Sukkid Yasothornsrikul

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

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of [Met]enkephalin by cathepsin L occurred by proteolytic processing at dibasic and monobasic prohormone-processing sites. Cellular studies showed the colocalization of cathepsin L with [Met]enkephalin in secretory vesicles of neuroendocrine chromaffin cells by immunofluorescent confocal and immunoelectron microscopy. Functional localization of cathepsin L to the regulated secretory pathway was demonstrated by its cosecretion with [Met]enkephalin. Finally, in cathepsin L gene knockout mice, [Met]enkephalin levels in brain were reduced significantly; this occurred with an increase in the relative amounts of enkephalin precursor. These findings indicate a previously uncharacterized biological role for secretory vesicle cathepsin L in the production of [Met]enkephalin, an endogenous peptide neurotransmitter.

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones

Abstract Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH[2]-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH[2]-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH[2-]terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisinlike prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.

Formation of the Catecholamine Release-inhibitory Peptide Catestatin from Chromogranin A DETERMINATION OF PROTEOLYTIC CLEAVAGE SITES IN HORMONE STORAGE GRANULES

The catestatin fragment of chromogranin A is an inhibitor of catecholamine release, but its occurrence in vivo has not yet been verified, nor have its precise cleavage sites been established. Here we found extensive processing of catestatin in chromogranin A, as judged by catestatin radioimmunoassay of size-fractionated chromaffin granules. On mass spectrometry, a major catestatin form was bovine chromogranin A332–364; identity of the peptide was confirmed by diagnostic Met346oxidation. Further analysis revealed two additional forms: bovine chromogranin A333–364 and A343–362. Synthetic longer (chromogranin A332–364) and shorter (chromogranin A344–364) versions of catestatin each inhibited catecholamine release from chromaffin cells, with superior potency for the shorter version (IC50 ∼2.01 versus∼0.35 μm). Radioimmunoassay demonstrated catestatin release from the regulated secretory pathway in chromaffin cells. Human catestatin was cleaved in pheochromocytoma chromaffin granules, with the major form, human chromogranin A340–372, bounded by dibasic sites. We conclude that catestatin is cleaved extensivelyin vivo, and the peptide is released by exocytosis. In chromaffin granules, the major form of catestatin is cleaved at dibasic sites, while smaller carboxyl-terminal forms also occur. Knowledge of cleavage sites of catestatin from chromogranin A may provide a useful starting point in analysis of the relationship between structure and function for this peptide.

β-Amyloid peptide in regulated secretory vesicles of chromaffin cells: evidence for multiple cysteine proteolytic activities in distinct pathways for β-secretase activity in chromaffin vesicles

A key factor in Alzheimers disease (AD) is the β‐secretase activity that is required for the production of beta‐amyloid (Aβ) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Aβ secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, β‐secretase activity in the regulated secretory pathway was examined for the production and secretion of Aβ in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Aβ(1–40) in APP‐containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse‐phase high‐performance liquid chromatography. These vesicles also contain Aβ(1–42), measured by RIA. Significantly, regulated secretion of Aβ(1–40) from chromaffin cells represented the majority of secreted Aβ (> 95% of total secreted Aβ), compared with low levels of constitutively secreted Aβ(1–40). These results indicate the importance of Aβ production and secretion in the regulated secretory pathway as a major source of extracellular Aβ. β‐secretase activity in isolated chromaffin vesicles was detected with the substrate Z‐Val‐Lys‐Met‐↓MCA (methylcoumarinamide) that contains the β‐secretase cleavage site. Optimum β‐secretase activity in these vesicles required reducing conditions and acidic pH (pH 5–6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z‐Val‐Lys‐Met‐MCA‐cleaving activity, and E64c inhibition of Aβ(1–40) production in isolated chromaffin vesicles. Chromatography resolved the β‐secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the β‐secretase site at Met‐↓Asp by two cysteine proteolytic activities represented by peaks II‐A and II‐B, and (ii) an aminopeptidase‐dependent pathway represented by peak I cysteine protease activity that cleaves between Lys‐↓Met, followed by Met‐aminopeptidase that would generate the β‐secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the β‐secretase product, a 12–14 kDa C‐terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total β‐secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to β‐secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as β‐secretase for Aβ production in the constitutive secretory pathway that provides basal secretion of Aβ into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Aβ levels as a potential therapeutic approach for AD.

