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Vivian Hook - One of the best experts on this subject based on the ideXlab platform.
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proteases for processing proneuropeptides into peptide neurotransmitters and hormones
Annual Review of Pharmacology and Toxicology, 2008Co-Authors: Vivian Hook, Lydiane Funkelstein, Douglas Lu, Steven J Bark, Jill L Wegrzyn, Shinrong HwangAbstract: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, this review discusses important areas of current and future biomedical neuropeptide research with respect to biological regulation, inhibitors, structural features of proneuropeptide and protease interactions, and peptidomics co...
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Cathepsin L and Arg/Lys aminopeptidase: a distinct Prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones
Biological Chemistry, 2004Co-Authors: Vivian Hook, Sukkid Yasothornsrikul, Thomas Toneff, Doron C. Greenbaum, Katalin F. Medzihradszky, Katie Troutner, Richard Bundey, Anna Logrinova, Thomas Reinheckel, Christoph PetersAbstract: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 NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-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 subtilisin-like 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.
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arginine and lysine aminopeptidase activities in chromaffin granules of bovine adrenal medulla relevance to Prohormone processing
Journal of Neurochemistry, 2002Co-Authors: Sukkid Yasothornsrikul, Shinrong Hwang, Thomas Toneff, Vivian HookAbstract: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.
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High-level expression of the Prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro Prohormone processing.
Protein expression and purification, 1997Co-Authors: Vivian Hook, Anahit V Azaryan, Martin R Schiller, Kim Moran, Rama Kannan, Andrea B. Kohn, Mark O. Lively, Kurt W. MillerAbstract:Prohormone substrates are required for investigation of the proteolytic processing of Prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of Prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of Prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the Prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant Prohormones in Escherichia coli. High-level expression of milligrams of Prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "Prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, Prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different Prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for Prohormone substrates illustrates the importance of expressing recombinant Prohormones for in vitro processing studies.
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the processing proteases Prohormone thiol protease pc1 3 and pc2 and 70 kda aspartic proteinase show preferences among proenkephalin proneuropeptide y and proopiomelanocortin substrates
Archives of Biochemistry and Biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate Prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease Prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for Prohormone substrates. The recombinant Prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular Prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the Prohormones tested. Importantly, these results indicate that Prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular Prohormone.
J. P. H. Burbach - One of the best experts on this subject based on the ideXlab platform.
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The Hormone Domain of the Vasopressin Prohormone is Required for the Correct Prohormone Trafficking Through the Secretory Pathway.
Journal of neuroendocrinology, 2003Co-Authors: F M De Bree, M. Nijenhuis, R. Zalm, A. A. M. Van Der Kleij, David Murphy, J. P. H. BurbachAbstract:It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same Prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr 2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the Prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct Prohormone folding in the endoplasmic reticulum.
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A diabetes insipidus vasopressin Prohormone altered outside the central core of neurophysin accumulates in the endoplasmic reticulum
Molecular and Cellular Endocrinology, 2000Co-Authors: M. Nijenhuis, R. Zalm, J. P. H. BurbachAbstract:Abstract Over 20 mutations affecting the neurophysin moiety of the vasopressin Prohormone, have been identified in families suffering from familial neurohypophysial diabetes insipidus (FNDI). Only one of these, NP87E→stop, is located outside the central conserved domain implicated in sorting of the vasopressin Prohormone. To obtain clues about the mechanism of induction of FNDI by this atypical mutant we stably expressed wild type and NP87E→stop vasopressin Prohormones in (neuro)endocrine cell lines. Metabolic labeling and immunoprecipitation demonstrated reduced processing of the mutant Prohormone to neurophysin. In addition, evoked secretion of neurophysin and vasopressin was diminished, suggesting that part of the mutant is retained in another intracellular compartment than the secretory granules. Indeed, immunofluorescence demonstrated accumulation of the truncated vasopressin Prohormone in the endoplasmic reticulum. We conclude that the presence of the vasopressin moiety and the central conserved core of the neurophysin domain suffices for sorting and processing, but not for efficient endoplasmic reticulum exit of the vasopressin-neurophysin molecule.
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Mutations in the Vasopressin Prohormone Involved in Diabetes Insipidus Impair Endoplasmic Reticulum Export but Not Sorting
The Journal of biological chemistry, 1999Co-Authors: M. Nijenhuis, R. Zalm, J. P. H. BurbachAbstract:Abstract Familial neurohypophysial diabetes insipidus is characterized by vasopressin deficiency caused by heterozygous expression of a mutated vasopressin Prohormone gene. To elucidate the mechanism of this disease, we stably expressed five vasopressin Prohormones with a mutation in the neurophysin moiety (NP14G→R, NP47E→G, NP47ΔE, NP57G→S, and NP65G→V) in the neuroendocrine cell lines Neuro-2A and PC12/PC2. Metabolic labeling demonstrated that processing and secretion of all five mutants was impaired, albeit to different extents (NP65G→V ≥ NP14G→R > NP47ΔE ≥ NP47E→G > NP57G→S). Persisting endoglycosidase H sensitivity revealed these defects to be due to retention of mutant Prohormone in the endoplasmic reticulum. Mutant Prohormones that partially passed the endoplasmic reticulum were normally targeted to the regulated secretory pathway. Surprisingly, this also included mutants with mutations in residues involved in binding of vasopressin to neurophysin, a process implicated in targeting of the Prohormone. To mimick the high expression in vasopressin-producing neurons, mutant vasopressin Prohormones were transiently expressed in Neuro-2A cells. Immunofluorescence displayed formation of large accumulations of mutant Prohormone in the endoplasmic reticulum, accompanied by redistribution of an endoplasmic reticulum marker. Our data suggest that prolonged perturbation of the endoplasmic reticulum eventually leads to degeneration of neurons expressing mutant vasopressin Prohormones, explaining the dominant nature of the disease.
