The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3.
A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.
Amour
A
A
School of Biological Sciences, University of East Anglia, Norwich, UK.
Knight
C G
CG
Webster
A
A
Slocombe
P M
PM
Stephens
P E
PE
Knäuper
V
V
Docherty
A J
AJ
Murphy
G
G
D051722:ADAM Proteins; D000072197:ADAM10 Protein; D053829:Amyloid Precursor Protein Secretases; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D023201:CD40 Ligand; D020134:Catalytic Domain; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D016564:Amyloid beta-Protein Precursor; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D004676:Myelin Basic Protein; D010455:Peptides; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D019717:Tissue Inhibitor of Metalloproteinase-3; D014409:Tumor Necrosis Factor-alpha; D023201:CD40 Ligand; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D008666:Metalloendopeptidases; C103778:ADAM 10 protein, Bos taurus; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human