Characterization of the catalytic activity of the membrane-anchored metalloproteinase ADAM15 in cell-based assays

2009 ◽  
Vol 420 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Thorsten Maretzky ◽  
Guangli Yang ◽  
Ouathek Ouerfelli ◽  
Christopher M. Overall ◽  
Susanne Worpenberg ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cancer Metastasis ◽  
Phorbol Esters ◽  
Catalytic Properties ◽  
Growth Factor Receptor ◽  
Ectodomain Shedding ◽  
Intact Cells ◽  
Peptide Substrates ◽  
Prostate Cancer Metastasis

ADAM15 (a disintegrin and metalloproteinase 15) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM15. Here, we purified soluble recombinant ADAM15 to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM15. Overexpression of full-length membrane-anchored ADAM15 or the catalytically inactive ADAM15E→A together with various membrane proteins resulted in increased release of the extracellular domain of the fibroblast growth factor receptor 2iiib (FGFR2iiib) by ADAM15, but not ADAM15E→A. This provided a robust assay for a characterization of the catalytic properties of ADAM15 in intact cells. We found that increased expression of ADAM15 resulted in increased FGFR2iiib shedding, but that ADAM15 was not stimulated by phorbol esters or calcium ionophores, two commonly used activators of ectodomain shedding. Moreover, ADAM15-dependent processing of FGFR2iiib was inhibited by the hydroxamate-based metalloproteinase inhibitors marimastat, TAPI-2 and GM6001, and by 50 nM TIMP-3 (tissue inhibitor of metalloproteinases 3), but not by 100 nM TIMP-1, and only weakly by 100 nM TIMP-2. These results define key catalytic properties of ADAM15 in cells and its response to stimulators and inhibitors of ectodomain shedding. A cell-based assay for the catalytic activity of ADAM15 could aid in identifying compounds, which could be used to block the function of ADAM15 in pathological neovascularization and cancer.

scholarly journals The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity

2005 ◽  
Vol 389 (3) ◽  
pp. 739-744 ◽  
Author(s):  
Zenda L. Woodman ◽  
Sylva L. U. Schwager ◽  
Pierre Redelinghuys ◽  
Adriana K. Carmona ◽  
Mario R. W. Ehlers ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Angiotensin Converting Enzyme ◽  
Catalytic Properties ◽  
Close Contact ◽  
Catalytic Efficiency ◽  
Ectodomain Shedding ◽  
Converting Enzyme ◽  
Peptide Substrates ◽  
Recognition Motif ◽  
Hydrolysis Of

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the kcat for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.

scholarly journals Characterization of 12-Oxophytodienoic Acid Reductases from Rose-scented Geranium (Pelargonium graveolens)

2016 ◽  
Vol 11 (12) ◽  
pp. 1934578X1601101
Author(s):  
Miu Iijima ◽  
Hiromichi Kenmoku ◽  
Hironobu Takahashi ◽  
Jung-Bum Lee ◽  
Masao Toyota ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Metabolic Engineering ◽  
Double Bond ◽  
Carbonyl Compound ◽  
Catalytic Properties ◽  
Biotechnological Applications ◽  
Pelargonium Graveolens ◽  
Double Bond Reductase ◽  
Herbal Plant

Pelargonium graveolens L'Hér, also referred to as rose geranium, is a popular herbal plant with typical rosy fragrance largely based on the blend of monoterpenoid constituents. Among them, citronellol, which is biosynthesized from geraniol via double bond reduction, is the most abundant scent compound. In this study, three 12-oxophytodienoic acid reductases (PgOPR1–3) have been cloned from P. graveolens, as possible candidates for the double-bond reductase involved in citronellol biosynthesis. The bacterially expressed recombinant PgOPRs did not reduce geraniol to citronellol, but stereoselectively converted citral into ( S)-citronellal in the presence of NADPH. Thus, the α,β-unsaturated carbonyl moiety in the substrate is essential for the catalytic activity of PgOPRs, as reported for OPRs from other plants and structurally related yeast old yellow enzymes. PgOPRs promiscuously accepted linear and cyclic α,β-unsaturated carbonyl substrates, including methacrolein, a typical reactive carbonyl compound. The possible biotechnological applications for PgOPRs in plant metabolic engineering, based on their catalytic properties, are discussed herein.

