The Experts below are selected from a list of 6342 Experts worldwide ranked by ideXlab platform
Gunnar Pejler - One of the best experts on this subject based on the ideXlab platform.
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Acidic pH is essential for maintaining mast cell Secretory Granule homeostasis
Cell Death & Disease, 2017Co-Authors: Gunnar Pejler, Jun Mei Hu Frisk, Daniel Sjöström, Aida Paivandy, Helena ÖhrvikAbstract:It has been recognized for a long time that the Secretory Granules of mast cells are acidic, but the functional importance of maintaining an acidic pH in the mast cell Granules is not fully understood. Here we addressed this issue by examining the effects of raising the pH of the mast cell Secretory Granules. Mast cells were incubated with bafilomycin A1, an inhibitor of the vacuolar-type ATPase proton pump. Supporting a role of vacuolar-type ATPase in mast cell Granule acidification, bafilomycin A1 treatment caused a robust increase in Granule pH. This was accompanied by marked effects on mast cell Granules, including swelling and acquisition of vacuole-like morphology. Moreover, bafilomycin A1 caused extensive, yet selective effects on the Granule content. These included aberrant processing of pro-carboxypeptidase A3 and a reduction in the level of intracellular histamine, the latter being accompanied by an increase in extracellular histamine. In contrast, the storage of β -hexosaminidase, a prototype lysosomal hydrolase known to be stored in mast cell Granules, was not affected by abrogation of Granule acidification. Moreover, bafilomycin A1 caused a reduction of tryptase enzymatic activity and appearance of tryptase degradation products. Tryptase inhibition prevented the formation of such degradation products, suggesting that the pH elevation causes tryptase to undergo autoproteolysis. Taken together, our findings reveal that mast cell Secretory Granule homeostasis is critically dependent on an acidic milieu.
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proteolytic histone modification by mast cell tryptase a serglycin proteoglycan dependent Secretory Granule protease
Journal of Biological Chemistry, 2014Co-Authors: Fabio R Melo, Beata Berentmaoz, Giuliano Zabucchi, Francesca Levischaffer, Francesca De Vita, Gunnar PejlerAbstract:A hallmark feature of mast cells is their high content of cytoplasmic Secretory Granules filled with various preformed compounds, including proteases of tryptase-, chymase-, and carboxypeptidase A3 type that are electrostatically bound to serglycin proteoglycan. Apart from participating in extracellular processes, serglycin proteoglycan and one of its associated proteases, tryptase, are known to regulate cell death by promoting apoptosis over necrosis. Here we sought to outline the underlying mechanism and identify core histones as primary proteolytic targets for the serglycin-tryptase axis. During the cell death process, tryptase was found to relocalize from Granules into the cytosol and nucleus, and it was found that the absence of tryptase was associated with a pronounced accumulation of core histones both in the cytosol and in the nucleus. Intriguingly, tryptase deficiency resulted in defective proteolytic modification of core histones even at baseline conditions, i.e. in the absence of cytotoxic agent, suggesting that tryptase has a homeostatic impact on nuclear events. Indeed, tryptase was found in the nucleus of viable cells and was shown to cleave core histones in their N-terminal tail. Moreover, it was shown that the absence of the serglycin-tryptase axis resulted in altered chromatin composition. Together, these findings implicate histone proteolysis through a Secretory Granule-derived serglycin-tryptase axis as a novel principle for histone modification, during both cell homeostasis and cell death.
