Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.
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PMID:Calcium-regulated exocytosis is required for cell membrane resealing. 855 42

Using digitonin-permeabilised bovine adrenal chromaffin cells, the effects of botulinum neurotoxin light chains on exocytosis triggered by Ca2+ or by GppNHp were examined. Botulinum neurotoxin D light chain, prepared as a His(6)-tagged recombinant protein, cleaved VAMP and substantially inhibited catecholamine release due to Ca2+ and GppNHp. Botulinum neurotoxin C1 and E light chains produced partial inhibition of both Ca(2+)- and GppNHp-induced catecholamine release. These results suggest that Ca(2+)-dependent exocytosis and Ca(2+)-independent exocytosis triggered by a non-hydrolysable GTP analogue occurs via a SNARE-dependent mechanism in chromaffin cells.
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PMID:Botulinum neurotoxin light chains inhibit both Ca(2+)-induced and GTP analogue-induced catecholamine release from permeabilised adrenal chromaffin cells. 864 68

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.
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PMID:Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. 898 82

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.
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PMID:Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25 for proteolysis. 941 82

SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.
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PMID:Targeting of SNAP-23 and SNAP-25 in polarized epithelial cells. 945 64

We demonstrated that botulinum neurotoxin attenuated the spontaneous beating rate of cultured cardiac myocytes. Primary cultured cardiac myocytes were prepared from the ventricles of neonatal Wistar rats (1-3 days old). On 7 days after cell seeding, botulinum toxin type A incorporated into liposomes was added to the culture medium. At a final concentration of 5.0 micrograms/ml, botulinum toxin markedly attenuated the beating rate of cardiac myocytes within 2-4 hours. These results demonstrated the effect of SNARE-complex proteins on the spontaneous beating of cardiac myocytes.
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PMID:Negative chronotropic effect of botulinum toxin on neonatal rat cardiac myocytes. 951 66

Botulinum neurotoxins type A and E (BoNT/A and /E) are metalloproteases with a unique specificity for SNAP-25 (synaptosomal-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It was proposed that this specificity is based on the recognition of a nine-residue sequence, termed SNARE motif, which is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we report on recent studies which provide evidence for the involvement of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and /E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and /E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. We also report on studies of poisoning human neuromuscular junctions with either BoNT/A or BoNT/E and describe the unexpected finding that the time of recovery of function after poisoning is much shorter in the case of type E with respect to type A intoxication. These data are discussed in terms of the different sites of action of the two toxins within SNAP-25.
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PMID:On the action of botulinum neurotoxins A and E at cholinergic terminals. 978 57

Rat parotid acinar cells secrete amylase through the stimulation of beta-adrenoceptors followed by accumulation of intracellular cAMP. However, it remains unclear at the molecular level how secretory granules fuse with the apical membranes. We have examined whether SNARE proteins are involved in exocytosis in the salivary glands, and have found that one of the SNARE proteins, VAMP-2, is localized at the secretory granule membrane of rat parotid acinar cells. Moreover, botulinum neurotoxin B, which has endoprotease activity that cleaves VAMP-2, inhibited cAMP-dependent amylase release but did not inhibit basal secretion in the absence of cAMP. These results suggest that VAMP-2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. In contrast, both neurotoxins A and C1 (endoproteases that cleave SNAP-25 and syntaxin 1 respectively) failed to inhibit cAMP-dependent amylase release. Therefore, neither SNAP-25 nor syntaxin 1 are involved in amylase secretion in the parotid glands. Clarification of the mechanism of secretion will require the identification of proteins that interact and function cooperatively with VAMP-2. This approach may also reveal details of the molecular mechanism by which the cAMP facilitates secretion in other systems, including neurotransmission.
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PMID:Snare proteins essential for cyclic AMP-regulated exocytosis in salivary glands. 982 92

Tricyclic antidepressants (e.g., imipramine, desipramine) are currently used in the treatment of mood disorders such as depression. At the cellular level they inhibit the re-uptake of the exocytosed monoamines serotonin and noradrenaline. However, they also stimulate phospholipase C activity and the production of the second messenger inositol 1,4,5-trisphosphate. Since phospholipase C activation can also lead to the production of the protein kinase C activator diacylglycerol, we have undertaken experiments to see whether acutely applied desipramine could change the synaptic strength of neurons in a protein kinase C-dependent manner. Experiments performed with cultured hippocampal neurons dissociated from neonatal rats revealed that desipramine rapidly enhanced the spontaneous vesicular release of glutamate. This was observed by measuring the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. Analysis of amplitude distribution histograms indicated a presynaptic site of action. The protein kinase inhibitor staurosporine and down-regulation of protein kinase C activity greatly reduced the desipramine-dependent enhancement of the frequency of tetrodotoxin-resistant spontaneous excitatory postsynaptic currents. This presynaptic modulation requires SNARE proteins because cleavage of SNAP-25 with the botulinum neurotoxin A strongly reduced the desipramine-induced glutamate release. Thus, acute applications of desipramine stimulated the ongoing neurotransmitter release pathway, probably by activating protein kinase C. Our data indicate that tricyclic antidepressant drugs not only act on serotoninergic and/or noradrenergic cells but can also modify the activity of glutamatergic neurons.
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PMID:Acute application of the tricyclic antidepressant desipramine presynaptically stimulates the exocytosis of glutamate in the hippocampus. 1021 74

The temporal sequence of SNARE protein interactions that cause exocytosis is unknown. Blockade of synaptic neurotransmitter release through cleavage of VAMP/synaptobrevin by tetanus toxin light chain (TeNT-LC) was accelerated by nerve stimulation. Botulinum/B neurotoxin light chain (BoNT/B-LC), which cleaves VAMP at the same site as TeNT-LC, did not require stimulation. Because TeNT-LC requires the N-terminal coil domain of VAMP for binding but BoNT/B-LC requires the C-terminal coil domain, it seems that, before nerve activity, the N-terminal domain is shielded in a protein complex, but the C-terminal domain is exposed. This N-terminal complex lasts until nerve activity occurs and may serve to cock synaptic vesicles for immediate exocytosis upon Ca2+ entry.
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PMID:Activity-dependent changes in partial VAMP complexes during neurotransmitter release. 1057 Apr 84


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