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.
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Cover Image
Cover Image
3D reconstructions of 4 individual Escherichia coli cells that have been serially sectioned and immunolabelled to unambiguously identify a membrane protein of interest (TatA, position marked by spheres). The reconstructions illustrate a linear clustering of TatA protein in the inner membrane of E. coli – a previously uncharacterised distribution for this protein. For more information, please see article by Sarah M. Smith et al, pages 1495–1508. Image provided by Sarah M. Smith.
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, Carl P. Blobel; Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays. Biochem J 1 May 2017; 474 (9): 1467–1479. doi: https://doi.org/10.1042/BCJ20170075
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