This laboratory's research program will investigate the biochemistry and molecular biology of proenkephalin (PE) processing enzymes required to synthesize active enkephalin opiate peptides. Specifically, the research plan will determine the relative importance of the novel 'prohormone thiol protease' (PTP) that is inhibited by endogenous alpha1-antichymotrypsin (ACT), as well as the roles of PC 1/3 and PC2 (PC - prohormone convertase), in PE processing. The first part of the project will compare PTP, PC 1/3, and PC2 processing of recombinant PE, obtained by high level expression in E. coli, with respect to PE products, cleavage sites, kinetics, and theoretical activities of each enzyme in vivo. Comparison of precursor selectivity of PTP, PC 1/3 and PC2 in processing recombinant pro-neuropeptide Y and beta-protachykinin precursors into NPY and substance P neuropeptides may suggest selective PE processing enzymes. Structural characterization of PTP through molecular cloning will test the hypothesis that PTP may be a unique cysteine protease. Preliminary NH2- terminal sequencing and a partial PTP cDNA indicate a novel sequence. Cloning of a full-length PTP cDNA will be accomplished by obtaining partial clones through RT-PCR (reverse transcriptase-polymerase chain reaction), and 5'- and 3'-RACE (rapid amplification of cDNA ends); partial clones will be used to screen cDNA libraries. Secondly, the project will characterize inhibition of PTP by endogenous alpha1-antichymotrypsin (ACT) with respect to Ki, complex formation, and biochemical properties. Full- length ACT cDNA clones will be obtained by screening cDNA libraries with partial clones. Production of specific PTP and ACT antibodies will allow studies of ACT and PTP colocalization in enkephalin-containing brain regions and endocrine tissues by immunofluorescence cell staining, immunoelectron microscopy, and western blots. Thirdly, the ability of the potent PTP inhibitor Ep453 to reduce PE processing in chromaffin cells will be assessed by pulse-chase labeling of PE and by determining cellular levels of (Met)enkephalin by RIA. Overall these studies will establish the relative importance of PTP, compared to PC 1/3 and PC2, in PE processing.