F M De Bree - One of the best experts on this subject based on the ideXlab platform.
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The Hormone Domain of the Vasopressin Prohormone is Required for the Correct Prohormone Trafficking Through the Secretory Pathway.
Journal of neuroendocrinology, 2003Co-Authors: F M De Bree, M. Nijenhuis, R. Zalm, A. A. M. Van Der Kleij, David Murphy, J. P. H. BurbachAbstract:It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same Prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr 2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the Prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct Prohormone folding in the endoplasmic reticulum.
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Trafficking of the Vasopressin and Oxytocin Prohormone Through the Regulated Secretory Pathway
Journal of neuroendocrinology, 2001Co-Authors: F M De BreeAbstract:The trafficking of Prohormones and of regulated secretory proteins in general has been studied extensively in the last decades of the last century. Prohormone trafficking starts with correct folding and subsequently efficient sorting into the secretory granule of the regulated secretory pathway. The vasopressin/oxytocin Prohormone is particularly interesting for studying protein trafficking, because the physicochemical properties and three-dimensional structure have been largely elucidated. In the case of pro-vasopressin and pro-oxytocin, folding and sorting depend completely on both intramolecular and intermolecular interactions. Proper folding is guided by the hormone-neurophysin association and the sorting event relies on the aggregative properties of the neurophysin domain in the Prohormone, as well as a specific sorting signal, which is revealed when the aggregative property of the neurophysin domain is deleted. A comprehensive mechanism for trafficking of the vasopressin/oxytocin Prohormone from the endoplasmic reticulum to the secretory granule is proposed.
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Structure–Function Relationships of the Vasopressin Prohormone Domains
Cellular and Molecular Neurobiology, 1998Co-Authors: F M De Bree, J. Peter H. BurbachAbstract:1. In this review the structure–function relationships of the different vasopressin Prohormone domains are dated and discussed, with special reference to the neurophysin and glycopeptide domains. 2. The primary structures of the currently known neurophysins and glycopeptide sequences are compared and discussed. 3. The hormone-binding and aggregational properties of neurophysin are reviewed and related to a possible function within the regulated secretory pathway. 4. It is proposed, based on the properties reviewed here as well as our own data shown here, that the sorting of the vasopressin Prohormone is initiated by hormone binding, which triggers aggregation of the Prohormone into the characteristic dense cores of the regulated secretory pathway. 5. This may suggest that Prohormone sorting into the regulated secretory pathway is, in general, determined by noncovalent, intramolecular interactions that promote aggregation.
Anahit V Azaryan - One of the best experts on this subject based on the ideXlab platform.
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High-level expression of the Prohormones proenkephalin, pro-neuropeptide Y, proopiomelanocortin, and beta-protachykinin for in vitro Prohormone processing.
Protein expression and purification, 1997Co-Authors: Vivian Hook, Anahit V Azaryan, Martin R Schiller, Kim Moran, Rama Kannan, Andrea B. Kohn, Mark O. Lively, Kurt W. MillerAbstract:Prohormone substrates are required for investigation of the proteolytic processing of Prohormones and proproteins into active peptide hormones and neurotransmitters. However, the lack of Prohormone proteins has been a limiting factor in elucidating proteolytic mechanisms for conversion of Prohormones into active peptides. Therefore, in this study, cloned cDNAs encoding the Prohormones proenkephalin (PE), pro-neuropeptide Y (pro-NPY), pro-opiomelanocortin (POMC), and beta-protachykinin (beta-PT) were utilized to express recombinant Prohormones in Escherichia coli. High-level expression of milligrams of Prohormones was achieved with the pET3c expression vector utilizing the T7 promoter for production of PE, pro-NPY, and POMC, as demonstrated by SDS-PAGE gel electrophoresis, Western blots, and 35S-methionine labeling. In addition, beta-PT was expressed at high levels as fusion proteins with the maltose-binding protein and glutathione S-transferase by the pMAL-c and pGEX-2T expression vectors, respectively. Relative rates of processing by the established processing proteases "Prohormone thiol protease" (PTP), 70-kDa aspartyl protease, and PC1/ 3 and PC2 (PC, Prohormone convertase) were examined with purified PE, pro-NPY, and POMC. Distinct preferences of processing enzymes for different Prohormones was demonstrated. PTP preferred PE and pro-NPY substrates, whereas little processing of POMC was detected. In contrast, the 70-kDa aspartyl protease cleaved POMC more readily than pro-NPY or PE. However, PC1/3 and PC2 prefer POMC as substrate. Demonstration of selectivity of processing enzymes for Prohormone substrates illustrates the importance of expressing recombinant Prohormones for in vitro processing studies.
