Processing of Proenkephalin-A in Bovine Chromaffin Cells

Goumon, Yannick; Lugardon, Karine; Gadroy, Patrice; Strub, Jean‐Marc; Welters, Ingeborg; Stefano, George B.; Aunis, Dominique; Metz‐Boutigue, Marie‐Hélène

doi:10.1074/jbc.m007557200

Cited by 30 publications

(10 citation statements)

References 71 publications

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“…52) and are co-stored with numerous proteolytic enzymes (4, 53). The chromaffin granule, which has been used as a model for the characterization of proteases and enzymes involved in neuropeptide maturation (53), contains the subtilisin-like prohormone-convertase family, prohormone-thiol-protease (cysteine protease), adrenorphin-Gly-generating enzyme, proopiomelanocortin-converting enzyme (or 70-kDa aspartic acid protease (54)) and serine proteases with trypsin-like activity (4,(55)(56)(57)(58). More recently, Parmer and colleagues (59) have shown that the plasmin-plasminogen system, which is present in the intragranular chromaffin matrix, seems to be implicated in chromogranin maturation.…”

Section: Discussionmentioning

confidence: 99%

“…These proteins are actively processed in the intragranular matrix to peptides with various molecular weight (1)(2)(3). Recently, using highly sensitive proteomic techniques we have characterized the maturation products of proenkephalin-A (4) and established the presence of other unexpected proteins (5).…”

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“…Several reports have also described the presence of PEBP immunoreactivity in biological fluids, including rat haploid testicular germ cell secretions (12), testicular interstitial fluid (13), the culture media of transfected Rat-1 fibroblasts (14), and more recently in the conditioned medium from adult rat hippocampal progenitors (15). PEBP and its N-terminal-derived peptide named hippocampal cholinergic neurostimulating peptide (HCNP), corresponding to the first eleven N-terminal amino acid residues (PEBP [1][2][3][4][5][6][7][8][9][10][11] ), were also found in synaptic vesicles after subcellular fractionation and were detected by immunohistochemistry in nerve cell terminals of neurons in rat brain (10) and small intestine (16). Interestingly, HCNP has also been detected in the cerebrospinal fluid of Alzheimer patients (17) and shown to be released from rat hippocampal neurons (10), as well as by murine adipocytes (18).…”

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The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Goumon

Angelone

Schoentgen

et al. 2004

Journal of Biological Chemistry

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Phosphatidylethanolamine-binding protein (PEBP), alternatively named Raf-1 kinase inhibitor protein, is the precursor of the hippocampal cholinergic neurostimulating peptide (HCNP) corresponding to its natural N-terminal fragment, previously described to be released by hippocampal neurons. PEBP is a soluble cytoplasmic protein, also associated with plasma and reticulum membranes of numerous cell types. In the present report, using biochemistry and cell biology techniques, we report for the first time the presence of PEBP in bovine chromaffin cell, a well described secretion model. We have examined its presence at the subcellular level and characterized this protein on both secretory granule membranes and intragranular matrix. In addition, its presence in bovine chromaffin cell and platelet exocytotic medium, as well as in serum, was reported showing that it is secreted. Like many other proteins that lack signal sequence, PEBP may be secreted through non-classic signal secretory mechanisms, which could be due to interactions with granule membrane lipids and lipid rafts. By two-dimensional liquid chromatography-tandem mass spectrometry, HCNP was detected among the intragranular matrix components. The observation that PEBP and HCNP were secreted with catecholamines into the circulation prompted us to investigate endocrine effects of this peptide on cardiovascular system. By using as bioassay an isolated and perfused frog (Rana esculenta) heart preparation, we show here that HCNP acts on the cardiac mechanical performance exerting a negative inotropism and counteracting the adrenergic stimulation of isoproterenol. All together, these data suggest that PEBP and HCNP might be considered as new endocrine factors involved in cardiac physiology.Chromaffin cells are derived from the neural crest and constitute the adrenal medulla. In the adrenal medullary chromaffin cell, secretory granules contain a complex mixture of proteins and peptides that are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation (1) . Among the high molecular mass water-soluble proteins, proenkephalin-A and the chromogranins family constitute the major constituents of chromaffin granules with other neuropeptides (neuropeptide Y, vasointestinal peptide, and others). These proteins are actively processed in the intragranular matrix to peptides with various molecular weight (1-3). Recently, using highly sensitive proteomic techniques we have characterized the maturation products of proenkephalin-A (4) and established the presence of other unexpected proteins (5).Phosphatidylethanolamine-binding protein (PEBP) 1 (6, 7)/ Raf-1 kinase inhibitor protein (8) has previously been described in brain and adrenal gland (9, 10). PEBP is a 21-kDa protein initially described as a cytoplasmic protein and later found to be associated with plasma or reticulum membranes (11). This protein has been found in numerous tissues of many species, including rat testis, liver, kidney, and human platelets (6, 9, 10), indicating its ubi...

