ENDOGENOUS INHIBITORS OF ADAMS/MDC PROTEINASES

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A new group of proteins termed "ADAMS" (A Disintegrin And Metal Metalloproteinase protein" or "MDC" (Metalloproteinase Disintegrin Cysteine-rich protein) have recently been identified. This group of proteins has been implicated in several important biological processes including fertilization, development and cell surface shedding. Additional physiological roles will likely be identified for the various members of the group. ADAMS are type-1 integral membrane proteins having a multiple domain structure comprised of metalloproteinase, disintegrin-like, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains. Most proteolytic enzyme systems have endogenous inhibitors that regulate the proteinases' biological regulation. Given the extensive distribution of the ADAMS, we hypothesize that there is a non-TIMP endogenous inhibitor/proteinase system for the metalloproteinase domain of the ADAMS. This hypothesis is supported by the identification of endogenous serum inhibitors of the snake venom metalloproteinases (homologs of the ADAMS) in opossum, woodrat, mongoose and certain snakes. We have preliminary data that supports the presence of a non-TIMP inhibitor(s) in human serum that inhibits ADAM 9, an ADAM family member we have cloned and sequenced from a human metastatic melanoma. We propose to isolate the ADAM proteinase inhibitor(s) from human serum and conditioned cell culture medium using conventional and affinity chromatography. The inhibitor will be partially sequenced by a combination of Edman and mass spectrometric techniques and the data used to clone and sequence its cDNA. The mechanism by which the inhibitor binds to ADAM 9 and blocks proteolytic activity and the kinetics of inhibition will be studied using surface plasmon resonance techniques. The structural requirements for proteinase inhibition by the inhibitor will be probed using reduced and alkylated inhibitor. Additionally, the inhibitor will be subjected to limited proteolysis and the fragments tested for inhibitory activity in order to identify regions of the inhibitor involved in proteinase interaction. Recombinant proteins representing the inhibitor and its domains will be expressed for functional studies. A second approach to identify ADAM 9 inhibitors will be to use a yeast two-hybrid screen. Characterization of ADAM binding proteins thus identified will be performed as described above. The discovery and characterization of endogenous inhibitors of ADAMs will mark a significant contribution to the understanding of the biology and biochemistry of the ADAMs. The broad-ranging biological attributes of members of the ADAMs family make it likely that pathologies associated with these proteins will be discovered in the near future. Isolation and characterization of these inhibits could therefore be of future importance in developing novel pharmacological agents for the regulation of ADAMs proteins' functions in pathological states.