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 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences. 317 18

Botulinum neurotoxin type A consists of a disulfide-linked light and heavy chain, with an intradisulfide present within the C-terminal half of the latter. The functional consequences of reducing these bonds and alkylating the thiols were investigated. Modification of free cysteine residues had no effect on the toxicity in mouse bioassays or on acetylcholine release in the mouse nerve-diaphragm and the buccal ganglion of Aplysia californica. However, reduction of the toxin prior to alkylation drastically decreased neuroparalytic potency; yet, this derivative inhibited transmitter release if injected directly into a presynaptic neuron in the Aplysia ganglion or added to bovine permeabilized adrenal chromaffin cells. Its antagonism of the action of botulinum neurotoxin A at mammalian motor nerve endings and Aplysia neurons indicates retention of the ability to bind to the toxin's productive ecto-acceptors. Thus, the abolition of the toxicity of extracellularly applied botulinum neurotoxin A by the cleavage of both disulfides, and the alkylation of the half-cystines involved, results from ineffective uptake. Modified forms of the isolated chains of botulinum neurotoxin A were utilized to determine which of the disulfides were necessary for internalization. Alkylation of the cysteines in the light and heavy chains, including those involved in the interchain bond but excluding those of the intact disulfide in the heavy chain, revealed that the intermolecular bond must be present, or the thiols concerned unmodified, for botulinum neurotoxin A to undergo membrane translocation into Aplysia neurons.
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PMID:A role for the interchain disulfide or its participating thiols in the internalization of botulinum neurotoxin A revealed by a toxin derivative that binds to ecto-acceptors and inhibits transmitter release intracellularly. 810 36

Syntaxin 1A has been identified previously as a neural-cell-specific, membrane-anchored receptor protein required for docking and fusion of synaptic vesicles with the presynaptic plasma membrane. Syntaxin 1A consists of 288 amino acid residues including a 265-residue N-terminal region exposed to the cytoplasm and a C-terminal hydrophobic stretch of 23 residues believed to anchor syntaxin to the plasma membrane. Using a human fat-cell library we have isolated a novel cDNA clone of syntaxin 1A containing an insert of 91 bp in codon 226. This insert and subsequent frame shift generated a cDNA that codes for a truncated protein of 260 residues without the C-terminal transmembrane domain characteristic of the syntaxin family. Analysis of the deduced amino acid sequence of the new cDNA clone, termed syntaxin 1C, showed that it was identical for the first 226 residues with the previously described neural syntaxin 1A, and diverged thereafter. The truncated protein lacked the botulinum neurotoxin C cleavage site (Lys253-Ala254), a feature of the syntaxin 1A protein, because of the novel C-terminal domain of 34 residues. The new C-terminal region contained a single cysteine residue and was moderately rich in proline, with three repeats of a PXP motif. The insert occurred within the region encoding the coiled-coil motifs required for interactions with synaptobrevin, alpha-SNAP (SNAP being soluble N-ethylmaleimide-sensitive factor attachment protein) and n-Sec1/Munc-18 (n-Sec1 being the rat brain homologue of yeast Sec1p and Munc-18 the mammalian homologue of Caenorhabditis elegans unc-18, but five residues outside the domain previously mapped as being required for binding SNAP-25. Interaction studies in vitro suggested that unlike syntaxin 1A, which binds to both Munc-18a and- 18b, syntaxin 1C binds only to Munc-18b. The new isoform syntaxin 1C, which might be generated by alternative splicing of the syntaxin 1 gene, was expressed in several human tissues, including brain. Immuno-precipitation and immunoblotting with the monoclonal antibody HPC-1 and a polyclonal antibody raised against a peptide corresponding to the unique C-terminal 35 residues of syntaxin 1C failed to detect syntaxin 1C at the protein level in extracts of muscle, fat or brain.
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PMID:Novel isoform of syntaxin 1 is expressed in mammalian cells. 900 14