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reduction with dithiothreitol causes serglycin specific defects in Secretory Granule integrity of bone marrow derived mast cells
Molecular Immunology, 2009Co-Authors: Tiago Braga, Magnus Åbrink, Maria Ringvall, Heidi Tveit, Gunnar PejlerAbstract:Mast cell Granule maturation and storage of Granule components has previously been shown to be critically dependent on serglycin (SG), a proteoglycan abundantly stored in mast cell Secretory Granules. The N-terminal portion of serglycin contains a conserved disulfide motif that is similar to motifs found in Secretory Granule compounds of neuroendocrine cells. Interference with such motifs of neuroendocrine cells with dithiothreitol (DTT) has previously been shown to cause cellular missorting. To investigate the implication for serglycin, serglycin(+/+) and serglycin(-/-) bone marrow derived mast cells (BMMCs) were treated with DTT followed by assessment of proteoglycan synthesis and Secretory Granule integrity. Treatment of serglycin(+/+) BMMCs with DTT almost completely abolished biosynthetic incorporation of (35)S-sulfate into proteoglycans, caused a dramatic reduction of granular staining with May Grunwald/Giemsa as well as disruption of Granule dense core formation as shown by transmission electron microscopy. In addition, the storage of carboxypeptidase A, a major Secretory Granule compound, was markedly reduced following DTT treatment. In contrast, none of these effects were seen after treatment of SG(-/-) BMMCs with DTT, indicating that they were serglycin-specific. Notably, DTT treated serglycin(+/+) BMMCs showed similar morphology as did the serglycin(-/-) BMMCs. DTT treatment affected neither the viability of the BMMCs nor the mRNA levels for serglycin or carboxypeptidase A. Together, these data indicate that DTT causes dramatic, serglycin-specific effects on mast cell Granule. These findings are thus in accordance with a role for the N-terminal disulfide motif in serglycin for regulation of mast cell Secretory Granule integrity.
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a role for serglycin proteoglycan in granular retention and processing of mast cell Secretory Granule components
FEBS Journal, 2006Co-Authors: Frida Henningsson, Magnus Åbrink, Sonja P Hergeth, Robert Cortelius, Gunnar PejlerAbstract:In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic Secretory Granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of Secretory Granules as well as the fate of the Secretory Granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional Secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell Granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into Secretory Granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into Secretory vesicles.
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Serglycin-deficient cytotoxic T lymphocytes display defective Secretory Granule maturation and granzyme B storage.
The Journal of biological chemistry, 2005Co-Authors: Mirjana Grujic, Gunnar Pejler, Tiago Braga, Agneta Lukinius, Maija-leena Eloranta, Stefan D. Knight, Magnus ÅbrinkAbstract:Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic Granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce Secretory Granule of high electron density, although Granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for Secretory Granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.
Peng Y Loh - One of the best experts on this subject based on the ideXlab platform.
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aquaporin 1 is important for maintaining Secretory Granule biogenesis in endocrine cells
Molecular Endocrinology, 2008Co-Authors: Irina Arnaoutova, Taeyoon Kim, Niamh X Cawley, Nimesh Patel, Trushar Rathod, Peng Y LohAbstract:Aquaporins (AQPs), a family of water channels expressed in epithelial cells, function to transport water in a bidirectional manner to facilitate transepithelial fluid absorption and secretion. Additionally, AQP1 and AQP5 are found in pancreatic zymogen Granules and synaptic vesicles and are involved in vesicle swelling and exocytosis in exocrine cells and neurons. Here, we show AQP1 is in dense-core Secretory Granule (DCSG) membranes of endocrine tissue: pituitary and adrenal medulla. The need for AQP1 in endocrine cell function was examined by stable transfection of AQP1 antisense RNA into AtT20 cells, a pituitary cell line, to down-regulate AQP1 expression. These AQP1-deficient cells showed more than 60% depletion of DCSGs and significantly decreased DCSG protein levels, including proopiomelanocotin/pro-ATCH and prohormone convertase 1/3, but not non-DCSG proteins. Pulse-chase studies revealed that whereas DCSG protein synthesis was unaffected, approximately 50% of the newly synthesized proopiomelanocortin was degraded within 1 h. Low levels of ACTH were released upon stimulation, indicating that the small number of DCSGs that were made in the presence of the residual AQP1 were functionally competent for exocytosis. Analysis of anterior pituitaries from AQP1 knockout mice showed reduced prohormone convertase 1/3, carboxypeptidase E, and ACTH levels compared to wild-type mice demonstrating that our results observed in AtT20 cells can be extended to the animal model. Thus, AQP1 is important for maintaining DCSG biogenesis and normal levels of hormone secretion in pituitary endocrine cells.
