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)

It is generally accepted that the SNARE proteins form the core of the machinery for intracellular membrane fusion and that formation of a SNARE complex is crucially important. Our aim is to dissect the molecular roles of the SNARE proteins and their regulators in physiological membrane fusion during exocytosis. We have developed approaches that allow us to manipulate protein expression in model secretory cells, PC12 and adrenal chromaffin cells, and to combine this with assay of exocytosis at high-time resolution using carbon-fiber amperometry. This technique allows us to assess the extent of exocytosis and to follow the kinetics of single secretory granule release events with millisecond time resolution. We established that manipulation of proteins involved in the exocytotic machinery can lead to detectable and interpretable changes in exocytosis kinetics that have revealed novel roles in late stages of exocytosis. Using this approach we have begun to analyze the function of SNAP-25B using a mutant resistant to the Clostridial neurotoxin BoNT/E. This SNAP-25 mutant can reconstitute exocytosis in BoNT/E-treated cells. With this construct it is possible to analyze the consequences of any introduced mutation in the absence of functional endogenous protein. We review here its use in the analysis of palmitoylated cysteines of SNAP-25 and the conserved residues of the 0 layer of the SNARE complex. The data suggest an important role of the cysteines, but not the 0 layer glutamines, in triggered exocytosis.
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PMID:Molecular analysis of SNAP-25 function in exocytosis. 1243 21

Free-energy terms that contribute to complex formation between the catalytic domain of botulinum neurotoxin type B (BoNT/B-L(C)) and a 36-residue synaptobrevin fragment were estimated by using a combination of microscopic simulations and continuum methods. The complex for a non-hydrolyzed substrate was calculated by optimizing an energy function applied to the X-ray co-crystal structure of BoNT/B-L(C) bound with reaction products from a cleaved synaptobrevin peptide, refined to high crystallographic thermal factors. The estimated absolute binding affinity of the simulation structure is in good qualitative agreement with the experimental free energy of Michaelis complex formation, given the approximations of the model calculations. The simulation structure revealed significant complex stabilization from the hydrophobic effect, while the electrostatic cost of releasing water molecules from the interface determined to be highly unfavorable. By partitioning the total electrostatic and hydrophobic terms into residue free-energy contributions, a binding-affinity 'signature' for synaptobrevin was developed from the optimized conformation. The results demonstrate the effect of substrate length on complex formation and identify a peripheral high-affinity binding site near the N-terminal region that might initiate cooperative activation responsible for the large minimal substrate length requirement. The so-called SNARE motif is observed to contribute negligible free energy of binding.
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PMID:Free-energy contributions to complex formation between botulinum neurotoxin type B and synaptobrevin fragment. 1245 72

Intoxication by the zinc protease botulinus neurotoxin A (BoNT-A) results from cleavage of a single Q-R bond in the neuronal protein SNAP-25, which disables the docking mechanism required for neurotransmitter release. In the present study, potential inhibitors of BoNT-A were assessed from their effects on the BoNT-A cleavage of a synthetic 17-mer peptide (SNAP-25, residues 187-203) spanning the Q-R cleavage site. Compounds that inhibited BoNT-A included thiols (zinc chelators) such as dithiothreitol, dimercaptopropanesulfonic acid, mercaptosuccinic acid and captopril. In addition, compounds containing multiple acidic functions, such as the SNARE motif V2 (ELDDRADALQ), the tripeptide Glu-Glu-Glu and the steroid glycoside glycyrrhizic acid, were effective inhibitors. 'Hinge' peptide mini-libraries (PMLs) having the structure acetyl-X(1)-X(2)-linker-X(3)-X(4)-NH(2) or X(1)-X(2)-linker-X(3), where X(1)-X(4) were mixtures of selected amino acids and the flexible linker was 4-aminobutyric acid, also provided effective inhibition. Targeted PMLs containing the acidic amino acids Asp and Glu, the scissile-bond amino acids Gln and Arg and the zinc chelators His and Cys produced pronounced inhibition of BoNT-A. Deconvolution of these libraries will provide novel ligands with improved inhibitory potency as leads in the design of peptide mimetics to treat BoNT poisoning.
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PMID:Discovery and design of novel inhibitors of botulinus neurotoxin A: targeted 'hinge' peptide libraries. 1251 30

Clostridial neurotoxins are potent inhibitors of synaptic function, with the zinc-dependent proteolytic light chain (LC) portion of the toxin cleaving one of three neural SNARE proteins. In nature, the LC is expressed as a part of a much larger toxin and hemagglutinin complex, protecting it from environmental degradation and preserving its catalytic activity. We developed forms of the LC of type A botulinum neurotoxin (BoNT-A) with parts of the larger toxin gene, for use as reagents in high-throughput assays to screen for potential LC antagonists, to further elucidate the toxin's mechanism of action, and to study immunological responses to the toxin. Three BoNT-A constructs were engineered and expressed: the LC, LC with translocation region (LC+H(n)), and the LC with the belt portion of the translocation region (LC+Belt). Purification was optimized to a two-step process, with relatively high yields of all three constructs obtained. Activity assays showed all three constructs to be active, with the LC being the most active. Immunogenic protection against native BoNT-A toxin challenge was observed for all three constructs, with the best protection observed with the LC+H(n) and LC+Belt proteins.
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PMID:Expression, purification, and efficacy of the type A botulinum neurotoxin catalytic domain fused to two translocation domain variants. 1272 73

