Metalloproteases play a leading role in maintenance of the extracellular matrix (ECM). We intend to define the nature, function, and regulation of collagenase and other members of this metalloprotease family at the molecular level. We plan to determine the structural organization of the normal human skin collagenase gene, including regulatory elements at the 5' end. The 5' end of the gene will be examined to identify the structural elements of conventional eucaryotic promoters, and chimeric genes will be used to identify regulatory elements needed for constitutive and inducible expression. The genomic arrangement and regulation of human skin collagenase, and its evolutionary relationship to other human and vertebrate collagenases and related members of this gene family, are major areas for continuing investigation. A knowledge of the normal human skin collagenase gene will enable us to determine the potential mutation site(s) responsible for the putative structurally altered RDEB collagenase. Any mutation will be confirmed by sequencing through the region of mutation in several RDEB cell strains. In vitro directed mutagenesis of normal collagenase would confirm that the mutation confers the phenotype of RDEB. Hydrated collagen lattices with embedded RDEB fibroblasts will be used to attempt to reconstruct the RDEB phenotype on the cellular level. Normal human skin collagenase and RDEB collagenase will be tested for catalytic effect on the type Vll collagen of anchoring fibrils, and their kinetic parameters determined. Stromelysin, another member of the gene family, with a broad range of substrate specificities, whose activity parallels that of collagenase in several instances, will also be assayed for its activity in RDEB. In addition, we have isolated a procollagenase activator molecule from skin explant culture medium, which may be an N-chloroamine. Future studies will focus on its range and mechanism of action. We have purified a secreted neutral metalloprotease from cultured human skin explants and fibroblasts which degrades gelatin and have isolated a cDNA clone coding for the complete progelatinase. Areas of investigation of this gelatinase molecule will focus on its structure, what cells (normal and tumor) produce it, whether its synthesis is coordinated with that of collagenase and stromelysin, what agents regulate its production, and its substrate and cleavage site specificities. This ECM metalloprotease gene family is crucial to our understanding of diseases of the skin and other organ systems and on basic cellular processes; proliferation, differentiation and tumorigenesis.
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