Enrichment of Presenilin 1 Peptides in Neuronal Large Dense‐Core and Somatodendritic Clathrin‐Coated Vesicles

Abstract: Presenilin 1 is an integral membrane protein specifically cleaved to yield an N‐terminal and a C‐terminal fragment, both membrane‐associated. More than 40 presenilin 1 mutations have been linked to early‐onset familial Alzheimer disease, although the mechanism by which these mutations induce the Alzheimer disease neuropathology is not clear. Presenilin 1 is expressed predominantly in neurons, suggesting that the familial Alzheimer disease mutants may compromise or change the neuronal function(s) of the wild‐type protein. To elucidate the function of this protein, we studied its expression in neuronal vesicular systems using as models the chromaffin granules of the neuroendocrine chromaffin cells and the major categories of brain neuronal vesicles, including the small clear‐core synaptic vesicles, the large dense‐core vesicles, and the somatodendritic and nerve terminal clathrin‐coated vesicles. Both the N‐ and C‐terminal presenilin 1 proteolytic fragments were greatly enriched in chromaffin granule and neuronal large dense‐core vesicle membranes, indicating that these fragments are targeted to these vesicles and may regulate the large dense‐core vesicle‐mediated secretion of neuropeptides and neurotransmitters at synaptic sites. The presenilin 1 fragments were also enriched in the somatodendritic clathrin‐coated vesicle membranes, suggesting that they are targeted to the somatodendritic membrane, where they may regulate constitutive secretion and endocytosis. In contrast, these fragments were not enriched in the small clear‐core synaptic vesicle or in the nerve terminal clathrin‐coated vesicle membranes. Taken together, our data indicate that presenilin 1 proteolytic fragments are targeted to specific populations of neuronal vesicles where they may regulate vesicular function. Although full‐length presenilin 1 was present in crude homogenates, it was not detected in any of the vesicles studied, indicating that, unlike the presenilin fragments, full‐length protein may not have a vesicular function.

Arginine and lysine aminopeptidase activities in chromaffin granules of bovine adrenal medulla: relevance to prohormone processing.

Abstract: Conversion of prohormones and neuropeptide precursors to smaller, biologically active peptides requires specific proteolytic processing at paired basic residues, which generates intermediate peptides with NH2 and COOH termini extended with Lys or Arg residues. These basic residues are then removed by aminopeptidase and carboxypeptidase activities, respectively. Among the proteases involved in prohormone processing, the basic residue aminopeptidase activity has not been well studied. This report demonstrates arginine and lysine aminopeptidase activities detected with Arg‐methylcoumarinamide (Arg‐MCA) and Lys‐MCA substrates in neurosecretory vesicles of bovine adrenal medulla [chromaffin granules (CG)], which contain endoproteolytic processing enzymes co‐localized with [Met]‐enkephalin and other neuropeptides. These arginine and lysine aminopeptidase activities showed many similarities and some differences. Both arginine and lysine aminopeptidase activities were stimulated by the reducing agent β‐mercaptoethanol (β‐ME) and inhibited by p‐hydroxymercuribenzoate, suggesting involvement of reduced cysteinyl residues. The arginine aminopeptidase activity was stimulated by NaCl (150 mM), but the lysine aminopeptidase activity was minimally affected. Moreover, characteristic β‐ME/NaCl‐stimulated Arg‐MCA cleaving activity and β‐ME‐stimulated Lys‐MCA cleaving activity were detected only in CG and not in other subcellular fractions; these findings indicate the localization of these particular basic residue aminopeptidase activities to secretory vesicles. The arginine and lysine aminopeptidase activities showed pH optima at 6.7 and 7.0, respectively. Km(app) values for the arginine and lysine aminopeptidase activities were 104 and 160 µM, respectively. Inhibition by the aminopeptidase inhibitors bestatin, amastatin, and arphamenine was observed for Arg‐MCA and Lys‐MCA cleaving activities. Inhibition by the metal ion chelators indicated that metalloproteases were involved; Co2+ stimulated the arginine aminopeptidase activity but was less effective in stimulating lysine aminopeptidase activity. In addition, the lysine aminopeptidase activity was partially inhibited by Ni2+ and Zn2+ (1 mM), whereas the arginine aminopeptidase activity was minimally affected. These results demonstrate the presence of related arginine and lysine thiol metalloaminopeptidase activities in CG that may participate in prohormone processing.