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abnormal sterols in cholesterol deficiency diseases cause Secretory Granule malformation and decreased membrane curvature
Journal of Cell Science, 2006Co-Authors: Marjorie C Gondrelewis, Horia I Petrache, Christopher A Wassif, Daniel Harries, Adrian Parsegian, Forbes D Porter, Peng Y LohAbstract:Cholesterol is an abundant lipid in eukaryotic membranes, implicated in numerous structural and functional capacities. Here, we have investigated the mechanism by which cholesterol affects Secretory Granule biogenesis in vivo using Dhcr7-/- and Sc5d-/- mouse models of the human diseases, Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis. These homozygous-recessive multiple-malformation disorders are characterized by the functional absence of one of the last two enzymes in the cholesterol biosynthetic pathway, resulting in the accumulation of precursors. Cholesterol-deficient mice exhibit a significant decrease in the numbers of Secretory Granules in the pancreas, pituitary and adrenal glands. Moreover, there was an increase in morphologically aberrant Granules in the exocrine pancreas of Dhcr7-/- acinar cells. Regulated Secretory pathway function was also severely diminished in these cells, but could be restored with exogenous cholesterol. Sterol precursors incorporated in artificial membranes resulted in decreased bending rigidity and intrinsic curvature compared with cholesterol, thus providing a cholesterol-mediated mechanism for normal Granule budding, and an explanation for Granule malformation in SLOS and lathosterolosis.
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protease nexin 1 promotes Secretory Granule biogenesis by preventing Granule protein degradation
Molecular Biology of the Cell, 2005Co-Authors: Taeyoon Kim, Peng Y LohAbstract:Dense-core Secretory Granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented Granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize Granule proteins against degradation.
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Secretory Granule biogenesis and neuropeptide sorting to the regulated Secretory pathway in neuroendocrine cells
Journal of Molecular Neuroscience, 2004Co-Authors: Peng Y Loh, Taeyoon Kim, Yazmin M Rodriguez, Niamh X CawleyAbstract:Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the Granules of the regulated Secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core Granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of Secretory Granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a domain on CPE (CPE254–273) containing two basic residues (R255 and K260) to effect sorting into immature Secretory Granules. Site-directed mutagenesis of the motif on POMC resulted in accumulation of the mutant in the Golgi, as well as high basal secretion, indicating that the mutant POMC was inefficiently sorted to the RSP. These results support the model that POMC is actively sorted to the RSP Granules for processing and secretion by a sorting signal-mediated mechanism.
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large dense core Secretory Granule biogenesis is under the control of chromogranin a in neuroendocrine cells
Annals of the New York Academy of Sciences, 2002Co-Authors: Taeyoon Kim, Junghwa Taocheng, Lee E Eiden, Peng Y LohAbstract:The large dense-core Secretory Granule is an organelle in neuroendocrine/endocrine cells, where prohormones and proneuropeptides are stored, processed, and secreted in a regulated manner. Here we present evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, regulates dense-core Secretory Granule biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of Secretory Granule formation. An exogenously expressed prohormone, pro-opiomelanocortin, was neither stored nor secreted in a regulated manner in CgA-deficient PC12 cells. Overexpression of bovine CgA into CgA-deficient PC12 cells rescued regulated secretion. Other Secretory Granule proteins, such as chromogranin B (CgB), carboxypeptidase E, and synaptotagmin, were rapidly degraded, whereas nonGranule proteins were not affected in CgA-deficient PC12 cells. Unlike CgA, another granin protein CgB could not substitute for the role of CgA in Secretory Granule biogenesis. Thus, we conclude that CgA is a master "on/off" switch regulating the formation of the dense-core Secretory Granule in neuroendocrine cells.
Taeyoon Kim - One of the best experts on this subject based on the ideXlab platform.
