Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Clostridal neurotoxins (CNTs) are the causative agents of the neuroparalytic diseases botulism and tetanus. CNTs impair neuronal exocytosis through specific proteolysis of essential proteins called SNAREs. SNARE assembly into a low-energy ternary complex is believed to catalyse membrane fusion, precipitating neurotransmitter release; this process is attenuated in response to SNARE proteolysis. Site-specific SNARE hydrolysis is catalysed by the CNT light chains, a unique group of zinc-dependent endopeptidases. The means by which a CNT properly identifies and cleaves its target SNARE has been a subject of much speculation; it is thought to use one or more regions of enzyme-substrate interaction remote from the active site (exosites). Here we report the first structure of a CNT endopeptidase in complex with its target SNARE at a resolution of 2.1 A: botulinum neurotoxin serotype A (BoNT/A) protease bound to human SNAP-25. The structure, together with enzyme kinetic data, reveals an array of exosites that determine substrate specificity. Substrate orientation is similar to that of the general zinc-dependent metalloprotease thermolysin. We observe significant structural changes near the toxin's catalytic pocket upon substrate binding, probably serving to render the protease competent for catalysis. The novel structures of the substrate-recognition exosites could be used for designing inhibitors specific to BoNT/A.
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PMID:Substrate recognition strategy for botulinum neurotoxin serotype A. 1559 54

Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.
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PMID:BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani. 1561 31

Renshaw cells receive a high density of inhibitory synapses characterized by large postsynaptic gephyrin clusters and mixed glycinergic/GABAergic inhibitory currents with large peak amplitudes and long decays. These properties appear adapted to increase inhibitory efficacy over Renshaw cells and mature postnatally by mechanisms that are unknown. We tested the hypothesis that heterosynaptic influences from excitatory motor axon inputs modulate the development of inhibitory synapses on Renshaw cells. Thus, tetanus (TeNT) and botulinum neurotoxin A (BoNT-A) were injected intramuscularly at postnatal day 5 (P5) to, respectively, elevate or reduce motor axon firing activity for approximately 2 weeks. After TeNT injections, the average gephyrin cluster areas on Renshaw cells increased by 18.4% at P15 and 28.4% at P20 and decreased after BoNT-A injections by 17.7% at P15 and 19.9% at P20. The average size differences resulted from changes in the proportions of small and large gephyrin clusters. Whole-cell recordings in P9-P15 Renshaw cells after P5 TeNT injections showed increases in the peak amplitude of glycinergic miniature postsynaptic currents (mPSCs) and the fast component of mixed (glycinergic/GABAergic) mPSCs compared with controls (60.9% and 78.9%, respectively). GABAergic mPSCs increased in peak amplitude to a smaller extent (45.8%). However, because of the comparatively longer decays of synaptic GABAergic currents, total current transfer changes after TeNT were similar for synaptic glycine and GABA(A) receptors (56 vs 48.9% increases, respectively). We concluded that motor axon excitatory synaptic activity modulates the development of inhibitory synapse properties on Renshaw cells, influencing recruitment of postsynaptic gephyrin and glycine receptors and, to lesser extent, GABA(A) receptors.
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PMID:Regulation of gephyrin cluster size and inhibitory synaptic currents on Renshaw cells by motor axon excitatory inputs. 1564 85

Botulinum neurotoxins comprise seven distinct serotypes (A-G) produced by Clostridium botulinum. The crystal structure of the binding domain of the botulinum neurotoxin type B (BBHc) has been determined to 2A resolution. The overall structure of BBHc is well ordered and similar to that of the binding domain of the holotoxin. However, significant structural changes occur at what would be the interface of translocation and binding domains of the holotoxin. The loop 911-924 shows a maximum displacement of 14.8A at the farthest point. The N-terminal helix reorients and moves by 19.5A from its original position. BBHc is compared with the binding domain of the holotoxin of botulinum type A and B, and the tetanus C-fragment to characterize the heavy chain-carbohydrate interactions. The probable reasons for different binding affinity of botulinum and tetanus toxins are discussed.
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PMID:N-terminal helix reorients in recombinant C-fragment of Clostridium botulinum type B. 1578 Dec 37

