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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.
D. Shields - One of the best experts on this subject based on the ideXlab platform.
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Prohormone processing in permeabilized cells: Endoproteolytic cleavage of prosomatostatin in the trans-Golgi network
Biochimie, 1994Co-Authors: D. ShieldsAbstract:Abstract Many peptide hormones are synthesized as larger precursors which undergo endoproteolytic cleavage at paired basic residues to generate a bioactive molecule. Morphological evidence has implicated either the trans-Golgi network (TGN) or immature secretory granules as the site of prohormone cleavage. To identify the site where Prehormone cleavage is initiated, we have used retrovirally infected rat anterior pituitary GH3 cells which express high levels of prosomatostatin, proSRIF, (Stoller TJ, Shields D (1988) J Cell Biol 107, 2087–2095). By incubating these cells at 20°C, a temperature that prevents exit from the Golgi apparatus, proSRIF accumulated quantitatively in the TGN and no proteolytic processing was evident. Following the 20°C block, the cells were permeabilized and proSRIF processing determined. Cleavage of proSRIF to the mature hormone was approximately 35–50% efficient, required incubation at 37°C and ATP hydrolysis, but was independent of GTP or cytosol. The in vitro ATP-dependent proSRIF processing was inhibited by inclusion of chloroquine, a weak base, CCCP, a protonophore, or by pre-incubating the permeabilized cells with low concentrations of N-ethylmaleimide or bafilomycin, both inhibitors of vacuolar-type ATP-dependent proton pumps. These data suggest that ATP is required for generation of an acidic pH in the lumen of the TGN which is necessary for the activity of prohormone processing enzymes. By exploiting a permeabilized cell system, we have demonstrated that proSRIF cleavage is initiated in the TGN, in a reaction which is facilitated by a Golgi-associated vacuolar type ATPase.
Jonathan V. Sweedler - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the prohormone complement in Amphiprion and related fish species integrating genome and transcriptome assemblies.
PloS one, 2020Co-Authors: Bruce R. Southey, Sandra L. Rodriguez-zas, Justin S. Rhodes, Jonathan V. SweedlerAbstract:The Amphiprion (anemonefish or clownfish) family of teleost fish, which is not a common model species, exhibits multiple unique characteristics, including social control of body size and protandrous sex change. The social changes in sex and body size are modulated by neuropeptide signaling pathways. These neuropeptides are formed from complex processing from larger prohormone proteins; understanding the neuropeptide complement requires information on complete prohormones sequences. Genome and transcriptome information within and across 22 teleost fish species, including 11 Amphiprion species, were assembled and integrated to achieve the first comprehensive survey of their prohormone genes. This information enabled the identification of 175 prohormone isoforms from 159 prohormone proteins across all species. This included identification of 9 CART prepropeptide genes and the loss of insulin-like 5B and tachykinin precursor 1B genes in Pomacentridae species. Transcriptome assemblies generally detected most prohormone genes but provided fewer prohormone genes than genome assemblies due to the lack of expression of prohormone genes or specific isoforms and tissue sampled. Comparisons between duplicate genes indicated that subfunctionalization, degradation, and neofunctionalization may be occurring between all copies. Characterization of the prohormone complement lays the foundation for future peptidomic investigation of the molecular basis of social physiology and behavior in the teleost fish.
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Identification of prohormones and pituitary neuropeptides in the African cichlid, Astatotilapia burtoni
BMC Genomics, 2016Co-Authors: Bruce R. Southey, Jonathan V. Sweedler, Karen P. Maruska, Elena V. Romanova, Russell D. FernaldAbstract:Background Cichlid fishes have evolved remarkably diverse reproductive, social, and feeding behaviors. Cell-to-cell signaling molecules, notably neuropeptides and peptide hormones, are known to regulate these behaviors across vertebrates. This class of signaling molecules derives from prohormone genes that have undergone multiple duplications and losses in fishes. Whether and how subfunctionalization, neofunctionalization, or losses of neuropeptides and peptide hormones have contributed to fish behavioral diversity is largely unknown. Information on fish prohormones has been limited and is complicated by the whole genome duplication of the teleost ancestor. We combined bioinformatics, mass spectrometry-enabled peptidomics, and molecular techniques to identify the suite of neuropeptide prohormones and pituitary peptide products in Astatotilapia burtoni , a well-studied member of the diverse African cichlid clade. Results Utilizing the A. burtoni genome, we identified 148 prohormone genes, with 21 identified as a single copy and 39 with at least 2 duplicated copies. Retention of prohormone duplicates was therefore 41 %, which is markedly above previous reports for the genome-wide average in teleosts. Beyond the expected whole genome duplication, differences between cichlids and mammals can be attributed to gene loss in tetrapods and additional duplication after divergence. Mass spectrometric analysis of the pituitary identified 620 unique peptide sequences that were matched to 120 unique proteins. Finally, we used in situ hybridization to localize the expression of galanin, a prohormone with exceptional sequence divergence in cichlids, as well as the expression of a proopiomelanocortin, prohormone that has undergone an additional duplication in some bony fish lineages. Conclusion We characterized the A. burtoni prohormone complement. Two thirds of prohormone families contain duplications either from the teleost whole genome duplication or a more recent duplication. Our bioinformatic and mass spectrometric findings provide information on a major vertebrate clade that will further our understanding of the functional ramifications of these prohormone losses, duplications, and sequence changes across vertebrate evolution. In the context of the cichlid radiation, these findings will also facilitate the exploration of neuropeptide and peptide hormone function in behavioral diversity both within A. burtoni and across cichlid and other fish species.