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aquaporin 1 is important for maintaining Secretory Granule biogenesis in endocrine cells
Molecular Endocrinology, 2008Co-Authors: Irina Arnaoutova, Taeyoon Kim, Niamh X Cawley, Nimesh Patel, Trushar Rathod, Peng Y LohAbstract:Aquaporins (AQPs), a family of water channels expressed in epithelial cells, function to transport water in a bidirectional manner to facilitate transepithelial fluid absorption and secretion. Additionally, AQP1 and AQP5 are found in pancreatic zymogen Granules and synaptic vesicles and are involved in vesicle swelling and exocytosis in exocrine cells and neurons. Here, we show AQP1 is in dense-core Secretory Granule (DCSG) membranes of endocrine tissue: pituitary and adrenal medulla. The need for AQP1 in endocrine cell function was examined by stable transfection of AQP1 antisense RNA into AtT20 cells, a pituitary cell line, to down-regulate AQP1 expression. These AQP1-deficient cells showed more than 60% depletion of DCSGs and significantly decreased DCSG protein levels, including proopiomelanocotin/pro-ATCH and prohormone convertase 1/3, but not non-DCSG proteins. Pulse-chase studies revealed that whereas DCSG protein synthesis was unaffected, approximately 50% of the newly synthesized proopiomelanocortin was degraded within 1 h. Low levels of ACTH were released upon stimulation, indicating that the small number of DCSGs that were made in the presence of the residual AQP1 were functionally competent for exocytosis. Analysis of anterior pituitaries from AQP1 knockout mice showed reduced prohormone convertase 1/3, carboxypeptidase E, and ACTH levels compared to wild-type mice demonstrating that our results observed in AtT20 cells can be extended to the animal model. Thus, AQP1 is important for maintaining DCSG biogenesis and normal levels of hormone secretion in pituitary endocrine cells.
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Dense-core Secretory Granule biogenesis.
Physiology (Bethesda Md.), 2006Co-Authors: Taeyoon Kim, Marjorie C. Gondré-lewis, Irina Arnaoutova, Y. Peng LohAbstract:The dense-core Secretory Granule is a key organelle for secretion of hormones and neuropeptides in endocrine cells and neurons, in response to stimulation. Cholesterol and granins are critical for the assembly of these organelles at the trans-Golgi network, and their biogenesis is regulated quantitatively by posttranscriptional and posttranslational mechanisms.
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protease nexin 1 promotes Secretory Granule biogenesis by preventing Granule protein degradation
Molecular Biology of the Cell, 2005Co-Authors: Taeyoon Kim, Peng Y LohAbstract:Dense-core Secretory Granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented Granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize Granule proteins against degradation.
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Secretory Granule biogenesis and neuropeptide sorting to the regulated Secretory pathway in neuroendocrine cells
Journal of Molecular Neuroscience, 2004Co-Authors: Peng Y Loh, Taeyoon Kim, Yazmin M Rodriguez, Niamh X CawleyAbstract:Neuropeptide precursors synthesized at the rough endoplasmic reticulum are transported and sorted at the trans-Golgi network (TGN) to the Granules of the regulated Secretory pathway (RSP) of neuroendocrine cells. They are then processed into active peptides and stored in large dense-core Granules (LDCGs) until secreted upon stimulation. We have studied the regulation of biogenesis of the LDCGs and the mechanism by which neuropeptide precursors, such as pro-opiomelanocortin (POMC), are sorted into these LDCGs of the RSP in neuroendocrine and endocrine cells. We provide evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, plays an important role in the regulation of LDCG biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of Secretory Granule formation. Exogenously expressed POMC was neither stored nor secreted in a regulated manner in these CgA-deficient PC12 cells. Overexpression of CgA in a CgA- and LDCG-deficient endocrine cell line, 6T3, restored regulated secretion of transfected POMC and the presence of immunoreactive CgA at the tips of the processes of these cells. Unlike CgA, CgB, another granin protein, could not substitute for the role of CgA in regulating LDCG biogenesis. Thus, we conclude that CgA is a key player in the regulation of the biogenesis of LDCGs in neuroendocrine cells. To examine the mechanism of sorting POMC to the LDCGs, we carried out site-directed mutagenesis, transfected the POMC mutants into PC12 cells, and assayed for regulated secretion. Our previous molecular modeling studies predicted a three-dimensional sorting motif in POMC that can bind to a sorting receptor, membrane carboxypeptidase E (CPE). The sorting signal consists of four conserved residues at the N-terminal loop structure of POMC: two acidic residues and two hydrophobic residues. The two acidic residues were predicted to bind to a domain on CPE (CPE254–273) containing two basic residues (R255 and K260) to effect sorting into immature Secretory Granules. Site-directed mutagenesis of the motif on POMC resulted in accumulation of the mutant in the Golgi, as well as high basal secretion, indicating that the mutant POMC was inefficiently sorted to the RSP. These results support the model that POMC is actively sorted to the RSP Granules for processing and secretion by a sorting signal-mediated mechanism.
