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Guy A Rutter - One of the best experts on this subject based on the ideXlab platform.
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5 amp activated protein kinase controls insulin containing Secretory Vesicle dynamics
Journal of Biological Chemistry, 2003Co-Authors: Takashi Tsuboi, Gabriela Da Silva Xavier, Isabelle Leclerc, Guy A RutterAbstract:Abstract Changes in 5′-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761–774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including Vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 β-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKα1, 1–312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mm) Vesicle movements, and decreased the number of Vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual Secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent Vesicle movements. These findings suggest that components of the Vesicle transport network, including Vesicle-associated motor proteins, may be targets of AMPK in β-cells, dephosphorylation of which is required for Vesicle mobilization at elevated glucose concentrations.
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glucose metabolism and glutamate analog acutely alkalinize ph of insulin Secretory Vesicles of pancreatic β cells
American Journal of Physiology-endocrinology and Metabolism, 2003Co-Authors: Kazuhiro Eto, Guy A Rutter, Tokuyuki Yamashita, Kenzo Hirose, Yoshiharu Tsubamoto, Edward K Ainscow, Satoshi Kimura, Mitsuhiko Noda, Masamitsu Iino, Takashi KadowakiAbstract:We studied acute changes of Secretory Vesicle pH in pancreatic β-cells with a fluorescent pH indicator, lysosensor green DND-189. Fluorescence was decreased by 0.66 ± 0.10% at 149 ± 16 s with 22.2 ...
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ryanodine receptor type i and nicotinic acid adenine dinucleotide phosphate receptors mediate ca2 release from insulin containing Vesicles in living pancreatic β cells min6
Journal of Biological Chemistry, 2003Co-Authors: Kathryn Mitchell, Guy A RutterAbstract:Abstract We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001)J. Cell Biol. 155, 41–51) that ryanodine receptors (RyR) are present on insulin-containing Secretory Vesicles. Here we show that pancreatic islets and derived β-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core Secretory Vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca2+ concentrations in the Secretory Vesicle compartment from 40.1 ± 6.7 to 90.4 ± 14.8 μm(n = 4, p < 0.01), while having no effect on ER Ca2+ concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing agent, decreased dense core Secretory Vesicle but not ER free Ca2+ concentrations in permeabilized MIN6 β-cells, and flash photolysis of caged NAADP released Ca2+ from a thapsigargin-insensitive Ca2+ store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 ± 0.51-fold stimulation to no significant glucose effect;n = 3, p < 0.01), we conclude that RyR I-mediated Ca2+-induced Ca2+ release from Secretory Vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.
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ryanodine receptor type i and nicotinic acid adenine dinucleotide phosphate receptors mediate ca2 release from insulin containing Vesicles in living pancreatic β cells min6
Journal of Biological Chemistry, 2003Co-Authors: Kathryn Mitchell, Anthony F Lai, Guy A RutterAbstract:We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing Secretory Vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core Secretory Vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the Secretory Vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core Secretory Vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from Secretory Vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.
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simultaneous evanescent wave imaging of insulin Vesicle membrane and cargo during a single exocytotic event
Current Biology, 2000Co-Authors: Takashi Tsuboi, Chao Zhao, Susumu Terakawa, Guy A RutterAbstract:The classical model of Secretory Vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves Secretory Vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single Secretory Vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing Vesicles, enhanced green fluorescent protein (EGFP) fused to the Vesicle membrane protein phogrin [3] was delivered to the Secretory Vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the Vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, Secretory Vesicles showed only slow Brownian movement. After a depolarizing pulse, most Vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, Secretory Vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing Vesicles occurred after this. Post-exocytotic retrieval of the Vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty Vesicles under the plasma membrane ('kiss and glide').
Kathryn Mitchell - One of the best experts on this subject based on the ideXlab platform.