Molecular Cloning of Endopin 1, a Novel Serpin Localized to Neurosecretory Vesicles of Chromaffin Cells INHIBITION OF BASIC RESIDUE-CLEAVING PROTEASES BY ENDOPIN 1

Serpins represent a diverse class of endogenous protease inhibitors that regulate important biological functions. In consideration of the importance of regulated proteolysis within secretory vesicles for the production of peptide hormones and neurotransmitters, this study revealed the molecular identity of a novel serpin, endopin 1, that is localized to neurosecretory vesicles of neuropeptide-containing chromaffin cells (chromaffin granules). Endopin 1 of 68–70 kDa was present within isolated chromaffin granules. Stimulated cosecretion of endopin 1 with chromaffin granule components, [Met]enkephalin and a cysteine protease known as “prohormone thiol protease,” demonstrated localization of endopin 1 to functional secretory vesicles. Punctate, discrete immunofluorescence cellular localization of endopin 1 in chromaffin cells was consistent with its secretory vesicle localization. Endopin 1 contains a unique reactive site loop with Arg as the predicted P1 residue, suggesting inhibition of basic residue-cleaving proteases; indeed, trypsin was potently inhibited (K i (app) of 5 nm), and plasmin was moderately inhibited. Although endopin 1 possesses homology with α1-antichymotrypsin, chymotrypsin was not inhibited. Moreover, endopin 1 inhibited the chromaffin granule prohormone thiol protease (involved in proenkephalin processing). These results suggest a role for the novel serpin, endopin 1, in regulating basic residue-cleaving proteases within neurosecretory vesicles of chromaffin cells.

THE KUNITZ PROTEASE INHIBITOR FORM OF THE AMYLOID PRECURSOR PROTEIN (KPI/APP) INHIBITS THE PRONEUROPEPTIDE PROCESSING ENZYME PROHORMONE THIOL PROTEASE (PTP): COLOCALIZATION OF KPI/APP AND PTP IN SECRETORY VESICLES

Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that “protease nexin 2 (PN2),” the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a K i ,app of 400 nm. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110–120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer’s disease.

The Local Chromaffin Cell Plasminogen/Plasmin System and the Regulation of Catecholamine Secretion

Abstract: Chromaffin cells express components of the plasminogen/plasmin system, including its major activator, tissue plasminogen activator (t‐PA), and high‐affinity cellular receptors for plasminogen, which promote local concentration and activation of plasminogen at the cell surface. Our studies suggest that plasmin participates in local neuroendocrine prohormone processing and that perturbation of this system profoundly affects the secretory characteristics of the cells. These results suggest the presence of a local, functionally active, chromaffin cell plasminogen/plasmin system that plays a major role in the regulation of catecholamine release from catecholaminergic cells.

α1-Antichymotrypsin-Like Proteins I and II Purified from Bovine Adrenal Medulla Are Enriched in Chromaffin Granules and Inhibit the Proenkephalin Processing Enzyme “Prohormone Thiol Protease”

Abstract: Proteolytic processing of inactive proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary α1‐antichymotrypsin (ACT), referred to as ACT‐like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56‐ and 60‐kDa ACT‐like proteins I and II, respectively, to enkephalin‐containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE‐Sepharose and chromatofocusing resulted in the separation of ACT‐like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56‐kDa ACT‐like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60‐kDa ACT‐like protein II was isolated by butyl‐Sepharose, Sephacryl S200, and concanavalin A‐Sepharose columns. The proenkephalin processing enzyme PTP was potently inhibited by ACT‐like protein I, with a Ki,app of 35 nM, but ACT‐like protein II was less effective. ACT‐like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT‐like proteins that differentially inhibit PTP. The colocalization of the ACT‐like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT‐like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited proenkephalin processing in adrenal medulla.