Proteolytic processing of inactive proenkephalin (PE) is required to produce active enkephalin opioid peptides that modulate analgesia and immune cell functions. Our studies of PE processing enzymes in the PI's laboratory have identified the novel """"""""prohormone thiol protease"""""""" (PTP) as the major PE processing enzyme. The prohormone convertases (PC) PC 1/3 and PC2 participate to a lesser extent. Of particular interest was the finding that PTP shows clear preference for processing PE compared to POMC (proopiomelanocortin), the precursor of the beta-endorphin opioid peptide. Therefore, the goal of this project will be to assess the role of PTP as a primary, specific PE processing enzyme. The first Specific Aim will obtain molecular and cellular characterization of PTP. Based on determined peptide sequences of PTP, molecular cloning of the bovine PTP cDNA will utilize PCR approaches (RT-PCR,5'- and 3' -RACE, and PCR of cDNA libraries) with complimentary oligonucleotides to obtain a partial PTP cDNA. Libraries for cDNAs will be screened with PCR- generated DNA probes, oligonucleotides, and anti -PTP antibodies in expression cloning. The deduced primary sequence of the PTP cDNA will demonstrate whether PTP is a unique protease, as indicated by peptide microsequencing. Expression in E. Coli and Sf9 cells will confirm the clone as PTP. In addition the colocalization of PTP, as well as the PC enzymes, with (Met)enkephalin will be assessed in cellular tissue distribution studies.
The second Aim will examine the co-regulation of PTP and PC enzymes during stimulated (Met)enkephalin production in chromaffin cells; these studies will assess enzymes with respect to activities, biosynthesis by 35s- pulse-chase analysis, and levels by RIA. Results will indicate the primary, regulated PE processing enzyme. In the third Aim, PTP will be evaluated as a specific, primary PE processing enzyme by tin vitro processing assays and cellular antisense studies. Comparison of in vitro processing of recombinant PE and POMC by PTP and PC enzymes will be accomplished by in vitro kinetic studies; results will indicate the most efficient processing enzymes for PE and POMC processing. The role of PTP as a primary PE processing enzyme in chromaffin cells will be analyzed in enzyme antisense studies to determine the effect of inhibiting enzyme gene expression on (Met)enkephalin production. Enzyme levels and PE processing will be analyzed by 35S-pulse-chase and RIAs. Parallel enzyme antisense experiments in pituitary cells will indicate the primary POMC processing enzyme(s). These studies will test the hypothesis that distinct proteases may be involved in processing PE compared to POMC. Results will provide new knowledge of the proteolytic mechanisms required for processing opioid precursors into active peptides. These studies may lead to novel therapeutics that modify the endogenous opioid system through precursor processing enzymes.
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