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)

The vesicular neuroexocytosis process consists of two important steps: fusion of transmitter-loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into the synaptic cleft. We previously reported that in nerve growth factor (NGF)-differentiated PC12 cells, arachidonic acid (AA) release is associated with acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits both processes and AA itself or a phospholipase A(2) (PLA(2)) activator can cause ACh release in BoNT/A-poisoned cells in which SNAP-25 has supposedly been hydrolyzed. In the present study, we examined the roles of two endogenous intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP-25. A PLA(2) activator, mastoparan, was used to induce the release of AA and ACh from NGF-differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca(2+) influx via the neuronal-type voltage-sensitive Ca(2+) channels. Release of ACh followed a rise in intracellular free Ca(2+) concentration; the increased Ca(2+) activated PLA(2) and, thereby, increased the AA level. Scanning and transmission electron microscopy confirmed that mastoparan-induced ACh and AA release were not due to simple diffusion through damaged plasma membranes. Treatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a specific monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K(+) induced ACh exocytosis. The results support the conclusion that PLA(2) and AA have important roles in neuroexocytosis that are independent of SNAP-25. Both PLA(2) and AA have been shown to be involved in actin cytoskeletal organization related to vesicle fusion and exocytosis. This mechanism may be an alternative target of BoNT/A other than SNAP-25.
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PMID:Phospholipaise A2 and arachidonic acid-mediated mechanism of neuroexocytosis: a possible target of botidinum neurotoxin A other then SNAP-25. 1059 96

Capsaicin, the pungent component of "hot" chili peppers, selectively activates a distinct population of primary sensory neurons responsive to noxious stimuli. Many of these fibres express neuropeptides including the tachykinin, substance P. Using cultured dorsal root ganglion neurons, we found that capsaicin (10 microM) stimulated a 2-fold increase in release of substance P in the absence of extracellular Ca(2+). Elevated potassium (75 mM) was unable to induce release under these conditions. The introduction of Ca(2+) enhanced capsaicin-induced release and brought about a robust response to potassium. Preincubation of cells with botulinum neurotoxin A (100 nM) completely blocked potassium-induced release but the capsaicin response, in the absence of Ca(2+), was unaffected. However, toxin treatment dramatically reduced capsaicin-stimulated release in the presence of Ca(2+). It is concluded that capsaicin induces release of substance P from dorsal root ganglion neurons via two mechanisms, one requiring extracellular Ca(2+) and the intact synaptosomal-associated protein 25 kDa (SNAP-25), and the other independent of extracellular Ca(2+) and not involving SNAP-25.
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PMID:Capsaicin-stimulated release of substance P from cultured dorsal root ganglion neurons: involvement of two distinct mechanisms. 1075 49

Several bacterial toxins are powerful and highly specific tools for studying basic mechanisms involved in cell biology. Whereas the clostridial neurotoxins are widely used by neurobiologists, many other toxins (i.e. toxins acting on small G-proteins or actin) are still overlooked. Botulinum neurotoxins (BoNT, serotypes A-G) and tetanus neurotoxin (TeNT), known under the generic term of clostridial neurotoxins, are characterized by their unique ability to selectively block neurotransmitter release. These proteins are formed of a light (Mr approximately 50) and a heavy (Mr approximately 100) chain which are disulfide linked. The cellular action of BoNT and TeNT involves several steps: heavy chain-mediated binding to the nerve ending membrane, endocytosis, and translocation of the light chain (their catalytic moiety) into the cytosol. The light chains each cleaves one of three, highly conserved, proteins (VAMP/synaptobrevin, syntaxin, and SNAP-25 also termed SNAREs) implicated in fusion of synaptic vesicles with plasma membrane at the release site. Hence, when these neurotoxins are applied extracellularly, they can be used as specific tools to inhibit evoked and spontaneous transmitter release from certain neurones whereas, when the membrane limiting steps are bypassed by the mean of intracellular applications, BoNTs orTeNT can be used to affect regulated secretion in various cell types. Several members of the Rho GTPase family have been involved in intracellular trafficking of synaptic vesicles and secretory organelles. As they are natural targets for several bacterial exoenzymes or cytotoxins, their role in neurotransmitter release can be probed by examining the action of these toxins on neurotransmission. Such toxins include: i) the non permeant C3 exoenzymes from C. botulinum or C. limosum which ADP-ribosylate and thereby inactivate Rho, ii) exoenzyme S from Pseudomonas aeruginosa which ADP-ribosylates different members of the Ras, Rab, Ral and Rap families, iii) toxin B from C. difficile which glucosylates Rho, Rac and CDC42, iv) lethal toxin from C. sordellii which glucosylates Rac, Ras and to a lesser extent, Rap and Ral, but not on Rho or CDC42, and v) CNF deamidases secreted by pathogenic strains of E. coli which activate Rho and, to a lesser extent, CDC42. Since these toxins or exoenzymes have no or little ability to enter into the neurones, they must be applied intraneuronally to bypass the membrane limiting steps. Injection of several of these toxins into Aplysia neurones allowed us to reveal a new role for Rac in the control of exocytosis. ADP-ribosylating enzymes, which specifically act on monomeric actin (C2 binary toxin from C. botulinum and iota toxin from C. perfringens), are potential tools to probe the role of actin filaments during secretion.
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PMID:[Analysis of synaptic neurotransmitter release mechanisms using bacterial toxins]. 1078 4

