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Janice E. A. Braun - One of the best experts on this subject based on the ideXlab platform.
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neurons export extracellular vesicles enriched in Cysteine String Protein and misfolded Protein cargo
Scientific Reports, 2017Co-Authors: Jingti Deng, Carolina Koutras, Julien Donnelier, Mana Alshehri, Maryam Fotouhi, Martine Girard, Steve Casha, Peter S Mcpherson, Stephen M Robbins, Janice E. A. BraunAbstract:The fidelity of synaptic transmission depends on the integrity of the Protein machinery at the synapse. Unfolded synaptic Proteins undergo refolding or degradation in order to maintain synaptic proteostasis and preserve synaptic function, and buildup of unfolded/toxic Proteins leads to neuronal dysfunction. Many molecular chaperones contribute to proteostasis, but one in particular, Cysteine String Protein (CSPα), is critical for proteostasis at the synapse. In this study we report that exported vesicles from neurons contain CSPα. Extracellular vesicles (EV's) have been implicated in a wide range of functions. However, the functional significance of neural EV's remains to be established. Here we demonstrate that co-expression of CSPα with the disease-associated Proteins, polyglutamine expanded Protein 72Q huntingtinex°n1 or superoxide dismutase-1 (SOD-1G93A) leads to the cellular export of both 72Q huntingtinex°n1 and SOD-1G93A via EV's. In contrast, the inactive CSPαHPD-AAA mutant does not facilitate elimination of misfolded Proteins. Furthermore, CSPα-mediated export of 72Q huntingtinex°n1 is reduced by the polyphenol, resveratrol. Our results indicate that by assisting local lysosome/proteasome processes, CSPα-mediated removal of toxic Proteins via EVs plays a central role in synaptic proteostasis and CSPα thus represents a potential therapeutic target for neurodegenerative diseases.
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correction Cysteine String Protein limits expression of the large conductance calcium activated k bk channel
PLOS ONE, 2015Co-Authors: Eva Ahrendt, Andrew P. Braun, Barry D Kyle, Janice E. A. BraunAbstract:Fig 1C is incorrect as it shows the wrong actin blot. The authors have provided a corrected version of Fig 1 here. Fig 1 CSPα alters BK channel expression. Additionally, there is a sentence missing from the caption for Fig 3. Please see the complete, correct Fig 3 caption here. The missing sentence is highlighted in bold. Fig 3 The J domain of CSPα reduces BK channel expression.
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Hilary Brown1,2, Olof Larsson2,
2015Co-Authors: Shao-nian Yang, Barbara Leibiger, Ingo Leibiger, Gabriel Fried, Tilo Moede, Jude T. Deeney, Graham R. Brown, Gunilla Jacobsson, Christopher J. Rhodes, Janice E. A. BraunAbstract:Cysteine String Protein (CSP) is an insulin secretory granule-associated Protein regulating b-cell exocytosi
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Correction: Cysteine String Protein Limits Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel
2015Co-Authors: Eva Ahrendt, Andrew P. Braun, Barry Kyle, Janice E. A. BraunAbstract:Correction: Cysteine String Protein Limits Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel
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Cysteine String Protein limits expression of the large conductance calcium activated k bk channel
PLOS ONE, 2014Co-Authors: Eva Ahrendt, Andrew P. Braun, Barry D Kyle, Janice E. A. BraunAbstract:Large-conductance, calcium-activated K+ (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in Cysteine String Protein (CSPα), a presynaptic J-Protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.
Luke H Chamberlain - One of the best experts on this subject based on the ideXlab platform.
