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)

Tetanus toxin (TeTx) and the various forms of botulinal neurotoxins (BoNT/A to BoNT/G) potently inhibit neurotransmission by means of their L chains which selectively proteolyze synaptic proteins such as synaptobrevin (TeTx, BoNT/B, BoNT/F), SNAP-25 (BoNT/A), and syntaxin (BoNT/C1). Here we show that BoNT/D cleaves rat synaptobrevin 1 and 2 in toxified synaptosomes and in isolated vesicles. In contrast, synaptobrevin 1, as generated by in vitro translation, is only a poor substrate for BoNT/D, whereas this species is cleaved by BoNT/F with similar potency. Cleavage by BoNT/D occurs at the peptide bond Lys59-Leu60 which is adjacent to the BoNT/F cleavage site (Gln58-Lys59) and again differs from the site hydrolyzed by TeTx and BoNT/B (Gln76-Phe77). Cellubrevin, a recently discovered isoform expressed outside the nervous system, is efficiently cleaved by all three toxins examined. For further characterization of the substrate requirements of BoNT/D, we tested amino- and carboxyl-terminal deletion mutants of synaptobrevin 2 as well as synthetic peptides. Shorter peptides containing up to 15 amino acids on either side of the cleavage site were not cleaved, and a peptide extending from Arg47 to Thr116 was a poor substrate for all three toxins tested. However, cleavability was restored when the peptide is further extended at the NH2 terminus (Thr27-Thr116) demonstrating that NH2 terminally located sequences of synaptobrevin which are distal from the respective cleavage sites are required for proteolysis. To further examine the isoform specificity, several mutants of rat synaptobrevin 2 were generated in which individual amino acids were replaced with those found in rat synaptobrevin 1. We show that a Met46 to Ile46 substitution drastically diminishes cleavability by BoNT/D and that the presence of Val76 instead of Gln76 dictates the reduced cleavability of synaptobrevin isoforms by TeTx.
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PMID:Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. 817 89

We have identified VAMP isoforms, VAMP-2 and cellubrevin, on GLUT4-containing vesicle membranes isolated from 3T3-Ll adipocytes. These proteins translocate from a low density microsomal fraction to the plasma membrane upon insulin stimulation in a fashion similar to GLUT4. VAMP-1 was not detected in this low density microsomal fraction nor on purified GLUT4-containing vesicles. In streptolysin-O permeabilized 3T3-L1 adipocytes, both VAMP-2 and cellubrevin were cleaved with botulinum neurotoxin isoform B, BoNTx/B. In addition, BoNTx/B partially inhibited insulin-stimulated GLUT4 translocation and glucose transport activity. We conclude that the synaptobrevin isoforms are important components of the insulin-dependent translocation of GLUT4 to the cell surface in adipocytes.
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PMID:Cleavage of vesicle-associated membrane protein (VAMP)-2 and cellubrevin on GLUT4-containing vesicles inhibits the translocation of GLUT4 in 3T3-L1 adipocytes. 860 35

Amylase exocytosis of the parotid gland is mediated by intracellular cAMP. To investigate whether cAMP-dependent secretion has a mechanism similar to that of regulated neuroexocytosis, we examined the expression of synaptosome-associated proteins. In rat parotid acinar cells, we found 25 (p25) and 18 kDa (p18) proteins reacted with antibodies against Rab3A and vesicle-associated membrane protein 2 (VAMP-2), respectively. On the other hand, syntaxin 1 and SNAP-25, which interact with VAMP-2 at synapses, were undetectable. Rab3A-like p25 and VAMP-2-like p18 were also expressed in other exocrine acinar cells. The latter was localized at secretory granule membranes, and the former was detected in secretory granule and cytosolic fractions. The antibody against VAMP-2 used in this study did not react with cellubrevin, and p18 was cleaved with botulinum neurotoxin B. Thus, we identified p18 as VAMP-2. Botulinum neurotoxin B inhibited the cAMP-induced amylase release from streptolysin O-permeabilized acinar cells. Therefore, VAMP-2 is required for cAMP-regulated amylase release in rat parotid acinar cells. This is the first report that VAMP-2 is involved in regulated exocytosis that is independent of Ca2+.
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PMID:Vesicle-associated membrane protein 2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. The inhibition of cAMP-dependent amylase release by botulinum neurotoxin B. 866 34