We have used carbon-fibre amperometry to examine the kinetics of individual secretory granule fusion/release events in bovine adrenal chromaffin cells. Transfection with plasmids encoding the light chains of botulinum neurotoxins (BoNTs) was used to investigate the effects of cleavage of syntaxin or SNAP-25 on exocytosis. Expression of BoNT/C1 or BoNT/E inhibited the extent of exocytosis that was evoked by application of digitonin/Ca(2+) to permeabilise and stimulate single chromaffin cells. Following neurotoxin expression, the residual release events were no different from those of control cells in their magnitude and kinetics from analysis of the amperometric spikes. In contrast, activation of protein kinase C (PKC) resulted in a modification of the kinetics of single granule release events. Following phorbol ester treatment, the amperometric spikes showed a significant decrease in their total charge due to a decrease in their mean half-width with increases in the rate of the initial rise and also the fall to baseline of the spikes. These changes were prevented by pre-treatment with the PKC inhibitor bisindolylmaleimide. These results suggest that PKC regulates the rate of fusion pore expansion and also subsequent pore closure or granule retrieval. A PKC-mediated regulation of kiss-and-run fusion may, therefore, control the extent of catecholamine release from single secretory granules. The experimental approach used here may provide further information on the protein constituents and regulation of the fusion pore machinery.
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PMID:Measurement of exocytosis by amperometry in adrenal chromaffin cells: effects of clostridial neurotoxins and activation of protein kinase C on fusion pore kinetics. 1086 33

The tSNARE SNAP-25 is expressed in pancreatic (beta)-cells and is involved in the regulated release of insulin. It has been shown previously that SNAP-25 associates with the plasma membrane consequent to palmitoylation of one or more cysteines in the central region of the molecule. The importance of palmitolyation in the biological function of SNAP-25 in exocytosis was not addressed. Furthermore, studies on both SNAP-25 and its non-palmitoylated homologues SNAP-29 and sec9, have suggested an alternative or complementary mechanism for membrane association involving interaction with syntaxin. To address these issues, we have now studied the behavior and biological activity of cysteine mutant SNAP-25 in insulin-secreting (HIT) cells. While 91% of native SNAP-25 was associated with the membrane, this value decreased to 56% for the single cysteine mutant C85/A and to 10% for the double (C85,88/A) and quadruple (C85,88,90,92/A) mutants. The mutant SNAP-25 forms were all found to bind syntaxin 1A with equal efficacy. Over-expression of syntaxin 1A in HIT cells allowed for partial relocalization of both the double and quadruple SNAP-25 cys mutants to the membrane. By introducing a further mutation to the SNAP-25 molecules to render them resistant to botulinum neurotoxin E, it was possible to study their ability to reconstitute regulated insulin secretion in toxin-treated HIT cells. Native SNAP-25 was able to fully reconstitute secretory activity in such cells. Despite the fact that the single cysteine mutant was significantly displaced to the cytosol, it still displayed 82% activity in the secretion reconstitution assay, and a similar discrepancy was seen for the double mutant. Even the quadruple mutant with no remaining cysteines was able to support a minimal level of secretion. It is concluded that both palmitoylation and binding to syntaxin are implicated in membrane association of SNAP-25. This as well as the discrepancy between membrane localization and biological activity of the cysteine mutants, suggests a complex, multi-component process for association of SNAP-25 with the membrane and its recruitment to a biologically productive state.
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PMID:Membrane localization and biological activity of SNAP-25 cysteine mutants in insulin-secreting cells. 1095 18

