Stimulation of “Prohormone Thiol Protease” (PTP) and [Met]Enkephalin by Forskolin.

Abstract

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

“…The cysteine protease activity termed PTP represents the major PE-cleaving activity in secretory vesicles of chromaffin cells (7)(8)(9)(10)(11). In this study affinity labeling with the activity-based probe DCG-04, combined with peptide sequencing by MS, provided molecular identification of secretory vesicle cathepsin L as the cysteine protease responsible for PE-processing activity.…”

“…In addition, the secretory vesicle cathepsin L is part of a high molecular mass complex of 180-200 kDa (10), whereas lysosomal cathepsin L is known as a single-chain form of Ϸ28 kDa (21). The high molecular mass components of the complex (11) have been identified by peptide sequencing with MS as ␤-mannosidase and dopamine ␤-monooxygenase. It will be of interest for future studies to examine the functional roles of the protein components of the secretory vesicle cathepsin L complex.…”

“…The cysteine protease activity cleaves PE and enkephalin-containing peptide substrates at paired basic residues, as well as at certain monobasic residues, to generate appropriate enkephalin-related peptide products. Cellular inhibition of PTP by a cysteine protease inhibitor results in reduced production of enkephalin (11). Molecular identification of the protease component responsible for this cysteine protease activity will facilitate our understanding of multiple proteolytic enzymes that produce active peptides including the opioid [Met]enkephalin (ME) (12,13).…”

Molecular Cloning of Endopin 1, a Novel Serpin Localized to Neurosecretory Vesicles of Chromaffin Cells

“…Specifically, secretory vesicles from chromaffin cells (known as chromaffin granules) contain proteases of several mechanistic classes that are involved in proneuropeptide processing. The cysteine protease "prohormone thiol protease" (PTP) has been characterized as a proenkephalin processing enzyme for the production of enkephalin-related opiate peptides (15)(16)(17). Chromaffin granules also contain the subtilisin-like PC1/3 and PC2 (PC ϭ prohormone convertase) enzymes that represent members of the subtilisin-like prohormone convertases that are involved in processing a variety of proneuropeptide and proprotein substrates (6 -8, 18).…”

Expression of recombinant pro‐neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by ‘prohormone thiol protease’ (PTP)

“…Peptide hormones and neurotransmitters are synthesized as protein precursors that require proteolytic processing at pairs of basic amino acid residues to generate active neuropeptides [I-3], In biochemical [4-7l and cell biological [8] studies of procnkephaUn procemng in adrenal medulla, the primary enkephalin precursor cleaving activity is represented by a novel cysteine pretense termed 'prohormone thiol protease' (PTP), The roi~ of PTP in enkephalin production in chromaffln cells was clearly demonstrated by complete blockade of cAMPstimulated enkephalin levels by a potent cysteine protease inhibitor of PTP [8], Proenkephalin processing also involves the sebtilisin-like PCil3 and PC2 proteases (PC=prohormone convertase) [9], and a 70 kDa aspartic proteinase [10].…”