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a cluster of palmitoylated Cysteines are essential for aggregation of Cysteine String Protein mutants that cause neuronal ceroid lipofuscinosis
Scientific Reports, 2017Co-Authors: Cinta Diezardanuy, Jennifer Greaves, Kevin R Munro, Nicholas C O Tomkinson, Luke H ChamberlainAbstract:Autosomal-dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is caused by mutation of the DNAJC5 gene encoding Cysteine String Protein alpha (CSPα). The disease-causing mutations, which result in substitution of leucine-115 with an arginine (L115R) or deletion of the neighbouring leucine-116 (∆L116) in the Cysteine-String domain cause CSPα to form high molecular weight SDS-resistant aggregates, which are also present in post-mortem brain tissue from patients. Formation and stability of these mutant aggregates is linked to palmitoylation of the Cysteine-String domain, however the regions of the mutant Proteins that drive aggregation have not been determined. The importance of specific residues in the Cysteine-String domain was investigated, revealing that a central core of palmitoylated Cysteines is essential for aggregation of ANCL CSPα mutants. Interestingly, palmitoylated monomers of ANCL CSPα mutants were shown to be short-lived compared with wild-type CSPα, suggesting that the mutants either have a faster rate of depalmitoylation or that they are consumed in a time-dependent manner into high molecular weight aggregates. These findings provide new insight into the features of CSPα that promote aggregation in the presence of L115R/∆L116 mutations and reveal a change in the lifetime of palmitoylated monomers of the mutant Proteins.
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Short Review Cysteine-String Protein: The Chaperone at the Synapse
2014Co-Authors: Luke H Chamberlain, Robert D BurgoyneAbstract:Abstract: Cysteine-String Protein (Csp) is a major syn-aptic vesicle and secretory granule Protein first discov-ered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendo-crine and endocrine cell types, and its distribution sug-gests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca21 channels. Csp, however, must have an additional function as a direct regulator of the exocy-totic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca21 in per-meabilised cells. Csps possess a Cysteine-String domain that is highly palmitoylated and confers membrane tar-geting. In addition, Csps have a conserved “J ” domain that mediates binding to an activation of the Hsp70/ Hsc70 chaperone ATPases. This and other evidence im-plicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic ma-chinery. Targets for Csp include the vesicle Protein VAMP/synaptobrevin and the plasma membrane Protein syntaxin 1, the significance of which is discussed in pos-sible models to account for current knowledge of Cs
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palmitoylation induced aggregation of Cysteine String Protein mutants that cause neuronal ceroid lipofuscinosis
Journal of Biological Chemistry, 2012Co-Authors: Jennifer Greaves, Oforiwa A Gorleku, Kimon Lemonidis, Carlos Cruchaga, Christopher Grefen, Luke H ChamberlainAbstract:Recently, mutations in the DNAJC5 gene encoding Cysteine-String Protein α (CSPα) were identified to cause the neurodegenerative disorder adult-onset neuronal ceroid lipofuscinosis. The disease-causing mutations (L115R or ΔL116) occur within the Cysteine-String domain, a region of the Protein that is post-translationally modified by extensive palmitoylation. Here we demonstrate that L115R and ΔL116 mutant Proteins are mistargeted in neuroendocrine cells and form SDS-resistant aggregates, concordant with the properties of other mutant Proteins linked to neurodegenerative disorders. The mutant aggregates are membrane-associated and incorporate palmitate. Indeed, co-expression of palmitoyltransferase enzymes promoted the aggregation of the CSPα mutants, and chemical depalmitoylation solubilized the aggregates, demonstrating that aggregation is induced and maintained by palmitoylation. In agreement with these observations, SDS-resistant CSPα aggregates were present in brain samples from patients carrying the L115R mutation and were depleted by chemical depalmitoylation. In summary, this study identifies a novel interplay between genetic mutations and palmitoylation in driving aggregation of CSPα mutant Proteins. We propose that this palmitoylation-induced aggregation of mutant CSPα Proteins may underlie the development of adult-onset neuronal ceroid lipofuscinosis in affected families.
