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)

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.
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PMID:Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex. 762 35

Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.
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PMID:Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers. 884 45

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N-ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is expressed in pancreatic B-cells and its cleavage by botulinum neurotoxin E (BoNT/E) abolishes stimulated secretion of insulin. In the nervous system, two SNAP-25 isoforms (a and b) have been described that are produced by alternative splicing. Here it is shown, using reverse transcriptase PCR, that messages for both SNAP-25 isoforms are expressed in primary pancreatic B and non-B cells as well as in insulin-secreting cell lines. After transfection, both isoforms can be detected at the plasma membrane as well as in an intracellular perinuclear region in the insulin-secreting cell line, HIT. To test for the functional role of the two isoforms in insulin secretion, mutant forms of SNAP-25a and b resistant against cleavage by BoNT/E were generated. Such mutant SNAP-25, when expressed in HIT cells, is not inactivated by BoNT/E and its ability to restore insulin secretion can thus be investigated. To obtain the toxin-resistant mutant isoforms, the sequence around the BoNT/E cleavage site (R176QIDRIM182) was changed to P176QIKRIT182. This is the sequence of the equivalent region of human SNAP-23 (P187-T194), which has been shown to be resistant to BoNT/E. The mutant SNAP-25 was resistant to BoNT/E in vitro and in vivo and both mutant isoforms were able to reconstitute insulin secretion from toxin-treated HIT cells.
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PMID:SNAP-25a and -25b isoforms are both expressed in insulin-secreting cells and can function in insulin secretion. 1008 40

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

The tSNARE (the target-membrane soluble NSF-attachment protein receptor, where NSF is N -ethylmaleimide-sensitive fusion protein) synaptosomal-associated protein of 25 kDa (SNAP-25) is implicated in regulated insulin secretion. In pheochromocytoma PC12 cells, SNAP-25 is phosphorylated at Ser(187), which lies in a region that is important for its function. The aims of the present study were to determine whether SNAP-25 is phosphorylated at Ser(187) in insulin-secreting cells and, if so, whether this is important for regulated insulin secretion. The major findings are: (i) SNAP-25 is rapidly and reversibly phosphorylated on Ser(187) in both rat insulinoma INS-1 cells and rat islets in response to the phorbol ester, PMA; (ii) less than 35% of SNAP-25 in INS-1 cells is phosphorylated in response to PMA, and phosphorylation is limited to plasma-membrane-associated SNAP-25; (iii) both SNAP-25 isoforms (a and b) are phosphorylated, with 1.8-fold greater phosphorylation for SNAP-25b in response to PMA; (iv) in rat islets, Ser(187) phosphorylation is stimulated by glucose or carbachol, albeit to a lesser extent than by PMA, but not by cAMP; (v) insulin secretion from botulinum neurotoxin E-treated hamster insulinoma tumour (HIT) cells, transfected with toxin-resistant Ser(187)-->Ala or Ser(187)-->Asp mutant SNAP-25, was similar to that of wild-type HIT cells. Furthermore, in rat islets no correlation was found between the extent of SNAP-25 phosphorylation at Ser(187) in response to secretagogues and stimulation of insulin release; (vi) use of protein kinase C (PKC) inhibitors suggests that glucose stimulates SNAP-25 phosphorylation via conventional and non-conventional PKC isoforms. In summary, although SNAP-25 phosphorylation at Ser(187) occurs in insulin-secreting cells and is mediated by PKC, it does not appear to play a major role in regulated insulin secretion.
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PMID:Phosphorylation of SNAP-25 on serine-187 is induced by secretagogues in insulin-secreting cells, but is not correlated with insulin secretion. 1216 83

Evidence indicates that accumulation of excitotoxic mediators, such as glutamate, contributes to neuronal damage after an ischaemic insult. It is not clear, however, whether this accumulation is due to excess synaptic release or to impaired uptake. To test a role for synaptic release, here we investigated the neuroprotective potential of the synaptic blocker botulinum neurotoxin E (BoNT/E), that prevents vesicle fusion via the cleavage of the SNARE (soluble NSF-attachment receptor) protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Focal ischaemia was induced in vivo by infusing the potent vasoconstricting peptide endothelin-1 (ET-1) into the CA1 area of the hippocampus in adult rats; BoNT/E or vehicle were administered into the same site 20 min later. Injection of ET-1 was found to produce a transient and massive increase in glutamate release that was potently antagonized by BoNT/E. To assess whether blocking transmitter release translates into neuroprotection, the extent of the ischaemic damage was determined 24 h and 6 weeks after the insult. We found that BoNT/E administration consistently reduced the loss of CA1 pyramidal neurons at 24 h. The neuroprotective effect of BoNT/E, however, was no longer significant at 6 weeks. These data provide evidence that blockade of synaptic transmitter release delays neuronal cell death following focal brain ischaemia, and underline the importance of assessing long-term neuroprotection in experimental stroke studies.
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PMID:Acute neuroprotection by the synaptic blocker botulinum neurotoxin E in a rat model of focal cerebral ischaemia. 2044 49

