Non-destructive and Selective Imaging of the Functionally Active, Pro-invasive Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Enzyme in Cancer Cells

Remacle, Albert G.; Shiryaev, Sergey A.; Golubkov, Vladislav S.; Freskos, John N.; Brown, Michael A.; Karwa, Amolkumar; Naik, Arati D.; Howard, Carol Pearcy; Sympson, Carolyn J.; Strongin, Alex Y.

doi:10.1074/jbc.m113.471508

Cited by 14 publications

(20 citation statements)

References 68 publications

(74 reference statements)

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“… Although the determined cell surface enzyme concentrations are only ~2–4 times greater than the K i value for the inhibitor (1.4 nM; Zucker et al., ), they are comparable to that determined by a different method for MT1‐MMP expressed in the same cell line (see Results; Remacle et al., ). …”

Section: Resultssupporting

confidence: 65%

Characterization and regulation of MT1‐MMP cell surface‐associated activity

et al. 2018

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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.

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Section: Resultssupporting

confidence: 65%

Characterization and regulation of MT1‐MMP cell surface‐associated activity

et al. 2018

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“…29 The purified samples (the purity >95%) were used in our subsequent studies. The concentration of the catalytically active MMPs we used in our study, including MT1–MMP, was measured by titration of the MMP samples against a standard GM6001 solution of known concentration and then measuring the residual activity against the MCA-PLGL-Dpa-AR-NH 2 substrate.…”

Section: Methodsmentioning

confidence: 99%

A monoclonal antibody interferes with TIMP-2 binding and incapacitates the MMP-2-activating function of multifunctional, pro-tumorigenic MMP-14/MT1–MMP

et al. 2013

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Matrix metalloproteinases (MMPs) and, especially membrane type 1 (MT1)-MMP/MMP-14, are promising drug targets in malignancies. In contrast with multiple small-molecule and protein pan-inhibitors of MT1–MMP cleavage activity, the murine 9E8 monoclonal antibody targets the MMP-2-activating function of cellular MT1–MMP alone, rather than the general proteolytic activity and the pro-migratory function of MT1–MMP. Furthermore, the antibody does not interact in any detectable manner with other members of the membrane type (MT)-MMP family. The mechanism of this selectivity remained unknown. Using mutagenesis, binding and activity assays, and modeling in silico, we have demonstrated that the 9E8 antibody recognizes the MT-loop structure, an eight residue insertion that is specific for MT–MMPs and that is distant from the MT1–MMP active site. The binding of the 9E8 antibody to the MT-loop, however, prevents tissue inhibitor of metalloproteinases-2 (TIMP-2) association with MT1–MMP. As a result, the 9E8 antibody incapacitates the TIMP-2-dependent MMP-2-activating function alone rather than the general enzymatic activity of human MT1–MMP. The specific function of the 9E8 antibody we determined directly supports an essential, albeit paradoxical, role of the protein inhibitor (TIMP-2) in MMP-2 activation via a unique membrane-tethered mechanism. In this mechanism, the formation of a tri-molecular MT1–MMPTIMP-2MMP-2 complex is required for both the capture of the soluble MMP-2 proenzyme by cells and then its well-controlled conversion into the mature MMP-2 enzyme. In sum, understanding of the structural requirements for the 9E8 antibody specificity may pave the way for the focused design of the inhibitory antibodies against other individual MMPs.

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“…Anthrax protective antigen-83 (PA83) was purchased from List Biological Laboratories. Recombinant human TIMP-2 was expressed in Madin-Darby canine kidney cells and then purified from conditioned medium as reported earlier (35). Human TIMP-1 and TIMP-3 were purchased from Invitrogen.…”

Section: Methodsmentioning

confidence: 99%

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

Shiryaev

Remacle

Chernov

et al. 2013

Journal of Biological Chemistry

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Background: Two distinct metalloproteinase types (fragilysin and metalloproteinase II/MPII) are encoded by the Bacteroides fragilis pathogenicity island. Results: Our assays determined substrate cleavage characteristics of fragilysin and MPII. Conclusion: MPII is the first zinc metalloproteinase with the dibasic cleavage preferences. Significance: Our results are important for understanding B. fragilis virulence and fundamental roles of the microbiome in human health and disease.

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Non-destructive and Selective Imaging of the Functionally Active, Pro-invasive Membrane Type-1 Matrix Metalloproteinase (MT1-MMP) Enzyme in Cancer Cells

Cited by 14 publications

References 68 publications

Characterization and regulation of MT1‐MMP cell surface‐associated activity

Characterization and regulation of MT1‐MMP cell surface‐associated activity

A monoclonal antibody interferes with TIMP-2 binding and incapacitates the MMP-2-activating function of multifunctional, pro-tumorigenic MMP-14/MT1–MMP

Substrate Cleavage Profiling Suggests a Distinct Function of Bacteroides fragilis Metalloproteinases (Fragilysin and Metalloproteinase II) at the Microbiome-Inflammation-Cancer Interface

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