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palmitoylation and testis enriched expression of the Cysteine String Protein beta isoform
Biochemistry, 2010Co-Authors: Oforiwa A Gorleku, Luke H ChamberlainAbstract:Cysteine-String Protein alpha (CSPalpha) is a DnaJ chaperone that is associated with secretory vesicles in diverse cell types. The Cysteine-String region is the signature domain of CSPalpha and contains 14 closely spaced Cysteines, the majority of which are palmitoylated; this post-translational modification mediates stable membrane attachment. CSPalpha has been proposed to function in regulated exocytosis pathways throughout the body and has an additional neuroprotective function. Two novel CSP isoforms, beta and gamma, were identified recently, although the expression profile, properties, and functions of these Proteins are not clear and in some cases are subject to debate. Here, we report that CSPbeta is enriched in rat testis and was not detected in any other tissue that was examined, including brain. Although the Cysteine-String domain of CSPbeta is distinct from that found in CSPalpha, the endogenous beta isoform expressed in testis is membrane-associated and palmitoylated. However, in agreement with earlier work, we find that the palmitoylation efficiency of CSPbeta is reduced compared with that of CSPalpha. Subsequent analysis of chimeric Proteins reveals that regions upstream of the Cysteine-String domains of CSPalpha and CSPbeta underlie this difference in palmitoylation efficiency between the two isoforms.
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palmitoylation and membrane interactions of the neuroprotective chaperone Cysteine String Protein
Journal of Biological Chemistry, 2008Co-Authors: Jennifer Greaves, Christine Salaun, Yuko Fukata, Masaki Fukata, Luke H ChamberlainAbstract:Cysteine-String Protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC Proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC Proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the Protein to locate its partner Golgi-localized DHHC Proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting.
Robert D Burgoyne - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of Cysteine String Protein triggers a major conformational switch
Structure, 2016Co-Authors: Pryank Patel, Robert D Burgoyne, Gerald R Prescott, Luyun Lian, A MorganAbstract:Cysteine String Protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client Proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its Protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoProteins.
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Cysteine String Protein csp and its role in preventing neurodegeneration
Seminars in Cell & Developmental Biology, 2015Co-Authors: Robert D Burgoyne, A MorganAbstract:Cysteine String Protein (CSP) is a member of the DnaJ/Hsp40 family of co-chaperones that localises to neuronal synaptic vesicles. Its name derives from the possession of a String of 12–15 Cysteine residues, palmitoylation of which is required for targeting to post-Golgi membranes. The DnaJ domain of CSP enables it to bind client Proteins and recruit Hsc70 chaperones, thereby contributing to the maintenance of Protein folding in the presynaptic compartment. Mutation of CSP in flies, worms and mice reduces lifespan and causes synaptic dysfunction and neurodegeneration. Furthermore, recent studies have revealed that the neurodegenerative disease, adult onset neuronal ceroid lipofuscinosis, is caused by mutations in the human CSPα-encoding DNAJC5 gene. Accumulating evidence suggests that the major mechanism by which CSP prevents neurodegeneration is by maintaining the conformation of SNAP-25, thereby facilitating its entry into the membrane-fusing SNARE complex. In this review, we focus on the role of CSP in preventing neurodegeneration and discuss how recent studies of this universal neuroprotective chaperone are being translated into potential novel therapeutics for neurodegenerative diseases.
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Short Review Cysteine-String Protein: The Chaperone at the Synapse
2014Co-Authors: Luke H Chamberlain, Robert D BurgoyneAbstract:Abstract: Cysteine-String Protein (Csp) is a major syn-aptic vesicle and secretory granule Protein first discov-ered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendo-crine and endocrine cell types, and its distribution sug-gests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca21 channels. Csp, however, must have an additional function as a direct regulator of the exocy-totic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca21 in per-meabilised cells. Csps possess a Cysteine-String domain that is highly palmitoylated and confers membrane tar-geting. In addition, Csps have a conserved “J ” domain that mediates binding to an activation of the Hsp70/ Hsc70 chaperone ATPases. This and other evidence im-plicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic ma-chinery. Targets for Csp include the vesicle Protein VAMP/synaptobrevin and the plasma membrane Protein syntaxin 1, the significance of which is discussed in pos-sible models to account for current knowledge of Cs
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Cysteine String Protein the chaperone at the synapse
Journal of Neurochemistry, 2008Co-Authors: Luke H Chamberlain, Robert D BurgoyneAbstract:Abstract: Cysteine-String Protein (Csp) is a major synaptic vesicle and secretory granule Protein first discovered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendocrine and endocrine cell types, and its distribution suggests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca2+ channels. Csp, however, must have an additional function as a direct regulator of the exocytotic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca2+ in permeabilised cells. Csps possess a Cysteine-String domain that is highly palmitoylated and confers membrane targeting. In addition, Csps have a conserved “J” domain that mediates binding to an activation of the Hsp70/Hsc70 chaperone ATPases. This and other evidence implicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic machinery. Targets for Csp include the vesicle Protein VAMP/synaptobrevin and the plasma membrane Protein syntaxin 1, the significance of which is discussed in possible models to account for current knowledge of Csp function.