TeNT is the causative agent of the neuroparalytic disease tetanus. A key component of TeNT is its light chain, a Zn(2+) endopeptidase that targets SNAREs. Recent structural studies of closely related BoNT endopeptidases indicate that substrate-binding exosites remote from a conserved active site are the primary determinants of substrate specificity. Here we report the 2.3 A X-ray crystal structure of TeNT-LC, determined by combined molecular replacement and MAD phasing. As expected, the overall structure of TeNT-LC is similar to the other known CNT light chain structures, including a conserved thermolysin-like core inserted between structurally distinct amino- and carboxy-terminal regions. Differences between TeNT-LC and the other CNT light chains are mainly limited to surface features such as unique electrostatic potential profiles. An analysis of surface residue conservation reveals a pattern of relatively high variability matching the path of substrate binding around BoNT/A, possibly serving to accommodate the variations in different SNARE targets of the CNT group.
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PMID:2.3 A crystal structure of tetanus neurotoxin light chain. 1589 88

Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.
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PMID:The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles. 1590 Jul 6

Clostridium neurotoxins, comprising the tetanus neurotoxin and the seven antigenically distinct botulinum neurotoxins (BoNT/A-G), are among the known most potent bacterial protein toxins to humans. Although they have similar function, sequences and three-dimensional structures, the substrate specificity and the selectivity of peptide bond cleavage are different and unique. Tetanus and botulinum type B neurotoxins enzymatically cleave the same substrate, vesicle-associated membrane protein, at the same peptide bond though the optimum length of substrate peptide required for cleavage by them is different. Here, we present the first experimentally determined three-dimensional structure of the catalytic domain of tetanus neurotoxin and analyze its active site. The structure provides insight into the active site of tetanus toxin's proteolytic activity and the importance of the nucleophilic water and the role of the zinc ion. The probable reason for different modes of binding of vesicle-associated membrane protein to botulinum neurotoxin type B and the tetanus toxin is discussed. The structure provides a basis for designing a novel recombinant vaccine or structure-based drugs for tetanus.
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PMID:Structural analysis of the catalytic domain of tetanus neurotoxin. 1590 88

Neurotransmitter release is triggered by calcium ions and depends critically on the correct function of three types of SNARE [soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor] proteins. With use of the large calyx of Held presynaptic terminal from rats, we found that cleavage of different SNARE proteins by clostridial neurotoxins caused distinct kinetic changes in neurotransmitter release. When elevating calcium ion concentration directly at the presynaptic terminal with the use of caged calcium, cleavage of SNAP-25 by botulinum toxin A (BoNT/A) produced a strong reduction in the calcium sensitivity for release, whereas cleavage of syntaxin using BoNT/C1 and synaptobrevin using tetanus toxin (TeNT) produced an all-or-nothing block without changing the kinetics of remaining vesicles. When stimulating release by calcium influx through channels, a difference between BoNT/C1 and TeNT emerged, which suggests that cleavage of synaptobrevin modifies the coupling between channels and release-competent vesicles.
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PMID:Distinct kinetic changes in neurotransmitter release after SNARE protein cleavage. 1602 Jul 41

Clostridial neurotoxins are comprised of botulinum (BoNT) and tetanus (TeNT), which share significant structural and functional similarity. Crystal structures of the binding domain of TeNT complexed with disialyllactose (DiSia) and a tri-peptide Tyr-Glu-Trp (YEW) have been determined to 2.3 and 2.2 A, respectively. Both DiSia and YEW bind in a shallow cleft region on the surface of the molecule in the beta-trefoil domain, interacting with a set of common residues, Asp1147, Asp1214, Asn1216, and Arg1226. DiSia and YEW binding at the same site in tetanus toxin provides a putative site that could be occupied either by a ganglioside moiety or a peptide. Soaking experiments with a mixture of YEW and DiSia show that YEW competes with DiSia, suggesting that YEW can be used to block ganglioside binding. A comparison with the TeNT binding domain in complex with small molecules, BoNT/A and /B, provides insight into the different modes of ganglioside binding.
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PMID:Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin. 1610 15

An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.
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PMID:Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments. 1614 75


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