OPIOID PEPTIDE PROCESSING ENZYMES

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DESCRIPTION (Applicant's Abstract): This renewal of a K02 award is requested in order to free the applicant from substantive teaching and administrative responsibilities to increase research productivity. During the past 4 years the PI has been able to devote more than 80% of her time to research activities, a level of effort which has resulted in a significantly enhanced ability to perform experiments, advise postdoctoral fellows and graduate students, and acquire new technologies both by collaboration as well as by hands-on experimentation. This enhancement of research effort is expected to continue during the renewal period of the application with the protection afforded by the K02 award; without it, teaching responsibilities are likely to increase dramatically, most notably by the assumption of course directorship in a major service course. The proposal is based upon two currently ongoing projects, one on the proteolytic processing of opioid peptide precursors, and one on the mechanism of action of the PC2 binding protein 7B2. The four specific aims are 1) to define the specificity of recombinant PC1 and PC2 using recombinant normal and mutant proenkephalins and related fluorogenic substrates; these experiments will delineate substrate preferences of the two PCs. 2) To identify novel PC1 inhibitors, study the biochemistry and cell biology of PC1 and PC2 inhibitors, and assess potential effects of targeted inhibitors on proenkephalin processing in vivo. These experiments will complement those described in the first specific aim as to enzyme specificity and should yield information on the design of synthetic enzyme inhibitors. 3) To determine the role of 7B2 in the regulation of proPC2 conversion in neuroendocrine cel1 lines, with a focus on the fate of the propeptide and the relationship of its cleavage/dissociation to that of 7B2 forms. 4) To explore the molecular interaction of 7B2 and PC2 through in vitro experiments. Taken together, these experiments should help us to understand the basic biochemistry of the prohormone convertases on opioid peptide precursors as well as the regulation of convertase activity.