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large dense core Secretory Granule biogenesis is under the control of chromogranin a in neuroendocrine cells
Annals of the New York Academy of Sciences, 2002Co-Authors: Taeyoon Kim, Junghwa Taocheng, Lee E Eiden, Peng Y LohAbstract:The large dense-core Secretory Granule is an organelle in neuroendocrine/endocrine cells, where prohormones and proneuropeptides are stored, processed, and secreted in a regulated manner. Here we present evidence that chromogranin A (CgA), one of the most abundant acidic glycoproteins ubiquitously present in neuroendocrine/endocrine cells, regulates dense-core Secretory Granule biogenesis. Specific depletion of CgA expression by antisense RNAs in PC12 cells led to a profound loss of Secretory Granule formation. An exogenously expressed prohormone, pro-opiomelanocortin, was neither stored nor secreted in a regulated manner in CgA-deficient PC12 cells. Overexpression of bovine CgA into CgA-deficient PC12 cells rescued regulated secretion. Other Secretory Granule proteins, such as chromogranin B (CgB), carboxypeptidase E, and synaptotagmin, were rapidly degraded, whereas nonGranule proteins were not affected in CgA-deficient PC12 cells. Unlike CgA, another granin protein CgB could not substitute for the role of CgA in Secretory Granule biogenesis. Thus, we conclude that CgA is a master "on/off" switch regulating the formation of the dense-core Secretory Granule in neuroendocrine cells.
Laurent Taupenot - One of the best experts on this subject based on the ideXlab platform.
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heritability and genome wide linkage in us and australian twins identify novel genomic regions controlling chromogranin a implications for secretion and blood pressure
Circulation, 2008Co-Authors: Daniel T Oconnor, Fangwen Rao, Madhusudan Das, Manjula Mahata, Laurent Taupenot, Sushil K Mahata, Gu Zhu, Maple M Fung, Lei Wang, Kuixing ZhangAbstract:Background— Chromogranin A (CHGA) triggers catecholamine Secretory Granule biogenesis, and its catestatin fragment inhibits catecholamine release. We approached catestatin heritability using twin p...
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biogenesis of the Secretory Granule chromogranin a coiled coil structure results in unusual physical properties and suggests a mechanism for Granule core condensation
Biochemistry, 2007Co-Authors: C Mosley, Laurent Taupenot, Nilima Biswas, Joseph P Taulane, Norman H Olson, Sucheta M Vaingankar, Gen Wen, Nicholas J Schork, Michael G Ziegler, Sushil K MahataAbstract:The Secretory pro-hormone chromogranin A (CHGA) is densely packed into storage Granules along with catecholamines, playing a catalytic role in Granule biogenesis. 3-Dimensional structural data on CHGA are lacking. We found a superfamily structural homology for CHGA in the tropomyosin family of alpha-helical coiled-coils, even in mid-molecule regions where primary sequence identity is only modest. The assignment was confirmed by an independent algorithm, suggesting approximately 6-7 such domains spanning CHGA. We provide additional physiochemical evidence (chromatographic, spectral, microscopic) consistent with this unusual structure. Alpha-helical secondary structure (at up to approximately 45%) was confirmed by circular dichroism. CHGA molecular mass was estimated by MALDI-TOF mass spectrometry at approximately 50 kDa and by denaturing gel filtration at approximately 50-61 kDa, while its native Stokes radius was approximately 84.8 A, as compared to an expected approximately 30 A; the increase gave rise to an apparent native molecular weight of approximately 578 kDa, also consistent with the extended conformation of a coiled-coil. Small-angle X-ray scattering (SAXS) on CHGA in solution best fit an elongated cylindrical conformation in the monodisperse region with a radius of gyration of the rod cross-section (Rt) of approximately 52 A, compatible with a coiled-coil in the hydrated, aqueous state, ormore » a multimeric coiled-coil. Electron microscopy with negative staining revealed an extended, filamentous CHGA structure with a diameter of approximately 