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ryanodine receptor type i and nicotinic acid adenine dinucleotide phosphate receptors mediate ca2 release from insulin containing Vesicles in living pancreatic β cells min6
Journal of Biological Chemistry, 2003Co-Authors: Kathryn Mitchell, Guy A RutterAbstract:Abstract We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001)J. Cell Biol. 155, 41–51) that ryanodine receptors (RyR) are present on insulin-containing Secretory Vesicles. Here we show that pancreatic islets and derived β-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core Secretory Vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca2+ concentrations in the Secretory Vesicle compartment from 40.1 ± 6.7 to 90.4 ± 14.8 μm(n = 4, p < 0.01), while having no effect on ER Ca2+ concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca2+-mobilizing agent, decreased dense core Secretory Vesicle but not ER free Ca2+ concentrations in permeabilized MIN6 β-cells, and flash photolysis of caged NAADP released Ca2+ from a thapsigargin-insensitive Ca2+ store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 ± 0.51-fold stimulation to no significant glucose effect;n = 3, p < 0.01), we conclude that RyR I-mediated Ca2+-induced Ca2+ release from Secretory Vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.
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ryanodine receptor type i and nicotinic acid adenine dinucleotide phosphate receptors mediate ca2 release from insulin containing Vesicles in living pancreatic β cells min6
Journal of Biological Chemistry, 2003Co-Authors: Kathryn Mitchell, Anthony F Lai, Guy A RutterAbstract:We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing Secretory Vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core Secretory Vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the Secretory Vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core Secretory Vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from Secretory Vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.
Wieland B. Huttner - One of the best experts on this subject based on the ideXlab platform.
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Cooperativity of phosphatidylinositol transfer protein and phospholipase D in Secretory Vesicle formation from the TGN--phosphoinositides as a common denominator?
FEBS letters, 1997Co-Authors: Oliver Tüscher, Christoph Lorra, Barend Bouma, Karel W. A. Wirtz, Wieland B. HuttnerAbstract:Phosphatidylinositol transfer protein (PITP) and phospholipase D (PLD) stimulate the formation of constitutive Secretory Vesicles (CSVs) and immature Secretory granules (ISGs) from the trans-Golgi network (TGN) in a cell-free system. The stimulatory effects of PITP and PLD are additive. Stimulation by either PITP or PLD is blocked by geneticin, a member of the aminoglycoside antibiotics known to bind to phosphoinositides. Since the PLD we used is insensitive to geneticin, our results suggest that phosphoinositides promote Secretory Vesicle formation as downstream effectors of both PITP and PLD, possibly via the recruitment of proteins mediating membrane budding and fission.
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a role for phosphatidylinositol transfer protein in Secretory Vesicle formation
Nature, 1995Co-Authors: Masato Ohashi, Vytas A Bankaitis, Karel W. A. Wirtz, Klaas Jan De Vries, Rainer Frank, Gerry T Snoek, Wieland B. HuttnerAbstract:Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of Vesicles from donor membranes and their fusion with acceptor membranes. With respect to Vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids. Here we report, using a previously established cell-free system derived from a neuroendocrine cell line, the purification of cytosolic factors that stimulate the formation of constitutive Secretory Vesicles and immature Secretory granules from the trans-Golgi network. One such factor, referred to as CAST1, was identified as the alpha and beta isoforms of the mammalian phosphatidylinositol transfer protein (PtdIns-TP) (refs 3-5). The yeast PtdIns-TP, SEC14p (ref. 6), which has no sequence homology to mammalian PtdIns-TP (refs 7,8), was able to substitute for the mammalian PtdIns-TP in Secretory Vesicle formation. Our results suggest a highly conserved role for phosphoinositides in Vesicle formation.
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an elevation of cytosolic protein phosphorylation modulates trimeric g protein regulation of Secretory Vesicle formation from the trans golgi network
Journal of Biological Chemistry, 1994Co-Authors: M Ohashi, Wieland B. HuttnerAbstract:The role of protein phosphorylation in the formation of Secretory Vesicles from the trans-Golgi network (TGN) and in the regulation of this process by TGN-associated trimeric G-proteins was investigated, using a previously established and a novel cell-free system derived from the neuroendocrine cell line PC12. In the absence of exogenous activators of trimeric G-proteins, okadaic acid, an inhibitor of protein serine/threonine phosphatase types 1, 2A, and PPX, had no significant effect on Secretory Vesicle formation as reconstituted in a postnuclear supernatant. However, okadaic acid antagonized the inhibition of Secretory Vesicle formation which occurred upon activation of trimeric G-proteins by either aluminum fluoride or guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S). Microcystin-LR, a protein phosphatase inhibitor structurally distinct from okadaic acid, also antagonized the trimeric G-protein-mediated inhibition of Secretory Vesicle formation but, in contrast to okadaic acid, alone was sufficient to stimulate this process. The antagonistic effect of the phosphatase inhibitors was abolished by a broad spectrum protein kinase inhibitor, staurosporine, which alone, however, did not affect Vesicle formation. The effect of okadaic acid was promoted by activators of protein kinase C (phorbol myristate acetate) and protein kinase A (cyclic AMP). To investigate the subcellular localization of the phosphoprotein that is involved in the antagonistic effect of protein phosphatase inhibitors, a novel cell-free system was established which reconstitutes the formation of Secretory Vesicles from TGN membranes supplemented with cytosol. Using this cell-free system, the relevant phosphoprotein was found to reside in the cytosol. In conclusion, our results suggest that serine/threonine protein phosphorylation is not required for Secretory Vesicle formation from the TGN but modulates, via a cytosolic phosphoprotein, the regulation of this process by TGN-associated trimeric G-proteins.
