We are investigating the in-vitro mechanism of activation of the matrix metalloproteinase Gelatinase A. Activation of the enzyme, normally secreted in a latent form, is a critical control step in determining the amount of matrix degredation contributed by this enzyme in vivo. By understanding, in detail, the in- vitro mechanism of activation, we hope to develop targets for therapeutic intervention. From previous studies we know that this enzyme is secreted as a latent pro-enzyme, often in complex with its specific inhibitor, tissue inhibitor of metalloproteinases-2, TIMP-2. Latency of the pro-enzyme is maintained by preservation of a covalent bond between a cysteine sulfhydryl and the active site zinc atom. Physical disruption (denaturation) or chemical cleavage (organomercurial) of this bond result in a series of events which end in loss of 80 amino acids from the amino-terminus and development of proteolytic activity. In vivo, activation occurs by a cell-surface associated mechanism that gives the same result as in vitro activation. Therefore, a clear understanding of the order and rate of events in- vitro will also allow better understanding of the in vivo mechanism. In parallel with the kinetic analysis of the activation mechanism we also seek to understand how inhibitors, including TIMPs, TIMP fragments or analogues and peptide inhibitors based on the activation locus as well as competing substrates inhibit the processes of activation and catalysis. We will use the results obtained with the analysis of the chemical activation mechanism to compare to the in- vitro activation of gelatinase A by the physiologic activator membrane- type matrix metalloproteinase-1 (MT-1-MMP). We have now pruified a recombinant soluble form of MT-1-MMP and have begun kinetic characterization of the role of this protease in pro-MMP-2 activation and the influence of TIMP-2 in this process. - extracellular matrix, proteases, protease inhibitors,
| Taraboletti, G; Sonzogni, L; Vergani, V et al. (2000) Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells. Exp Cell Res 258:384-94 |
| Marchetti, C; Cornaglia, I; Casasco, A et al. (1999) Immunolocalization of gelatinase-A (matrix metalloproteinase-2) in damaged human temporomandibular joint discs. Arch Oral Biol 44:297-304 |