94 +/- 4.5 A. Extended, coiled-coil conformation is likely to permit protein 'packing' in the Secretory Granule at approximately 50% higher density than a globular/spherical conformation. Natural allelic variation in the catestatin region was predicted to disrupt the coiled-coil. Chromaffin Granule ultrastructure revealed a approximately 108 +/- 6.3 A periodicity of electron density, suggesting nucleation of a binding complex by the CHGA core. Inhibition of CHGA expression, by siRNA, disrupted regulated Secretory protein traffic by approximately 65%, while targeted ablation of the CHGA gene in the mouse reduced chromaffin Granule cotransmitter concentrations by approximately 40-80%. These results suggest new roles for Secretory protein tertiary structure in hormone and transmitter storage, with implications for Secretory cargo condensation (or dense core 'packing' structure) within the regulated pathway.« less
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Secretory Granule biogenesis in sympathoadrenal cells identification of a granulogenic determinant in the Secretory prohormone chromogranin a
Journal of Biological Chemistry, 2006Co-Authors: Maité Courel, Daniel T Oconnor, Carrie Rodemer, Susan T Nguyen, Alena Pance, Antony P Jackson, Laurent TaupenotAbstract:Chromogranin A (CgA) may be critical for Secretory Granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of Secretory Granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated Secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The Secretory Granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive Secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense Granules approximately 180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated Secretory pathway and thereby rescue the sorting of other Secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of Secretory Granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.
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Secretory Granule biogenesis in sympathoadrenal cells identification of a granulogenic determinant in the Secretory prohormone chromogranin a
Journal of Biological Chemistry, 2006Co-Authors: Maité Courel, Daniel T Oconnor, Laurent Taupenot, Carrie Rodemer, Susan T Nguyen, Alena Pance, Antony P JacksonAbstract:Chromogranin A (CgA) may be critical for Secretory Granule biogenesis in sympathoadrenal cells. We found that silencing the expression of CgA reduced the number of Secretory Granules in normal sympathoadrenal cells (PC12), and we therefore questioned whether a discrete domain of CgA might promote the formation of a regulated Secretory pathway in variant sympathoadrenal cells (A35C) devoid of such a phenotype. The Secretory Granule-forming activity of a series of human CgA domains labeled with a hemagglutinin epitope, green fluorescent protein, or embryonic alkaline phosphatase was assessed in A35C cells by deconvolution and electron microscopy and by secretagogue-stimulated release assays. Expression of CgA in A35C cells induced the formation of vesicular organelles throughout the cytoplasm, whereas two constitutive Secretory pathway markers accumulated in the Golgi complex. The lysosome-associated membrane protein LGP110 did not co-localize with CgA, consistent with non-lysosomal targeting of the granin in A35C cells. Thus, CgA-expressing A35C cells showed electron-dense Granules ∼180-220 nm in diameter, and secretagogue-stimulated exocytosis of CgA from A35C cells suggested that expression of the granin may be sufficient to restore a regulated Secretory pathway and thereby rescue the sorting of other Secretory proteins. We show that the formation of vesicular structures destined for regulated exocytosis may be mediated by a determinant located within the CgA N-terminal region (CgA-(1-115), with a necessary contribution of CgA-(40-115)), but not the C-terminal region (CgA-(233-439)) of the protein. We propose that CgA promotes the biogenesis of Secretory Granules by a mechanism involving a granulogenic determinant located within CgA-(40-115) of the mature protein.
Magnus Åbrink - One of the best experts on this subject based on the ideXlab platform.