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multiple trimeric g proteins on the trans golgi network exert stimulatory and inhibitory effects on Secretory Vesicle formation
The EMBO Journal, 1992Co-Authors: Anja Leyte, Francis A Barr, Ralph H Kehlenbach, Wieland B. HuttnerAbstract:The role of heterotrimeric G-proteins on the formation of constitutive Secretory Vesicles (CSVs) and immature Secretory granules (ISGs) from the trans-Golgi network (TGN) of PC12 cells was investigated. Using immunofluorescence and subcellular fractionation in conjunction with immunoblotting or ADP-ribosylation by either pertussis toxin or cholera toxin, TGN membranes were found to contain not only several alpha i/alpha o G-protein subunits including apparently alpha i3, but also alpha s. Pertussis toxin treatment of cells, which resulted in the stoichiometric ADP-ribosylation of alpha i/alpha o, a modification known to prevent their coupling to receptors, led to the stimulation of cell-free CSV and ISG formation, suggesting the presence of a guanine nucleotide exchange factor for alpha i/alpha o on the TGN. Mastoparan-7, a peptide known to mimic an activated receptor and to stimulate nucleotide exchange on alpha i/alpha o, inhibited cell-free Vesicle formation, an effect abolished by pertussis toxin. In contrast, activation of alpha s by cholera toxin treatment of cells resulted in a stimulation of cell-free CSV and ISG formation. This stimulation could be reversed when the alpha subunits not activated by cholera toxin, i.e. alpha i/alpha o, were activated by GTP gamma S and [AIF4]-. Our results show that both inhibitory and stimulatory trimeric G-proteins on the TGN participate in the regulation of Secretory Vesicle formation.
Vivian Hook - One of the best experts on this subject based on the ideXlab platform.
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cysteine cathepsins in the Secretory Vesicle produce active peptides cathepsin l generates peptide neurotransmitters and cathepsin b produces beta amyloid of alzheimer s disease
Biochimica et Biophysica Acta, 2012Co-Authors: Vivian Hook, Steven J Bark, Lydiane Funkelstein, Jill L Wegrzyn, Mark S Kindy, Gregory HookAbstract:Abstract Recent new findings indicate significant biological roles of cysteine cathepsin proteases in Secretory Vesicles for production of biologically active peptides. Notably, cathepsin L in Secretory Vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell–cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in Secretory Vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These Secretory Vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique Secretory Vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the Secretory Vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
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proteomics of neuroendocrine Secretory Vesicles reveal distinct functional systems for biosynthesis and exocytosis of peptide hormones and neurotransmitters
Journal of Proteome Research, 2007Co-Authors: Jill L Wegrzyn, Jean Lee, John M Neveu, William S Lane, Vivian HookAbstract:Regulated Secretory Vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such Secretory Vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core Secretory Vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these Vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (microLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel Secretory Vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARE-related proteins and annexins that are involved in trafficking and exocytosis of Secretory Vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these Secretory Vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates Secretory Vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions.
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Secretory Vesicle aminopeptidase b related to neuropeptide processing molecular identification and subcellular localization to enkephalin and npy containing chromaffin granules
Journal of Neurochemistry, 2007Co-Authors: Shinrong Hwang, Thierry Foulon, Vivian Hook, Audrey K Oneill, Steven J BarkAbstract:Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by Secretory Vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH2-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in Secretory Vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in ‘model’ neuropeptide-containing Secretory Vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in Secretory Vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5–6.5 that is similar to the internal pH of Secretory Vesicles. The significant finding of the Secretory Vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.
