EXTRACELLULAR REGULATION OF MATRIX METALLOPROTEINASES

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The long range goal of this research is to elucidate the mechanisms by which the matrix metalloproteinases (NM) are regulated extracellularly. The NM are a homologous group of zinc proteinases that play a major role in the normal and pathological breakdown of the extracellular matrix. The MMP are produced by resident and inflammatory-cells as zymogens. The basis for the latency of these zymogens and the means by which they are activated extracellularly are incompletely understood. The proposed experiments will examine various aspects of the "cysteine-switch" model for the latency and activation of pro-MMP recently proposed by us. The model attributes the inactivity of the pro-MMP to a novel complex between a cysteine residue in their propeptide domains and the active-site zinc atom in their catalytic domains. The pro-MMP exhibit multiple modes of activation that share the property that they disrupt this complex. To investigate this new hypothesis, the metal contents of five major human MMP will be measured and the functional role of each metal ion examined. In particular, the presence of zinc at the active site will be investigated. Active-site metal-substituted pro-MMP will be prepared and the presence of a cysteine-zinc bond in the pro-MMP investigated by EXAFS studies on the native species, 113Cd NMR studies on the Cd-substituted enzymes and optical studies of the Co(II)-substituted species. The, efficiency of a variety of known activation routes (proteases, oxidants, disulfide exchange, heavy metals, etc.) will be quantitated and correlated with the dissociation of the zinc-cysteine bond. These events will also be correlated with the autolytic loss of the propeptide domain in these MMP. The source of the small amount of "residual" activity in the pro-MMP will be carefully studied to assess whether it is due to a conformational equilibrium in these zymogens that could lead to their activation by substrates. To provide a structural model for the zinc site in pro-MMP, model peptides will be synthesized that mimic both the cysteine propeptide and zinc binding regions. Their structures and that of a possible complex between them will be elucidated by two dimensional (1)H-NMR experiments.