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the processing proteases Prohormone thiol protease pc1 3 and pc2 and 70 kda aspartic proteinase show preferences among proenkephalin proneuropeptide y and proopiomelanocortin substrates
Archives of Biochemistry and Biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate Prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease Prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for Prohormone substrates. The recombinant Prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular Prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the Prohormones tested. Importantly, these results indicate that Prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular Prohormone.
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The processing proteases Prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.
Archives of biochemistry and biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate Prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease Prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for Prohormone substrates. The recombinant Prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular Prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the Prohormones tested. Importantly, these results indicate that Prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular Prohormone.
M. Nijenhuis - One of the best experts on this subject based on the ideXlab platform.
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The Hormone Domain of the Vasopressin Prohormone is Required for the Correct Prohormone Trafficking Through the Secretory Pathway.
Journal of neuroendocrinology, 2003Co-Authors: F M De Bree, M. Nijenhuis, R. Zalm, A. A. M. Van Der Kleij, David Murphy, J. P. H. BurbachAbstract:It has long been known that under intracellular conditions vasopressin associates tightly to neurophysin, which is present in the same Prohormone. As the association has been suggested to play a role during hormone biosynthesis, its role was studied in a cellular context by expressing mutant vasopressin precursors in Neuro2A cells. Mutant vasopressin precursors, in which the association between the vasopressin and neurophysin domains was prevented either by deleting the vasopressin domain from the precursor or by substitution of the essential Tyr 2 residue in vasopressin for Gly, were neither processed nor targeted into secretory granules. Rather, both provasopressin mutants were retained in the endoplasmic reticulum. Our results demonstrate that the vasopressin domain is crucial for correct trafficking of the Prohormone through the secretory pathway, and suggest that vasopressin-neurophysin association provides correct Prohormone folding in the endoplasmic reticulum.
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A diabetes insipidus vasopressin Prohormone altered outside the central core of neurophysin accumulates in the endoplasmic reticulum
Molecular and Cellular Endocrinology, 2000Co-Authors: M. Nijenhuis, R. Zalm, J. P. H. BurbachAbstract:Abstract Over 20 mutations affecting the neurophysin moiety of the vasopressin Prohormone, have been identified in families suffering from familial neurohypophysial diabetes insipidus (FNDI). Only one of these, NP87E→stop, is located outside the central conserved domain implicated in sorting of the vasopressin Prohormone. To obtain clues about the mechanism of induction of FNDI by this atypical mutant we stably expressed wild type and NP87E→stop vasopressin Prohormones in (neuro)endocrine cell lines. Metabolic labeling and immunoprecipitation demonstrated reduced processing of the mutant Prohormone to neurophysin. In addition, evoked secretion of neurophysin and vasopressin was diminished, suggesting that part of the mutant is retained in another intracellular compartment than the secretory granules. Indeed, immunofluorescence demonstrated accumulation of the truncated vasopressin Prohormone in the endoplasmic reticulum. We conclude that the presence of the vasopressin moiety and the central conserved core of the neurophysin domain suffices for sorting and processing, but not for efficient endoplasmic reticulum exit of the vasopressin-neurophysin molecule.
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Mutations in the Vasopressin Prohormone Involved in Diabetes Insipidus Impair Endoplasmic Reticulum Export but Not Sorting
The Journal of biological chemistry, 1999Co-Authors: M. Nijenhuis, R. Zalm, J. P. H. BurbachAbstract:Abstract Familial neurohypophysial diabetes insipidus is characterized by vasopressin deficiency caused by heterozygous expression of a mutated vasopressin Prohormone gene. To elucidate the mechanism of this disease, we stably expressed five vasopressin Prohormones with a mutation in the neurophysin moiety (NP14G→R, NP47E→G, NP47ΔE, NP57G→S, and NP65G→V) in the neuroendocrine cell lines Neuro-2A and PC12/PC2. Metabolic labeling demonstrated that processing and secretion of all five mutants was impaired, albeit to different extents (NP65G→V ≥ NP14G→R > NP47ΔE ≥ NP47E→G > NP57G→S). Persisting endoglycosidase H sensitivity revealed these defects to be due to retention of mutant Prohormone in the endoplasmic reticulum. Mutant Prohormones that partially passed the endoplasmic reticulum were normally targeted to the regulated secretory pathway. Surprisingly, this also included mutants with mutations in residues involved in binding of vasopressin to neurophysin, a process implicated in targeting of the Prohormone. To mimick the high expression in vasopressin-producing neurons, mutant vasopressin Prohormones were transiently expressed in Neuro-2A cells. Immunofluorescence displayed formation of large accumulations of mutant Prohormone in the endoplasmic reticulum, accompanied by redistribution of an endoplasmic reticulum marker. Our data suggest that prolonged perturbation of the endoplasmic reticulum eventually leads to degeneration of neurons expressing mutant vasopressin Prohormones, explaining the dominant nature of the disease.
