This proposal is to study the regulation of opioid peptide biosynthesis. Bio-active opioid peptide hormones are processed from prohormones by post-translational modifications, such as proteolytic cleavage, oligosaccharide addition. Prohormone convertase 2 (PC2) is involved in the proteolytic cleavage of proenkephalin. Presently the biosynthesis and regulation of activity of PC2 is not fully understood. Recent studies have shown that the 7B2 protein, which like PC2 is selectively present in the central nervous system and in endocrine tissues, is involved in PC2 regulation. Research has shown that this interesting molecule is bi- functional: it chaperones PC2 maturation and inhibits the enzymatic activity of PC2. The specific goals of the experiments proposed here are: 1). Separate the chaperone domain from the inhibitory domain and assess their effects on opioid peptide production; 2). Determine the minimum requirement for the chaperone activity; and 3) Determine the biosynthetic pathway of the inhibitory peptide, which will be essential to a complete understanding regulation of PC2. The following methods will be employed: 1) PCR mediated mutagenesis will be applied to generate desired truncations, chimeras and point directed mutations; 2) eukaryotic expression vectors, such as pRc/CMV and pCEP 4 will be used to clone the mutated DNA fragments and transfect neuroendocrine cell lines, such as AtT-20 cells and rat insulinoma cells; 3) kinetics of peptide and PC2 biosynthesis will be analyzed by pulse-chase, radioimmunoassay, and HPLC/HPGPC. A thorough knowledge of peptide biosynthesis would help us to understand the biochemical basis for opiate addiction.