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Recently, several SNARE proteins were found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homologue cellubrevin, and syntaxin-4. In this report we show that treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibited the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Furthermore, treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively blocked insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues.
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PMID:Insulin-stimulated translocation of GLUT4 glucose transporters requires SNARE-complex proteins. 898 82

Recombinant DNA techniques were used to develop an expression system for a 51-amino acid peptide fragment that encompasses residues 44-94 of human synaptobrevin 2. This protein is associated with secretory vesicles of nerve terminals and is a substrate for four of the seven serotypes of botulinum neurotoxin (BoNT). The DNA for the recombinant peptide was amplified by the polymerase chain reaction and cloned into the pTrxFus vector. The resulting synaptobrevin peptide was expressed as a thioredoxin fusion protein in E. coli and released into the medium by osmotic lysis. The 18.7-kDa thioredoxin-synaptobrevin protein, designated as TSB-51, is intended for use in a cell-free assay to test potential inhibitors of BoNT/B-mediated proteolysis of synaptobrevin with the ultimate aim of developing clinically effective therapeutic agents to counteract botulism. Incubation of TSB-51 with the purified light chain of BoNT/B resulted in proteolysis which was evident within 30 min and increased with time until completion (approximately 4 hr). Cleavage of TSB-51 appeared to be at the appropriate BoNT/B cleavage site as indicated by a reduced intensity of the 18.7-kDa band and the appearance of a band at 16.4 kDa on Tris-tricene polyacrylamide gradient gels. The concentration of free Zn2+ had a significant effect on the cleavage rate; low Zn2+ concentrations stimulated substrate cleavage, whereas high concentrations were inhibitory. Cleavage was not significantly depressed by the naturally occurring metalloprotease inhibitor phosphoramidon when tested at concentrations up to 5 mM. TSB-51 appears to be a useful substrate for studying BoNT/B and is expected to aid in the discovery of effective BoNT inhibitors.
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PMID:Production of an expression system for a synaptobrevin fragment to monitor cleavage by botulinum neurotoxin B. 971 40

Rat parotid acinar cells secrete amylase through the stimulation of beta-adrenoceptors followed by accumulation of intracellular cAMP. However, it remains unclear at the molecular level how secretory granules fuse with the apical membranes. We have examined whether SNARE proteins are involved in exocytosis in the salivary glands, and have found that one of the SNARE proteins, VAMP-2, is localized at the secretory granule membrane of rat parotid acinar cells. Moreover, botulinum neurotoxin B, which has endoprotease activity that cleaves VAMP-2, inhibited cAMP-dependent amylase release but did not inhibit basal secretion in the absence of cAMP. These results suggest that VAMP-2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. In contrast, both neurotoxins A and C1 (endoproteases that cleave SNAP-25 and syntaxin 1 respectively) failed to inhibit cAMP-dependent amylase release. Therefore, neither SNAP-25 nor syntaxin 1 are involved in amylase secretion in the parotid glands. Clarification of the mechanism of secretion will require the identification of proteins that interact and function cooperatively with VAMP-2. This approach may also reveal details of the molecular mechanism by which the cAMP facilitates secretion in other systems, including neurotransmission.
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PMID:Snare proteins essential for cyclic AMP-regulated exocytosis in salivary glands. 982 92

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.
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PMID:Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell. 1042 73

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.
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PMID:Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment. 1055 89

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.
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PMID:Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line. 1102 Mar 78

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).
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PMID:Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization. 1182 15


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