Botulinum neurotoxin serotypes A (BoNT/A) and E (BoNT/E) inhibit neurotransmitter release from peripheral cholinergic nerve terminals by cleaving different sites on SNAP-25, a protein involved in synaptic vesicle docking and exocytosis. Since recovery from BoNT/A is protracted, but reversal of BoNT/E intoxication is relatively rapid, it was of interest to determine whether sequential exposure to BoNT/A and BoNT/E could provide insight into the factors responsible for persistence of BoNT action. Extensor digitorum longus (EDL) muscles from rats were injected locally with 5 mouse LD(50) units of BoNT/A or 20 mouse LD(50) units of BoNT/E; these doses were selected to produce total paralysis of EDL muscles within 48 hr. Additional groups of rats were injected sequentially with either BoNT/A followed 48 h later by BoNT/E or with BoNT/E followed 48 h later by BoNT/A. Muscle tensions were elicited in situ in response to supramaximal stimulation of the peroneal nerve to monitor recovery from BoNT intoxication. Tensions returned to 53% and 94% of control, respectively, 7 and 15 days after injection of BoNT/E. In contrast, tensions in muscles injected with BoNT/A returned to only 2% and 12% of control at these time points. Preparations injected sequentially with BoNT/A followed by BoNT/E or with BoNT/E followed by BoNT/A exhibited slow recovery times resembling those recorded in the presence of BoNT/A alone. Pronounced atrophy of the EDL muscle was observed in rats injected with BoNT/A or in those receiving serotype combinations in either sequence, whereas no loss of muscle mass was observed in animals treated with BoNT/E alone. Data suggesting that BoNT/E can enter BoNT/A-treated preparations was obtained by findings that 3,4-diaminopyridine, which readily reversed muscle paralysis after BoNT/A exposure, lost this ability within 1 h of BoNT/E addition. Evidence that BoNT/E was able to cleave SNAP-25 at its characteristic site during sequential neurotoxin exposure was demonstrated by western blot analysis of cultured primary cortical neurons. Since the sequential exposure studies indicate that recovery from BoNT intoxication is lengthened by exposure to serotype A, but not shortened by exposure to serotype E, the duration of BoNT/A intoxication appears to be determined predominantly by the intracellular stability of catalytically active BoNT/A light chain.
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PMID:Persistence of botulinum neurotoxin A demonstrated by sequential administration of serotypes A and E in rat EDL muscle. 1097 41

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4 degrees C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency kcat/Km was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn2+ to a Zn2+-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form.
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PMID:Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active. 1119 72

Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.
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PMID:Protein kinase C modulates NMDA receptor trafficking and gating. 1127 28

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca(2+). In this case, vesicular release is restored by increasing intracellular Ca(2+). These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca(2+).
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PMID:The role of the synaptic protein snap-25 in the potency of botulinum neurotoxin type A. 1127 7

The calcium (Ca(2+)) regulation of neurotransmitter release is poorly understood. Here we investigated several aspects of this process in PC12 cells. We first showed that osmotic shock by 1 m sucrose stimulated rapid release of neurotransmitters from intact PC12 cells, indicating that most of the vesicles were docked at the plasma membrane. Second, we further investigated the mechanism of rescue of botulinum neurotoxin E inhibition of release by recombinant SNAP-25 COOH-terminal coil, which is known to be required in the triggering stage. We confirmed here that Ca(2+) was required simultaneously with the SNAP-25 peptide, with no significant increase in release if either the peptide or Ca(2+) was present during the priming stage as well as the triggering, suggesting that SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex assembly was involved in the final Ca(2+)-triggered event. Using this rescue system, we also identified a series of acidic surface SNAP-25 residues that rescued better than wild-type when mutated, due to broadened Ca(2+) sensitivity, suggesting that this charged patch may interact electrostatically with a negative regulator of membrane fusion. Finally, we showed that the previously demonstrated stimulation of exocytosis in this system by calmodulin required calcium binding, since calmodulin mutants defective in Ca(2+)-binding were not able to enhance release.
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PMID:Calcium regulation of exocytosis in PC12 cells. 1135 85


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