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Protein kinase b akt is a novel Cysteine String Protein kinase that regulates exocytosis release kinetics and quantal size
Journal of Biological Chemistry, 2006Co-Authors: Gareth J O Evans, Robert D Burgoyne, Gerald R Prescott, Jeff W Barclay, Morris J Birnbaum, A MorganAbstract:Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of Cysteine String Protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.
Cameron B Gundersen - One of the best experts on this subject based on the ideXlab platform.
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Cysteine String Protein beta is prominently associated with nerve terminals and secretory organelles in mouse brain
Brain Research, 2010Co-Authors: Cameron B Gundersen, Sirus A Kohan, Puneet Souda, Julian P Whitelegge, Joy A UmbachAbstract:Cysteine String Proteins (CSPs) are associated with regulated secretory organelles in organisms ranging from fruit flies to man. Mammals have three csp genes (alpha, beta and gamma), and previous work indicated that expression of the csp-beta and -gamma genes was restricted to the testes. For the current investigation, antibodies specific for CSP-beta were developed. Unexpectedly, immunoblot analysis indicated that CSP-beta was prominently expressed throughout mouse brain. Upon sub-cellular fractionation, CSP-beta was enriched in synaptosomes and synaptic vesicle fractions. Interestingly, CSP-beta existed almost exclusively as part of a high mass complex both in testis and brain. This complex required aggressive denaturation to release monomeric CSP-beta. By Northern analysis CSP-beta mRNA was present at very low abundance as a approximately 1.0kb species in mouse brain. Collectively, the enrichment of CSP-beta in synaptosomes and the association of CSP-beta with synaptic vesicles suggest that CSP-beta, like CSP-alpha, may be an important component of the regulated secretory machinery in mouse brain.
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antipeptide antibodies against a torpedo Cysteine String Protein
Journal of Neurochemistry, 2008Co-Authors: Alessandro Mastrogiacomo, Christopher J Evans, Cameron B GundersenAbstract:Abstract: An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo Cysteine-String Protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane Proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single Protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel Proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane Protein.
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interaction between constitutively expressed heat shock Protein hsc 70 and Cysteine String Protein is important for cortical granule exocytosis in xenopus oocytes
Journal of Biological Chemistry, 2005Co-Authors: Geoffrey B Smith, Joy A Umbach, Arlene Hirano, Cameron B GundersenAbstract:In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the Protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of Cysteine String Protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by ∼80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a Protein that is important for regulated exocytosis. Because the NH2-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock Proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.
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dietary lithium induces regional increases of mrna encoding Cysteine String Protein in rat brain
Journal of Neuroscience Research, 2003Co-Authors: Mara L Cordeiro, Cameron B Gundersen, Joy A UmbachAbstract:Lithium salts are used to treat manic-depressive disorders; however, the mechanism by which lithium produces its therapeutic benefit remains obscure. The action of lithium may involve alterations of Proteins important for regulating synaptic function. In this context, we observed recently that lithium at therapeutically relevant concentrations enhanced expression of Cysteine String Protein (csp) at the level of both mRNA and Protein, in cell culture and in rat brain. Several lines of evidence have shown that csps are vital components of the regulated secretory pathway. We were interested whether lithium modulates expression of csp in specific brain regions. To study this issue, we analyzed the effects of chronic lithium administration (21 days) on csp mRNA levels in rat brain using in situ hybridization. Densitometric analysis revealed that lithium upregulated csp mRNA in several brain areas that are important for mood and behavior. This effect may be germane to understanding the beneficial action of lithium in mood disorders. © 2003 Wiley-Liss, Inc.
