Purification and Characteristics of the Candidate Prohormone Processing Proteases PC2 and PC1/3 from Bovine Adrenal Medulla Chromaffin Granules

Azaryan, Anahit V.; Krieger, Timothy J.; Hook, Vivian

doi:10.1074/jbc.270.14.8201

Cited by 55 publications

(53 citation statements)

References 58 publications

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“…Notably, cleavage site studies with peptide-MCA substrates containing dibasic residue-processing sites indicated that cathepsin L prefers to cleave at the N-terminal side of dibasic residues as well as between the dibasic residues (28). In contrast, similar evaluation of PC1/3 and PC2 with dipeptide-MCA substrates showed that these PC proteases prefer to cleave at the C-terminal side of dibasic residue-processing sites (29). These data indicate that, following cathepsin L cleavage, removal of the remaining basic residue extensions at the N terminus of peptide products can be accomplished by Arg/Lys aminopeptidase activity.…”

Section: Discussionmentioning

confidence: 63%

Major Role of Cathepsin L for Producing the Peptide Hormones ACTH, β-Endorphin, and α-MSH, Illustrated by Protease Gene Knockout and Expression

Funkelstein

Toneff

Mosier

et al. 2008

Journal of Biological Chemistry

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The pituitary hormones adrenocorticotropic hormone (ACTH), ␤-endorphin, and ␣-melanocyte stimulating hormone (␣-MSH) are synthesized by proteolytic processing of their common proopiomelanocortin (POMC) precursor. Key findings from this study show that cathepsin L functions as a major proteolytic enzyme for the production of POMC-derived peptide hormones in secretory vesicles. Specifically, cathepsin L knockout mice showed major decreases in ACTH, ␤-endorphin, and ␣-MSH that were reduced to 23, 18, and 7% of wild-type controls (100%) in pituitary. These decreased peptide levels were accompanied by increased levels of POMC consistent with proteolysis of POMC by cathepsin L. The peptide hormones ACTH 2 , ␤-endorphin, and ␣-MSH are produced by proteolytic processing of their common proopiomelanocortin (POMC) prohormone precursor (1, 2). The mature peptide hormones are stored in pituitary secretory vesicles for regulated secretion and control of physiological functions in target organs. ACTH regulates the production of glucocorticoids in the adrenal cortex for control of metabolism (3, 4). ␣-MSH is implicated in the regulation of appetite and the production of melanin (5, 6). ␤-Endorphin is an endogenous opioid peptide involved in pain regulation (7,8). The proteolytic mechanisms that generate these functionally distinct peptide hormones from POMC are essential for these hormones to exert their biological functions.The role of cysteine protease activity for POMC processing has been implicated by several studies (9 -11), but the identity of the protease has not yet been achieved. In early studies of POMC processing activity in pituitary secretory vesicles, cysteine protease activity represented a significant portion of POMC cleaving activity, based on its inhibition by the thiol reagent p-chloromercuribenzoate (9). In anterior and intermediate pituitary cells in culture, treatment of cells with the cysteine protease inhibitor E64d reduced cell levels of POMC-derived ACTH, ␤-endorphin, and ␣-MSH (10, 11). Thus, it is likely that a cysteine protease participates in POMC processing. Participation of a cysteine protease in POMC processing would represent a new protease pathway, in addition to the subtilisinlike prohormone convertase enzymes (PC2 and PC1/3) that participate in processing POMC (12)(13)(14)(15). Therefore, the goal of this study was to identify the cysteine protease that produces ACTH, ␤-endorphin, and ␣-MSH peptide hormones derived from POMC.Herein we provide evidence indicating a key role for the cysteine protease cathepsin L in the biosynthesis of ACTH, ␤-endorphin, and ␣-MSH in secretory vesicles. Cathepsin L gene knock-out mice showed major reductions in pituitary tissue levels of ACTH, ␤-endorphin, and ␣-MSH. Furthermore, cathepsin L KO mouse pituitaries displayed accumulation of POMC, consistent with proteolysis of POMC by cathepsin L. In vivo cellular localization of cathepsin L in pituitary cells demonstrated a high degree of colocalization with ␤-endorphin, ␣-MSH, and ACTH in secretory vesicles. Ex...