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protein trafficking to chromaffin granules and proteolytic processing within regulated Secretory Vesicles of neuroendocrine chromaffin cells
Annals of the New York Academy of Sciences, 2002Co-Authors: Vivian Hook, Mariehelene MetzboutigueAbstract:: Proteolytic processing within Secretory Vesicles is required for the production and secretion of biologically active peptide neurotransmitters and hormones, known collectively as neuropeptides. This chapter addresses several new aspects of proteolysis in Secretory Vesicles, chromaffin granules, with respect to sorting proneuropeptides or prohormones into such regulated Secretory Vesicles that use specific prohormone sorting signals. Concomitant with prohormone sorting, evidence for the role of chromogranin A in Secretory granule biogenesis is presented. Secretory Vesicle function involves endogenous serpin protease inhibitors for the regulation of proteolysis. The novel serpins endopin 1 and endopin 2 possess high homology to a1-antichymotrypsin, yet they possess distinct target protease specificities. The serpins PAI-1 and neuroserpin are also localized to chromaffin granules. In addition, regulation of Secretory Vesicle function involves cytochrome b561 that regulates reducing equivalents to maintain the intravesicular redox state. These studies demonstrate multiple components as regulatory factors in the control of Secretory Vesicle function for the biosynthesis and secretion of neuropeptides and catecholamines.
Robert D Burgoyne - One of the best experts on this subject based on the ideXlab platform.
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The cysteine-string domain of the Secretory Vesicle cysteine-string protein is required for membrane targeting
Biochemical Journal, 1998Co-Authors: Luke H Chamberlain, Robert D BurgoyneAbstract:The post-translational addition of palmitic acid residues to cysteine-string protein (Csp) was originally thought to form the basis for membrane association of this Secretory-Vesicle protein. However, subsequent work showed that chemical depalmitoylation of Csp does not result in its release from membranes. We have confirmed these findings and employed [3H]palmitate labelling of PC12 cells to demonstrate that Csp1 remains associated with membranes following the complete removal of palmitic acid residues. Although palmitoylation is not essential for the stable membrane association of Csp, its role in membrane targeting has not been assessed. To examine this, we constructed a Csp mutant protein with seven cysteines replaced by serines in the cysteine-string domain. In contrast to wild-type Csps, this mutant protein was not targeted to membranes when expressed in PC12 or HeLa cells. We conclude that although a palmitoylated cysteine-string domain is not required for stable membrane association of Csp, it is essential for initial membrane targeting.
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the molecular chaperone function of the Secretory Vesicle cysteine string proteins
Journal of Biological Chemistry, 1997Co-Authors: Luke H Chamberlain, Robert D BurgoyneAbstract:The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s. The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs. We have characterized mutations in the HPD motif of the synaptic Vesicle protein cysteine-string protein (Csp). Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70. The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent. The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70. Thus, the HPD motif of Csp is required for binding to Hsc70. We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase. Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation. In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins. Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein. These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones. This suggests that Csps may have a general chaperone function in regulated exocytosis.
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ca2 and Secretory Vesicle dynamics
Trends in Neurosciences, 1995Co-Authors: Robert D Burgoyne, Alan MorganAbstract:Abstract Exocytosis in neurones and neuroendocrine cells is triggered by an increase in the cytosolic concentration of Ca 2+ , and is followed by endocytotic membrane retrieval. Electrophysiological studies have characterized the nature of the Ca 2+ signal that is required for exocytosis, and have defined the Ca 2+ -dependent steps in exocytotic and endocytotic Vesicle cycling. In parallel, biochemical approaches have led to the discovery of a range of proteins that appears to function in synaptic- and Secretory-Vesicle dynamics. The nature of the Ca 2+ -binding proteins, and how they interact with the identified components of the exocytotic and endocytotic machinery, remain key unresolved issues. However, it is apparent that exocytosis involves multiple Ca 2+ -binding proteins with different affinities, and that the Ca 2+ sensor involved in the final membrane-fusion step has different affinities for Ca 2+ in synapses and neuroendocrine cells.