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Section: Discussionmentioning

confidence: 99%

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The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

Goumon

Angelone

Schoentgen

et al. 2004

Journal of Biological Chemistry

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“…The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5,6), with relevant prohormone convertases for converting PE into active enkephalin.…”

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

Yasothornsrikul

Greenbaum

Medzihradszky

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

198

223

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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 T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1-4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5, 6), with relevant prohormone convertases for converting PE into active enkephalin.The primary PE-cleaving activity in chromaffin granules has been characterized as a cysteine protease complex known as ''prohormone thiol protease'' (PTP) (7-10). 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).In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14, 15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7-10). Cathepsin L generated the active peptide ME by cleaving enkephalin-containing peptide substrates at native dibasic and monobasic sites. Notably, cathepsin L colocalized with ME in the regulated secretory pathway of chromaffin cells. In cathepsin L gene knockout (KO) mice (16-1...

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“…3) (23), it is likely that the PEBP⅐M6G complex exists after secretion and is present in the blood. A large variety of proteolytic enzymes are present in chromaffin secretory granules and act to process precursor proteins such as PEA and chromogranins/secretogranins (36). In contrast, intragranular PEBP is highly resistant to proteolytic degradation (23).…”

Section: Discussionmentioning

confidence: 99%

Identification of Morphine-6-glucuronide in Chromaffin Cell Secretory Granules

Goumon

Muller²,

Glattard³

et al. 2006

Journal of Biological Chemistry

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We report for the first time that morphine-6-glucuronide, a highly analgesic morphine-derived molecule, is present in adrenal chromaffin granules and secreted from chromaffin cells upon stimulation. We also demonstrate that phosphatidylethanolamine-binding protein (alternatively named Raf-1 kinase inhibitor protein or RKIP) acts as an endogenous morphine-6-glucuronide-binding protein. An UDPglucuronosyltransferase 2B-like enzyme, described to transform morphine into morphine-6-glucuronide, has been immunodetected in the chromaffin granule matrix, and morphine-6-glucuronide de novo synthesis has been characterized, demonstrating the possible involvement of intragranular UDP-glucuronosyltransferase 2B-like enzyme in morphine-6-glucuronide metabolism. Once secreted into the circulation, morphine-6-glucuronide may mediate several systemic actions (e.g. on immune cells) based on its affinity for -opioid receptors. These activities could be facilitated by phosphatidylethanolamine-binding protein (PEBP), acting as a molecular shield and preventing morphine-6-glucuronide from rapid clearance. Taken together, our data represent an important observation on the role of morphine-6-glucuronide as a new endocrine factor.For 20 years, cerebral endogenous morphine has been isolated and characterized, and its structure has been shown to be identical to morphine from poppy (1, 2). In the 1990s, few groups succeeded in characterizing endogenous morphine from the organs and fluids of vertebrates, including bovine brain and adrenal gland, rat heart and adrenal gland, and human heart and urine (for review, Refs. 1-3); invertebrates (4 -6); and parasites (7,8). In mammals, the endogenous morphine synthetic pathway is associated with the dopamine and catecholamine biosynthetic pathway (for review, see Refs. 1, 3, and 9). Very recently, several crucial steps were reached when Zenk and co-workers (10, 11) demonstrated that morphine can be formed using a multistep biosynthetic route, and Zhu et al. (12) showed that human primary polymorphonuclear cells are able to synthesize morphine. These authors have shown morphine de novo synthesis in human and animal primary and cancer cell cultures.Chromaffin cells are neuroendocrine cells originating from the neural crest and are the predominant cell type in the adrenal medulla (for review, see Ref. 13). These cells possess the catecholamine biosynthetic pathway, leading to dopamine and adrenaline/noradrenaline synthesis (13). Chromaffin secretory granules contain a complex mixture of peptides and proteins that are co-released with catecholamines into the circulation in response to splanchnic nerve stimulation (13). Based on the morphine biosynthetic pathway and on the presence of morphine in bovine and rat adrenal glands (14, 15), rat pheochromocytoma PC-12 cells (10, 16), and eel chromaffin cells (17), we hypothesized that mammalian chromaffin cells might have the capacity to synthesize morphine and that their secretory granules could potentially release this alkaloid into the blood.Several mole...

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Processing of Proenkephalin-A in Bovine Chromaffin Cells

Cited by 30 publications

References 71 publications

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

The Hippocampal Cholinergic Neurostimulating Peptide, the N-terminal Fragment of the Secreted Phosphatidylethanolamine-binding Protein, Possesses a New Biological Activity on Cardiac Physiology

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

Identification of Morphine-6-glucuronide in Chromaffin Cell Secretory Granules

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