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Section: Discussionmentioning

confidence: 63%

Major Role of Cathepsin L for Producing the Peptide Hormones ACTH, β-Endorphin, and α-MSH, Illustrated by Protease Gene Knockout and Expression

Funkelstein

Toneff

Mosier

et al. 2008

Journal of Biological Chemistry

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“…Endogenous CgA may act differently in the various tissues, depending on the presence or absence of different proteolytic mechanisms. PC1 and PC2 prohormone convertases and cathepsin L have been identified as cleavage enzymes in chromaffin vesicles (4,5); specifically, cathepsin L acts at CgA cleavage sites to synthesize Cts.…”

Section: )mentioning

confidence: 99%

Catestatin in rat RVLM is sympathoexcitatory, increases barosensitivity, and attenuates chemosensitivity and the somatosympathetic reflex

Gaede

Pilowsky

2010

American Journal of Physiology-Regulatory, Integrative and Comp

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Gaede AH, Pilowsky PM. Catestatin in rat RVLM is sympathoexcitatory, increases barosensitivity, and attenuates chemosensitivity and the somatosympathetic reflex. Am J Physiol Regul Integr Comp Physiol 299: R1538 -R1545, 2010. First published October 6, 2010 doi:10.1152/ajpregu.00335.2010.-The fundamental role and corollary effects of neuropeptides that govern cardiorespiratory control in the brain stem are poorly understood. One such regulatory peptide, catestatin [Cts,], noncompetitively inhibits nicotinic-cholinergic-stimulated catecholamine release. Previously, we demonstrated the presence of chromogranin A mRNA in brain stem neurons that are important for the maintenance of arterial pressure. In the present study, using immunofluorescence histochemistry, we show that Cts immunoreactivity is colocalized with tyrosine hydroxylase in C1 neurons of the rostral ventrolateral medulla (RVLM, n ϭ 3). Furthermore, we investigated the effects of Cts on resting blood pressure, splanchnic sympathetic nerve activity, phrenic nerve activity, heart rate, and adaptive reflexes. Cts (1 mM in 50 nl or 100 M in 50 -100 nl) was microinjected into the RVLM in urethaneanesthetized, vagotomized, ventilated Sprague-Dawley rats (n ϭ 19). Cardiovascular responses to stimulation of carotid baroreceptors, peripheral chemoreceptors, and the sciatic nerve (somatosympathetic reflex) were analyzed. Cts (1 mM in 50 nl) increased resting arterial pressure (28 Ϯ 3 mmHg at 2 min postinjection), sympathetic nerve activity (15 Ϯ 3% at 2 min postinjection), and phrenic discharge amplitude (31 Ϯ 4% at 10 min postinjection). Cts increased sympathetic barosensitivity 40% (slope increased from Ϫ0.05 Ϯ 0.01 before Cts to Ϫ0.07 Ϯ 0.01 after Cts) and attenuated the somatosympathetic reflex [1st peak: 36% (from 132 Ϯ 32.1 to 84.0 Ϯ 17.0 V); 2nd peak: 44% (from 65.1 Ϯ 21.4 to 36.6 Ϯ 14.1 V)] and chemoreflex (blood pressure response to anoxia decreased 55%, sympathetic response decreased 46%). The results suggest that Cts activates sympathoexcitatory bulbospinal neurons in the RVLM and plays an important regulatory role in adaptive reflexes. baroreflex; hypoxic chemoreflex; Sprague-Dawley rat; chromogranin A THE CATECHOLAMINE release-inhibitory cleavage product of chromogranin A (CgA) was identified and named catestatin (Cts) in 1997 (23). Cts inhibits ACh-and nicotine-induced catecholamine secretion in cell culture (21, 23), and studies in humans suggest that it may play an important role in hypertension (38).Cts is unusual in its ability to function as an antagonist at neuronal nicotinic ACh receptors (nAChRs) and, possibly, other receptors, such as ␤-adrenoceptors and histamine H 1 receptors (8,14,25), as opposed to directly activating specific "Cts" receptors, as seen with other neurotransmitters and neuropeptides. Here we investigate the distribution of Cts in catecholamine-containing neurons of the brain stem and the physiological effects of Cts following microinjection into the rostral ventrolateral medulla (RVLM). The RVLM comprises a heterogeneo...

