title: Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme.
abstract: Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.
authors.affiliation: Department of Biomedical Science, Florida Atlantic University, Boca Raton, Florida 33431, USA.
authors.forename: Shuo
authors.initials: S
authors.lastname: Wei
authors.forename: Masahide
authors.initials: M
authors.lastname: Kashiwagi
authors.forename: Smitha
authors.initials: S
authors.lastname: Kota
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Xie
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Keith
authors.initials: K
authors.lastname: Brew
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D000072198:ADAM17 Protein; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010703:Phorbol Esters / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D010703:Phorbol Esters; D019717:Tissue Inhibitor of Metalloproteinase-3; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D020782:Matrix Metalloproteinases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human