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First survey and functional annotation of prohormone and convertase genes in the pig
BMC genomics, 2012Co-Authors: Kenneth I. Porter, Jonathan V. Sweedler, Bruce R. Southey, Sandra L. Rodriguez-zasAbstract:Background The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted.
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First survey and functional annotation of prohormone and convertase genes in the pig
BMC Genomics, 2012Co-Authors: Kenneth I. Porter, Jonathan V. Sweedler, Bruce R. Southey, Sandra L. Rodriguez-zasAbstract:Background The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted. Results We identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin , cortistatin , insulin-like 5 , orexigenic neuropeptide QRFP , prokineticin 2 , prolactin-releasing peptide , parathyroid hormone 2 , urocortin , urocortin 2 , urocortin 3 , and urotensin 2-related peptide . In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the production of the bioactive neuropeptides urocortin 2 , urocortin 3 , torsin family 2 member A , tachykinin 4 , islet amyloid polypeptide , and calcitonin receptor-stimulating peptide 2 in the pig. Conclusions The present genomic and functional characterization supports the use of the pig as an effective animal model to gain a deeper understanding of prohormones, prohormone convertases and neuropeptides in biomedical and agricultural research.
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Genome-wide census and expression profiling of chicken neuropeptide and prohormone convertase genes
Neuropeptides, 2009Co-Authors: Kristin Delfino, Jonathan V. Sweedler, Bruce R. Southey, Sandra L. Rodriguez-zasAbstract:Neuropeptides regulate cell-cell signaling and influence many biological processes in vertebrates, including development, growth, and reproduction. The complex processing of neuropeptides from prohormone proteins by prohormone convertases, combined with the evolutionary distance between the chicken and mammalian species that have experienced extensive neuropeptide research, has led to the empirical confirmation of only 18 chicken prohormone proteins. To expand our knowledge of the neuropeptide and prohormone convertase gene complement, we performed an exhaustive survey of the chicken genomic, EST, and proteomic databases using a list of 95 neuropeptide and 7 prohormone convertase genes known in other species. Analysis of the EST resources and 22 microarray studies offered a comprehensive portrait of gene expression across multiple conditions. Five neuropeptide genes (apelin, cocaine-and amphetamine-regulated transcript protein, insulin-like 5, neuropeptide S, and neuropeptide B) previously unknown in chicken were identified and 62 genes were confirmed. Although most neuropeptide gene families known in human are present in chicken, there are several gene not present in the chicken. Conversely, several chicken neuropeptide genes are absent from mammalian species, including C-RF amide, c-type natriuretic peptide 1 precursor, and renal natriuretic peptide. The prohormone convertases, with one exception, were found in the chicken genome. Bioinformatic models used to predict prohormone cleavages confirm that the processing of prohormone proteins into neuropeptides is similar between species. Neuropeptide genes are most frequently expressed in the brain and head, followed by the ovary and small intestine. Microarray analyses revealed that the expression of adrenomedullin, chromogranin-A, augurin, neuromedin-U, platelet-derived growth factor A and D, proenkephalin, relaxin-3, prepronociceptin, and insulin-like growth factor I was most susceptible (P-value
Richard H. Scheller - One of the best experts on this subject based on the ideXlab platform.
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Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.
The Journal of cell biology, 1993Co-Authors: Linda J. Jung, T Kreiner, Richard H. SchellerAbstract:Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.
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Prohormone structure governs proteolytic processing and sorting in the Golgi complex.
Recent progress in hormone research, 1993Co-Authors: Linda J. Jung, Thane Kreiner, Richard H. SchellerAbstract:Publisher Summary Prohormone structure governs proteolytic processing and sorting in the Golgi complex. In the trans-Golgi network (TGN), neuropeptides are segregated from other secretory proteins and are packaged into dense-core vesicles (DCVs). These vesicles are then transported to the nerve terminal and their contents are released by exocytosis when the appropriate signal is received by the neuron. Processing begins with the removal of the signal sequence as the nascent peptide enters the endoplasmic reticulum . Hence, elucidating the molecular and cellular mechanisms governing these processing events is the key to fully understanding the regulation of neuropeptide biosynthesis. This chapter discusses (1) the structural features of the prohormone important for processing, (2) the processing enzymes, and (3) how the processing sequence of the egg-laying hormone (ELH) prohormone influences the sorting of peptides derived from this molecule. Using the ELH prohormone to study the relationship between processing and sorting has opened up new avenues of thought. In the past, it was learned that cellular mechanisms can control the relative levels and spatial localization of different neuropeptides derived from a common precursor. However, recent results indicate that perturbation of a processing site can cause processing in a different locale in the cell and result in altered pathways of sorting. Defining the hierarchy of processing enzymes and their localization will be crucial to future work in this area and to understand how the design of prohormones complements the repertoire of processing enzymes.