Metalloproteases play an important role in the remodeling of extracellular matrix (ECM). This proposal is designed to address the biologic role of one particular enzyme, human 92 kDa type IV collagenase. The 92 kda metalloprotease is a major secreted product of keratinocytes, mononuclear phagocytes, and many transformed cell lines. We have recently purified this enzyme to homogeneity and determined its complete primary structure. To further study the biologic role of this enzyme, we propose to investigate the molecular mechanism of proenzyme-inhibitor complex formation. We will also examine the role of 92 kDa type IV collagenase in extracellular matrix degradation by studying interaction of the secreted enzyme with ECM and cell surface. Finally, the role of the 92 kDa type IV collagenase in tissue remodeling will be studied and compared to that of other secreted metalloproteases by transfection of cDNA clones with wild type versus mutant enzymes into parental tumor and E1A transfected cells. Our experimental approaches will include mutagenesis, characterization of intermolecular interactions on the protein level, crosslinking and competition experiments using synthetic peptides. Site-directed mutagenesis of wild type enzyme will be used to generate point mutants unable to interact with TIMP. The studies of 92 kDa enzyme interactions with matrix and/or cell surfaces will utilize a """"""""suicide"""""""" ligand technology recently developed in our laboratory. Synthetic peptides able to compete with the enzyme-ECM and/or enzyme-cell surface interactions will be chemically modified to incorporate affinity and radiolabeled photoactivatable crosslinking groups to specifically tag the sites of intermolecular associations. Transfection of tumor cells with the adenovirus E1A gene results in transcriptional repression of the endogenous interstitial and type IV collagenase promoters. By allowing the expression of each of the enzymes in parental HT1080 and E1A transfectants of human tumor cells, under control of a promoter not subject to E1A repression, we can gain insights into specific effects of individual enzyme expression on cell phenotype. The information obtained from these studies will provide a further understanding of the molecular mechanisms involved in secreted metalloprotease action in normal and pathologic conditions.