scholarly journals Selective Hydration of Nitriles to Corresponding Amides in Air with Rh(I)-N-Heterocyclic Complex Catalysts

Catalysts ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 125 ◽  
Author(s):  
Csilla Enikő Czégéni ◽  
Sourav De ◽  
Antal Udvardy ◽  
Nóra Judit Derzsi ◽  
Gergely Papp ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Solid State ◽  
Nmr Spectroscopy ◽  
Catalytic Properties ◽  
13C Nmr Spectroscopy ◽  
Synthetic Method ◽  
X Ray Diffraction ◽  
Nitrile Hydration ◽  
X Ray

A new synthetic method for obtaining [RhCl(cod)(NHC)] complexes (1–4) (cod = η4-1,5-cyclooctadiene, NHC = N-heterocyclic carbene: IMes, SIMes, IPr, and SIPr, respectively) is reported together with the catalytic properties of 1–4 in nitrile hydration. In addition to the characterization of 1–4 in solution by 13C NMR spectroscopy, the structures of complexes 3, and 4 have been established also in the solid state with single-crystal X-ray diffraction analysis. The Rh(I)-NHC complexes displayed excellent catalytic activity in hydration of aromatic nitriles (up to TOF = 276 h−1) in water/2-propanol (1/1 v/v) mixtures in air.

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

2017 ◽  
Vol 474 (9) ◽  
pp. 1467-1479 ◽  
Author(s):  
Thorsten Maretzky ◽  
Steven Swendeman ◽  
Elin Mogollon ◽  
Gisela Weskamp ◽  
Umut Sahin ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Phorbol Esters ◽  
Catalytic Properties ◽  
Negative Control ◽  
Ectodomain Shedding ◽  
Molecular Fingerprint ◽  
Ephrin Receptor ◽  
New Information ◽  
Metalloprotease Inhibitors ◽  
20 Nm

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.

Abstract 5183: Identification and characterization of wild type kinases driving prostate cancer metastasis

2015 ◽  
Author(s):  
Claire Faltermeier ◽  
Justin Drake ◽  
Peter Clark ◽  
Bryan Smith ◽  
Colleen Mathis ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Cancer Metastasis ◽  
Wild Type ◽  
Prostate Cancer Metastasis ◽  
Identification And Characterization

scholarly journals Biochemical correlation of activity of the α-dystroglycan-modifying glycosyltransferase POMGnT1 with mutations in muscle-eye-brain disease

2011 ◽  
Vol 436 (2) ◽  
pp. 447-455 ◽  
Author(s):  
Josef Voglmeir ◽  
Sara Kaloo ◽  
Nicolas Laurent ◽  
Marco M. Meloni ◽  
Lisa Bohlmann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Homology Modelling ◽  
Severe Disease ◽  
Brain Disease ◽  
Muscular Dystrophies ◽  
Peptide Substrates ◽  
Human Enzyme

Congenital muscular dystrophies have a broad spectrum of genotypes and phenotypes and there is a need for a better biochemical understanding of this group of diseases in order to aid diagnosis and treatment. Several mutations resulting in these diseases cause reduced O-mannosyl glycosylation of glycoproteins, including α-dystroglycan. The enzyme POMGnT1 (protein-O-mannose N-acetylglucosaminyltransferase 1; EC 2.4.1.-) catalyses the transfer of N-acetylglucosamine to O-linked mannose of α-dystroglycan. In the present paper we describe the biochemical characterization of 14 clinical mutants of the glycosyltransferase POMGnT1, which have been linked to muscle-eye-brain disease or similar conditions. Truncated mutant variants of the human enzyme (recombinant POMGnT1) were expressed in Escherichia coli and screened for catalytic activity. We find that three mutants show some activity towards mannosylated peptide substrates mimicking α-dystroglycan; the residues affected by these mutants are predicted by homology modelling to be on the periphery of the POMGnT1 surface. Only in part does the location of a previously described mutated residue on the periphery of the protein structure correlate with a less severe disease mutant.

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