase; Protease Inhibitor

PMID:
30480376
DOI:
10.1111/cbdd.13450

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