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Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays
Journal article   Peer reviewed

Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays

Thorsten Maretzky, Steven Swendeman, Elin Mogollon, Gisela Weskamp, Umut Sahin, Karina Reiss and Carl P Blobel
The Biochemical journal, Vol.474(9), pp.1467-1479
04/13/2017
DOI: 10.1042/BCJ20170075
PMID: 28264989
url
https://www.ncbi.nlm.nih.gov/pmc/articles/8606101View
Open Access

Abstract

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.
 ADAM Proteins - antagonists & inhibitors Chlorocebus aethiops Enzyme Inhibitors - pharmacology Dose-Response Relationship, Drug Cell Membrane - enzymology ADAM Proteins - metabolism Animals Membrane Proteins - antagonists & inhibitors Membrane Proteins - metabolism Mice Catalysis COS Cells Cell Membrane - drug effects

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