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lithium ions enhance Cysteine String Protein gene expression in vivo and in vitro
Journal of Neurochemistry, 2002Co-Authors: Mara L Cordeiro, Joy A Umbach, Cameron B GundersenAbstract:Lithium is a well established pharmacotherapy for the treatment of recurrent manic-depressive illness. However, the mechanism by which lithium exerts its therapeutic action remains elusive. Here we report that lithium at 1 mM significantly increased the expression of Cysteine String Proteins (CSPs) in a pheochromocytoma cell line (PC12 cells) differentiated by nerve growth factor. These cells concomitantly exhibited increased expression of CSPs in their cell bodies and boutons. Enhanced CSP expression was also observed in the brain of rats fed a lithium-containing diet, which elevated serum lithium to a therapeutically relevant concentration of approximately 1.0 mM. However, both in vitro and in vivo, the expression of another synaptic vesicle Protein, synaptophysin, and the t-SNARE, synaptosomal-associated Protein of 25 kDa (SNAP-25), was not significantly altered by lithium. These observations indicate that lithium-induced changes of CSP gene expression may contribute to the therapeutic efficacy of this monovalent cation.
A Morgan - One of the best experts on this subject based on the ideXlab platform.
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phosphorylation of Cysteine String Protein triggers a major conformational switch
Structure, 2016Co-Authors: Pryank Patel, Robert D Burgoyne, Gerald R Prescott, Luyun Lian, A MorganAbstract:Cysteine String Protein (CSP) is a member of the DnaJ/Hsp40 chaperone family that localizes to neuronal synaptic vesicles. Impaired CSP function leads to neurodegeneration in humans and model organisms as a result of misfolding of client Proteins involved in neurotransmission. Mammalian CSP is phosphorylated in vivo on Ser10, and this modulates its Protein interactions and effects on neurotransmitter release. However, there are no data on the structural consequences of CSP phosphorylation to explain these functional effects. We show that Ser10 phosphorylation causes an order-to-disorder transition that disrupts CSP's extreme N-terminal α helix. This triggers the concomitant formation of a hairpin loop stabilized by ionic interactions between phosphoSer10 and the highly conserved J-domain residue, Lys58. These phosphorylation-induced effects result in significant changes to CSP conformation and surface charge distribution. The phospho-switch revealed here provides structural insight into how Ser10 phosphorylation modulates CSP function and also has potential implications for other DnaJ phosphoProteins.
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Cysteine String Protein csp and its role in preventing neurodegeneration
Seminars in Cell & Developmental Biology, 2015Co-Authors: Robert D Burgoyne, A MorganAbstract:Cysteine String Protein (CSP) is a member of the DnaJ/Hsp40 family of co-chaperones that localises to neuronal synaptic vesicles. Its name derives from the possession of a String of 12–15 Cysteine residues, palmitoylation of which is required for targeting to post-Golgi membranes. The DnaJ domain of CSP enables it to bind client Proteins and recruit Hsc70 chaperones, thereby contributing to the maintenance of Protein folding in the presynaptic compartment. Mutation of CSP in flies, worms and mice reduces lifespan and causes synaptic dysfunction and neurodegeneration. Furthermore, recent studies have revealed that the neurodegenerative disease, adult onset neuronal ceroid lipofuscinosis, is caused by mutations in the human CSPα-encoding DNAJC5 gene. Accumulating evidence suggests that the major mechanism by which CSP prevents neurodegeneration is by maintaining the conformation of SNAP-25, thereby facilitating its entry into the membrane-fusing SNARE complex. In this review, we focus on the role of CSP in preventing neurodegeneration and discuss how recent studies of this universal neuroprotective chaperone are being translated into potential novel therapeutics for neurodegenerative diseases.