Nerve sprouts emerge from motor nerve terminals following blockade of exo-endocytosis for more than 3 days by botulinum neurotoxin (BoNT), and form functional synapses, albeit temporary. Upon restoration of synaptic activity to the parent terminal 7 and 90 days after exposure to BoNT/F or A respectively, a concomitant retraction of the outgrowths was observed. BoNT/E caused short-term neuroparalysis, and dramatically accelerated the recovery of BoNT/A-paralyzed muscle by further truncation of SNAP-25 and its replenishment with functional full-length SNARE. The removal of 9 C-terminal residues from SNAP-25 by BoNT/A leads to persistence of the inhibitory product due to the formation of a nonproductive SNARE complex(es) at release sites, whereas deletion of a further 17 amino acids permits replenishment and a speedy recovery.
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PMID:Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation. 1272 43

Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.
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PMID:VAMP/synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes. 1272 12

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release by cleaving the 206-amino-acid SNARE protein, SNAP-25. For each BoNT serotype, cleavage of SNAP-25 results in the loss of intact protein, the production of an N-terminal truncated protein, and the generation of a small C-terminal peptide. Peptides that mimic the C-terminal fragments of SNAP-25 following BoNT/A or BoNT/E cleavage were shown to depress transmitter release in bovine chromaffin cells and in Aplysia buccal ganglion cells. Similarly, the N-terminal-truncated SNAP-25 resulting from BoNT/A or BoNT/E cleavage has been found to inhibit transmitter exocytosis in various systems. With one exception, however, the inhibitory action of truncated SNAP-25 has not been demonstrated at a well-defined cholinergic synapse. The goal of the current study was to determine the level of inhibition of neurotransmitter release by N-terminal BoNT/A- or BoNT/E-truncated SNAP-25 in two different neuronal systems: cholinergically coupled Aplysia neurons and rat hippocampal cell cultures. Both truncated SNAP-25 products inhibited depolarization-dependent glutamate release from hippocampal cultures and depressed synaptic transmission in Aplysia buccal ganglion cells. These results suggest that truncated SNAP-25 can compete with endogenous SNAP-25 for binding with other SNARE proteins involved in transmitter release, thus inhibiting neurotransmitter exocytosis.
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PMID:Inhibition of neurotransmitter release by peptides that mimic the N-terminal domain of SNAP-25. 1276 Apr 19

The refinement and plasticity of neuronal connections require synaptic activity and neurotrophin signalling; their specific contributions and interplay are, however, poorly understood. We show here that brain-derived neurotrophic factor (BDNF) increased spine density in apical dendrites of CA1 pyramidal neurones in organotypic slice cultures prepared from postnatal rat hippocampal slices. This effect was observed also in the absence of action potentials, and even when miniature synaptic transmission was inhibited with botulinum neurotoxin C (BoNT/C). There were, however, marked differences in the morphology of individual spines induced by BDNF across these different levels of spontaneous ongoing synaptic activity. During both normal synaptic transmission, and when action potentials were blocked with TTX, BDNF increased the proportion of stubby, type-I spines. However, when SNARE-dependent vesicular release was inhibited with BoNT/C, BDNF increased the proportion of thin, type-III spines. Our results indicate that BDNF increases spine density irrespective of the levels of synaptic transmission. In addition, miniature synaptic transmission provides sufficient activity for the functional translation of BDNF-triggered spinogenesis into clearly defined morphological spine types, favouring those spines potentially responsible for coordinated Ca2+ transients thought to mediate synaptic plasticity. We propose that BDNF/TrkB signalling represents a mechanism of expression of both morphological and physiological homeostatic plasticity in the hippocampus, leading to a more efficient synaptic information transfer across widespread levels of synaptic activity.
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PMID:Miniature synaptic transmission and BDNF modulate dendritic spine growth and form in rat CA1 neurones. 1450 Jul 67

Phorbol esters, activators of protein kinase C (PKC), have been shown to enhance synaptic transmission. One potential downstream target of PKC in the presynaptic terminal is the soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor (SNARE) SNAP-25, which has a PKC phosphorylation site in its C-terminal coil centered at serine 187 (S187/Ser187). We examined the role of S187 in hippocampal synaptic transmission. After proteolytic cleavage of native SNAP-25 by botulinum neurotoxin E (BoNT/E), synaptic transmission was restored in a subset of transfected CA3 pyramidal cells with a toxin-resistant form of SNAP-25 containing unaltered S187 (Swt), S187 mutated to alanine (SA) or S187 mutated to glutamate (SE). We observed that phorbol-12,13-diacetate (PDAc, 10 microM) induced potentiation of neurotransmission to a similar degree for both Swt and SA (2.4-fold and 3.1-fold increase, respectively). Furthermore, basal levels of transmission mediated by SE were reduced relative to that of Swt (failure rates of 72% and 41%, respectively). Together, these data suggest that phosphorylation of SNAP-25 S187 does not mediate the observed enhancement of neurotransmission by phorbol esters at hippocampal synapses.
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PMID:SNAP-25 Ser187 does not mediate phorbol ester enhancement of hippocampal synaptic transmission. 1452 23

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.
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PMID:High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E. 1455 Nov 99


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