Rat pinealocytes accumulate glutamate in microvesicles and secrete it through exocytosis so as to transmit signals intercellularly. Glutamate is involved in the negative regulation of norepinephrine-stimulated melatonin production. In this study, we found that aspartate is also released from cultured rat pinealocytes during the exocytosis of glutamate. The release of aspartate was triggered by addition of KCI or A23187 (a Ca2+ ionophore) in the presence of Ca2+ and was proportional to the amount of L-glutamate released. Furthermore, the release of aspartate was inhibited by both botulinum neurotoxin type E and L- or N-type voltage-gated Ca2+ channel blockers. Bay K 8644, an agonist for the L-type Ca2+ channel, stimulated the release of aspartate 2.1-fold. Immunohistochemical analyses with antibodies against aspartate and synaptophysin revealed that aspartate is colocalized with synaptophysin in a cultured pinealocyte. HPLC with fluorometric detection indicated that the released aspartate is of the L form, although pinealocytes also contain the D form in their cytoplasm, corresponding to approximately 30% of the total free aspartate. Radiolabeled L-aspartate was taken up by the microsomal fraction from bovine pineal glands in a Na+-dependent manner. The Na+-dependent uptake of L-aspartate was strongly inhibited by L-cysteine sulfinate, beta-hydroxyaspartate, and L-serine-O-sulfate, inhibitors for the Na+-dependent glutamate/aspartate transporter on the plasma membrane. Na+-dependent sequestration of L-aspartate was also observed in cultured rat pinealocytes, which was inhibited similarly by these transporter inhibitors. These results strongly suggest that L-aspartate is released through microvesicle-mediated exocytosis from pinealocytes and is taken up again through the Na+-dependent transporter at the plasma membrane. The possible role of L-aspartate as an intercellular chemical transmitter in the pineal gland is discussed.
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PMID:L-aspartate but not the D form is secreted through microvesicle-mediated exocytosis and is sequestered through Na+-dependent transporter in rat pinealocytes. 920 28

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.
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PMID:Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite. 975 59

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 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

Serine-, cysteine-, and metalloproteases are widely spread in many pathogenic bacteria, where they play critical functions related to colonization and evasion of host immune defenses, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection. Since all the antibiotics used clinically at the moment share a common mechanism of action, acting as inhibitors of the bacterial cell wall biosynthesis or affecting protein synthesis on ribosomes, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using new generation of antibiotics, possessing a different mechanism of action. Bacterial protease inhibitors constitute an interesting such possibility, due to the fact that many specific as well as ubiquitous proteases have recently been characterized in some detail in both gram-positive as well as gram-negative pathogens. Few potent, specific inhibitors for such bacterial proteases have been reported at this moment except for some signal peptidase, clostripain, Clostridium histolyticum collagenase, botulinum neurotoxin, and tetanus neurotoxin inhibitors. No inhibitors of the critically important and ubiquitous AAA proteases, degP or sortase have been reported, although such compounds would presumably constitute a new class of highly effective antibiotics. This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy.
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PMID:Bacterial protease inhibitors. 1211 49

SNAP-25 is an integral protein of the plasma membrane involved in neurotransmission and hormone secretion. The cysteine-rich domain of SNAP-25 is essential for membrane binding and plasma-membrane targeting. However, this domain is not required for SNARE complex formation and fusion of membranes in vitro. In this paper, we describe an 'intact-cell'-based system designed to compare the effect of similar amounts of membrane-bound and soluble SNAP-25 proteins on regulated exocytosis. In transfected neuroblastoma cells, Botulinum neurotoxin E (BoNT/E), a protease that cleaves SNAP-25, blocks regulated release of hormone. However, hormone release is rescued by expressing a wild-type SNAP-25 protein resistant to the toxin. BoNT/E-resistant SNAP-25 proteins lacking the cysteine-rich domain or with all the cysteines substituted by alanines do not form SNARE complexes or rescue regulated exocytosis when expressed at the same level as membrane-bound SNAP-25, which is approximately four-fold higher than the endogenous protein. We conclude that the cysteine-rich domain of SNAP-25 is essential for Ca(2+)-dependent hormone release because, by targeting SNAP-25 to the plasma membrane, it increases its local concentration, leading to the formation of enough SNARE complexes to support exocytosis.
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PMID:Plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation and regulated exocytosis. 1214 Feb 65

The peptide N-acetyl-CRATKML-amide is an effective inhibitor of type A botulinum neurotoxin (BoNT A) protease activity [Schmidt et al., FEBS Lett. 435 (1998) 61-64]. To improve inhibitor binding, the peptide was modified by replacing cysteine with other sulfhydryl-containing compounds. Ten peptides were synthesized. One peptide adapted the structure of captopril to the binding requirements of BoNT A, but it was a weak inhibitor, suggesting that angiotensin-converting enzyme is not a good model for BoNT A inhibitor development. However, replacing cysteine with 2-mercapto-3-phenylpropionyl yielded a peptide with K(i) of 330 nM, the best inhibitor of BoNT A protease activity reported to date. Additional modifications of the inhibitor revealed structural elements important for binding and supported our earlier findings that, with the exception of P1' arginine, subsites on BoNT A are not highly specific for particular amino acid side chains.
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PMID:A high-affinity competitive inhibitor of type A botulinum neurotoxin protease activity. 1248 5


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