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“…These chromaffin granules were utilized to identify the primary proenkephalincleaving prohormone processing activity as cathepsin L using biochemistry and chemical biology approaches, as well as for studies of native in vivo prohormone convertases (38)(39)(40)(41).…”

Section: Cysteine and Serine Protease Pathways For Processing Proneurmentioning

confidence: 99%

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

Hook

Funkelstein

et al. 2008

Annu. Rev. Pharmacol. Toxicol.

220

304

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Summary Points List:1. Proteases are essential for proteolytic processing of proneuropeptide precursors into active peptide neurotransmitters and hormones.2. Secretory vesicles represent the primary subcellular site of neuropeptide biosynthesis, which are produced, stored, and secreted to mediate cell-cell communication. 3.Protease pathways for proneuropeptide processing have been elucidated consisting of (a) the newly identified cysteine protease cathepsin L with aminopeptidase B in secretory vesicles, and (b) the well-established, proprotein convertase family that include the neuroendocrine-specific prohormone convertases 1 and 2 (PC1/3 and PC2) with carboxypeptidase E. 4.Protease gene knockout experiments have validated the roles of PC1/3, PC2, as well as cathepsin L for the production of neuropeptides in nervous and endocrine tissues. 5.Endogenous regulators consisting of inhibitors and activators participate in the in vivo control of processing enzyme functions.6. Structural biology of protease and proneuropeptides will be important to understand interacting mechanisms for proneuropeptide processing. 7.Neuropeptidomics has recently been applied to investigations of neuropeptide systems for their primary sequence and structural identification, as well as quantitation by LC-MS/MS tandem mass spectrometry. 8.Proteomic studies have revealed functional protein families that participate in secretory vesicle functions for the production, storage, and secretion of neuropeptides.9. Pharmacological evaluation of unique specificities among neuropeptide processing systems will be valuable for design of future strategies to develop selective small molecule modulators of processing enzymes for therapeutic applications in health and disease.Future Issues: Areas of Neuropeptide Research for Exploration. 1.How are cathepsin L and prohormone convertase protease pathways coordinately regulated? 2.What is the proteolytic basis for tissue-specific processing of proneuropeptides, such as that for POMC?3. Selective and potent inhibitors of protease components for processing prohormones should be developed to facilitate basic and pharmacological research. 4.What are the structural features of prohormone and protease interactions for functional processing? Peptide neurotransmitters and peptide hormones, collectively known as neuropeptides, are required for cell-cell communication in neurotransmission and for regulation of endocrine functions. Neuropeptides are synthesized from protein precursors (termed proneuropeptides or prohormones) that require proteolytic processing primarily within secretory vesicles that store and secrete the mature neuropeptides to control target cellular and organ systems. This review describes interdisciplinary strategies that have elucidated two primary protease pathways for prohormone processing consisting of the cysteine protease pathway mediated by secretory vesicle cathepsin L and the well-known subtilisin-like proprotein convertase pathway that together support neuropeptide biosynthesis. Importantly...

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Purification and Characteristics of the Candidate Prohormone Processing Proteases PC2 and PC1/3 from Bovine Adrenal Medulla Chromaffin Granules

Cited by 55 publications

References 58 publications

Major Role of Cathepsin L for Producing the Peptide Hormones ACTH, β-Endorphin, and α-MSH, Illustrated by Protease Gene Knockout and Expression

Major Role of Cathepsin L for Producing the Peptide Hormones ACTH, β-Endorphin, and α-MSH, Illustrated by Protease Gene Knockout and Expression

Catestatin in rat RVLM is sympathoexcitatory, increases barosensitivity, and attenuates chemosensitivity and the somatosympathetic reflex

Proteases for Processing Proneuropeptides into Peptide Neurotransmitters and Hormones

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