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phosphorylation of Cysteine String Protein on serine 10 triggers 14 3 3 Protein binding
Biochemical and Biophysical Research Communications, 2008Co-Authors: Gerald R Prescott, Rosalind E Jenkins, Ciara M Walsh, A MorganAbstract:Abstract Cysteine String Protein (CSP) is a neuronal chaperone that maintains normal neurotransmitter exocytosis and is essential for preventing presynaptic neurodegeneration. CSP is phosphorylated in vivo on a single residue, Ser10, and this phosphorylation regulates its cellular functions, although the molecular mechanisms involved are unclear. To identify novel phosphorylation-specific binding partners for CSP, we used a pull-down approach using synthetic peptides and recombinant Proteins. A single Protein band was observed to bind specifically to a Ser10-phosphorylated CSP peptide (residues 4–14) compared to a non-phosphorylated peptide. This band was identified as 14-3-3 Protein of various isoforms using mass spectrometry and Western blotting. PKA phosphorylation of full-length CSP Protein stimulated 14-3-3 binding, and this was abolished in a Ser10-Ala mutant CSP, confirming the binding site as phospho-Ser10. As both CSP and 14-3-3 Proteins are implicated in neurotransmitter exocytosis and neurodegeneration, this novel phosphorylation-dependent interaction may help maintain the functional integrity of the synapse.
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Protein kinase b akt is a novel Cysteine String Protein kinase that regulates exocytosis release kinetics and quantal size
Journal of Biological Chemistry, 2006Co-Authors: Gareth J O Evans, Robert D Burgoyne, Gerald R Prescott, Jeff W Barclay, Morris J Birnbaum, A MorganAbstract:Protein kinase B/Akt has been implicated in the insulin-dependent exocytosis of GLUT4-containing vesicles, and, more recently, insulin secretion. To determine if Akt also regulates insulin-independent exocytosis, we used adrenal chromaffin cells, a popular neuronal model. Akt1 was the predominant isoform expressed in chromaffin cells, although lower levels of Akt2 and Akt3 were also found. Secretory stimuli in both intact and permeabilized cells induced Akt phosphorylation on serine 473, and the time course of Ca2+-induced Akt phosphorylation was similar to that of exocytosis in permeabilized cells. To determine if Akt modulated exocytosis, we transfected chromaffin cells with Akt constructs and monitored catecholamine release by amperometry. Wild-type Akt had no effect on the overall number of exocytotic events, but slowed the kinetics of catecholamine release from individual vesicles, resulting in an increased quantal size. This effect was due to phosphorylation by Akt, because it was not seen in cells transfected with kinase-dead mutant Akt. As overexpression of Cysteine String Protein (CSP) results in a similar alteration in release kinetics and quantal size, we determined if CSP was an Akt substrate. In vitro 32P-phosphorylation studies revealed that Akt phosphorylates CSP on serine 10. Using phospho-Ser10-specific antisera, we found that both transfected and endogenous cellular CSP is phosphorylated by Akt on this residue. Taken together, these findings reveal a novel role for Akt phosphorylation in regulating the late stages of exocytosis and suggest that this is achieved via the phosphorylation of CSP on serine 10.
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Phosphorylation of Cysteine String Protein in the brain: developmental, regional and synaptic specificity.
The European journal of neuroscience, 2005Co-Authors: Gareth J O Evans, A MorganAbstract:Protein phosphorylation modulates regulated exocytosis in most cells, including neurons. Cysteine String Protein (CSP) has been implicated in this process because its phosphorylation on Ser10 alters its interactions with syntaxin and synaptotagmin, and because the effect of CSP overexpression on exocytosis kinetics in chromaffin cells requires phosphorylatable Ser10. To characterize CSP phosphorylation in the brain, we raised phosphospecific antibodies to Ser10. Western blotting revealed that the proportion of phosphorylated CSP (P-CSP) varies between distinct brain regions and also exhibits developmental regulation, with P-CSP highest early in development. Immunohistochemical analysis of the cerebellar cortex revealed a novel pool of P-CSP that did not colocalize with synaptic vesicle markers during early development. Strikingly, in the adult cerebellar granular layer P-CSP was highly enriched in a subset of glutamatergic synapses but undetectable in neighbouring GABA-ergic synapses. In view of the functional consequences of CSP phosphorylation, such differences could contribute to the synapse-specific regulation of neurotransmitter release.