Clostridium botulinum is a gram-positive anaerobic rod that forms endospores. This bacterium produces large molecular toxin complexes, namely botulinum toxin complexes (progenitor toxins). It (L toxin complex) is composed of a single neurotoxin molecule (BoNT with a molecular weight of 150 kDa), a single nontoxic nonhemagglutinin molecule (NTNHA), and a hemagglutinin complex (HA). On food-borne botulism, nontoxic components have the roles of protecting toxin protein from the degeneration and degradation action of acids and proteases existing in the gastrointestinal tract. The HA facilitates transport when progenitor toxins cross the intestinal epithelial barrier to enter the systemic circulation. BoNT disassociates from the toxin complexes in the systemic circulation. BoNT is immunologically classified into 7 serotypes, A to G. Serotypes A, B, E, and F are the causative agents of human botulism. The active BoNT molecules are composed of 2 chains that are termed the heavy chain (c. 100 kDa) and the light chain (c. 50 kDa); these are covalently connected by a disulfide bond. The light chains have a tetrahedral zinc binding motif conteining a consensus HExxH amino acid sequence, and exhibit metalloprotease activity. After BoNTs reach the neuromuscular junction (the peripheral nerve ending), these are endocytosed in lipid vesicles (synaptic vesicles), and the light chain is released into the cytosol of a nerve cell via a translocation event through the phospholipid vesicle membrane. The light chains of BoNTs (zinc endopeptidases) cleave core proteins involved in the trafficking and release of neurotransmitters (acetylcholine), including synaptobrevin, SNAP-25, and syntaxin. These proteins comprise the synaptic members of the SNARE complex (soluble NSF (N-ethylmaleimide-sensitive fusionprotein) attachment protein) that have a central role in membrane fusion events. The selective proteolysis of these SNARE proteins inhibits neurotransmitter release from neurons. Botulism occurs via a series of processes that cause muscular paralysis in human and animals.
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PMID:[Clostridium botulinum and botulinum neurotoxin]. 2174 46

Although migraine is a common, paroxysmal, highly disabling disorder, the primary cause and the pathomechanism of migraine attacks are enigmatic. Experimental results suggest that activation of the trigeminovascular system is crucial in its pathogenesis. This activation leads to the release of vasoactive neuropeptides (calcitonin gene-related peptide - CGRP, and substance P - SP) and to neurogenic inflammation, and peripheral and central sensitisation are expressed. Botulinum neurotoxin-A (BoNT-A), a potent toxin produced by Clostridium botulinum, affects the nervous system through specific cleavage of the soluble NSF-attachment protein receptor complex (SNARE), like synaptosomal-associated protein of 25 kDa (SNAP-25). The result of this multistage process is blockade of the presynaptic release of pain neurotransmitters such as CGRP, SP and glutamate. A pooled analysis of the data from two programmes of Phase 3 Research Evaluating Migraine Prophylaxis Therapy (PREEMPT 1 and 2) with BoNT-A in chronic migraine demonstrated significant benefit of BoNT-A over placebo with regard to the numbers of headache days and migraine episodes. BoNT-A diminished the frequency of acute headache pain medication intake, and resulted in reductions in headache impact and improvements in scores on the Migraine-Specific Quality of Life Questionnaire. The treatments with BoNT-A proved safe and were well tolerated.
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PMID:[Botulinum neurotoxin--a therapy in migraine]. 2313 25

The detection of catalytically active botulinum neurotoxins (BoNTs) can be achieved by monitoring the enzymatic cleavage of soluble NSF (N-ethylmaleimide-sensitive-factor) attachment protein receptor (SNARE) proteins by the toxins' light chains (LC) in cleavage-based assays. Thus, for sensitive BoNT detection, optimal cleavage conditions for the clinically relevant A-F serotypes are required. Until now, a systematic evaluation of cleavage conditions for the different BoNT serotypes is still lacking. To address this issue, we optimized cleavage conditions for BoNT/A-F using the Taguchi design-of-experiments (DoE) method. To this aim, we analyzed the influence of buffer composition (pH, Zn2+, DTT (dithiothreitol), NaCl) as well as frequently used additives (BSA (bovine serum albumin), Tween 20, trimethylamine N-oxide (TMAO)) on BoNT substrate cleavage. We identified major critical factors (DTT, Zn2+, TMAO) and were able to increase the catalytic efficiency of BoNT/B, C, E, and F when compared to previously described buffers. Moreover, we designed a single consensus buffer for the optimal cleavage of all tested serotypes. Our optimized buffers are instrumental to increase the sensitivity of cleavage-based assays for BoNT detection. Furthermore, the application of the Taguchi DoE approach shows how the method helps to rationally improve enzymatic assays.
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PMID:Optimization of SNAP-25 and VAMP-2 Cleavage by Botulinum Neurotoxin Serotypes A-F Employing Taguchi Design-of-Experiments. 3161 66