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reduction with dithiothreitol causes serglycin specific defects in Secretory Granule integrity of bone marrow derived mast cells
Molecular Immunology, 2009Co-Authors: Tiago Braga, Magnus Åbrink, Maria Ringvall, Heidi Tveit, Gunnar PejlerAbstract:Mast cell Granule maturation and storage of Granule components has previously been shown to be critically dependent on serglycin (SG), a proteoglycan abundantly stored in mast cell Secretory Granules. The N-terminal portion of serglycin contains a conserved disulfide motif that is similar to motifs found in Secretory Granule compounds of neuroendocrine cells. Interference with such motifs of neuroendocrine cells with dithiothreitol (DTT) has previously been shown to cause cellular missorting. To investigate the implication for serglycin, serglycin(+/+) and serglycin(-/-) bone marrow derived mast cells (BMMCs) were treated with DTT followed by assessment of proteoglycan synthesis and Secretory Granule integrity. Treatment of serglycin(+/+) BMMCs with DTT almost completely abolished biosynthetic incorporation of (35)S-sulfate into proteoglycans, caused a dramatic reduction of granular staining with May Grunwald/Giemsa as well as disruption of Granule dense core formation as shown by transmission electron microscopy. In addition, the storage of carboxypeptidase A, a major Secretory Granule compound, was markedly reduced following DTT treatment. In contrast, none of these effects were seen after treatment of SG(-/-) BMMCs with DTT, indicating that they were serglycin-specific. Notably, DTT treated serglycin(+/+) BMMCs showed similar morphology as did the serglycin(-/-) BMMCs. DTT treatment affected neither the viability of the BMMCs nor the mRNA levels for serglycin or carboxypeptidase A. Together, these data indicate that DTT causes dramatic, serglycin-specific effects on mast cell Granule. These findings are thus in accordance with a role for the N-terminal disulfide motif in serglycin for regulation of mast cell Secretory Granule integrity.
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a role for serglycin proteoglycan in granular retention and processing of mast cell Secretory Granule components
FEBS Journal, 2006Co-Authors: Frida Henningsson, Magnus Åbrink, Sonja P Hergeth, Robert Cortelius, Gunnar PejlerAbstract:In the absence of serglycin proteoglycans, connective tissue-type mast cells fail to assemble mature metachromatic Secretory Granules, and this is accompanied by a markedly reduced ability to store neutral proteases. However, the mechanisms behind these phenomena are not known. In this study, we addressed these issues by studying the functionality and morphology of Secretory Granules as well as the fate of the Secretory Granule proteases in bone marrow-derived mast cells from serglycin(+/+) and serglycin(-/-) mice. We show that functional Secretory vesicles are formed in both the presence and absence of serglycin, but that dense core formation is defective in serglycin(-/-) mast cell Granules. The low levels of mast cell proteases present in serglycin(-/-) cells had a granular location, as judged by immunohistochemistry, and were released following exposure to calcium ionophore, indicating that they were correctly targeted into Secretory Granules even in the absence of serglycin. In the absence of serglycin, the fates of the serglycin-dependent proteases differed, including preferential degradation, exocytosis or defective intracellular processing. In contrast, beta-hexosaminidase storage and release was not dependent on serglycin. Together, these findings indicate that the reduced amounts of neutral proteases in the absence of serglycin is not caused by missorting into the constitutive pathway of secretion, but rather that serglycin may be involved in the retention of the proteases after their entry into Secretory vesicles.
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Serglycin-deficient cytotoxic T lymphocytes display defective Secretory Granule maturation and granzyme B storage.
The Journal of biological chemistry, 2005Co-Authors: Mirjana Grujic, Gunnar Pejler, Tiago Braga, Agneta Lukinius, Maija-leena Eloranta, Stefan D. Knight, Magnus ÅbrinkAbstract:Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic Granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce Secretory Granule of high electron density, although Granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for Secretory Granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.
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serglycin is essential for maturation of mast cell Secretory Granule
Journal of Biological Chemistry, 2004Co-Authors: Magnus Åbrink, Mirjana Grujic, Gunnar PejlerAbstract:To address the biological function of the scarcely studied intracellular proteoglycans, we targeted the gene for serglycin (SG), the only known committed intracellular proteoglycan. SG-/- mice developed normally and were fertile, but their mast cells (MCs) were severely affected. In peritoneum there was a complete absence of normal granulated MCs. Furthermore, peritoneal cells and ear tissue from SG-/- animals were devoid of the various MC-specific proteases. However, mRNA for the proteases was present in SG+/+, SG+/-, and SG-/- tissues, indicating that SG is essential for the storage, but not expression, of the MC proteases. Experiments, in which the differentiation of bone marrow stem cells into mature MCs was followed, showed that Secretory Granule maturation was compromised in SG-/- cells. Moreover, SG+/+ and SG+/- cells, but not SG-/- cells, synthesized proteoglycans of high anionic charge density. Taken together, we demonstrate a key role for SG proteoglycan in MC function.