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)

Rat islet beta-cells spread in response to glucose when attached on the matrix produced by a rat bladder carcinoma cell line (804G). Furthermore, in a mixed population of cells, it has been observed previously that spread cells secrete more insulin acutely in response to glucose, compared with cells that remain rounded. These results suggest bi-directional signaling between the islet beta-cell and the extracellular matrix. In the present study, the role of increased intracellular free Ca2+ concentration [Ca2+]i as an intracellular step linking glucose stimulation and beta-cell spreading (inside-out signaling) was investigated. Purified rat beta-cells were attached to this matrix and incubated under various conditions known to affect [Ca2+]i. The effect of glucose on beta-cell spreading was mimicked by 25 mmol/l KCl (which induces calcium influx) and inhibited by diazoxide (which impairs depolarization and calcium entry) and by the L-type Ca2+ channel blocker SR-7037. When a 24-h incubation at 16.7 glucose was followed by 24 h at 2.8 mmol/l, beta-cells that had first spread regained a round phenotype. In the presence of thapsigargin, spreading progressed throughout the experiment, suggesting that capture of calcium by the endoplasmic reticulum is involved in the reversibility of spreading previously induced by glucose. Spreading was still observed in degranulated beta-cells and in botulinum neurotoxin E-expressing beta-cells when exocytosis was prevented. In summary, the results indicate that increased [Ca2+]i is required for the glucose-induced spreading of beta-cells on 804G matrix and that it is not a consequence of exocytotic processes that follow elevation of [Ca2+]i.
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PMID:Increased intracellular calcium is required for spreading of rat islet beta-cells on extracellular matrix. 1133 6

Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting beta-cells inhibited Ca(2+)-evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca(2+) (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40-50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca(2+)-evoked exocytosis by membrane depolarization to -10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca(2+) influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca(2+)-evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca(2+) or cAMP potentiation.
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PMID:Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis. 1150 51

Delayed-rectifier K(+) channels (K(DR)) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K(+) channels results in membrane repolarization, leading to the closure of the Ca(2+) channels and cessation of insulin secretion in neuroendocrine islet beta cells. Using patch clamp techniques, we have demonstrated that the activity of the K(DR) channel subtype, K(V)1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 beta cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the K(V)1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of K(DR). This inhibitory effect of SNAP-25 is mediated by the N terminus of K(V)1.1, likely by direct interactions with K(Valpha)1.1 and/or K(V)beta subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with K(DR) channels.
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PMID:The 25-kDa synaptosome-associated protein (SNAP-25) binds and inhibits delayed rectifier potassium channels in secretory cells. 1192 39

Cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are now known to associate the secretory vesicle with both the target plasma membrane and Ca(2+) channels in order to mediate the sequence of events leading to exocytosis in neurons and neuroendocrine cells. Neuroendocrine cells, particularly insulin-secreting islet beta-cells, t-SNARE proteins, 25-kDa synaptosomal-associated protein (SNAP-25), and syntaxin 1A, independently inhibit the L-type Ca(2+) channel (L(Ca)). However, when both are present, they actually exhibit stimulatory actions on the L(Ca). This suggests that the positive regulation of the L(Ca) is conferred by a multi-SNARE protein complex. We hypothesized an alternate explanation, which is that each of these SNARE proteins possess distinct inhibitory and stimulatory domains that act on the L(Ca). These SNARE proteins were recently shown to bind the Lc(753-893) domain corresponding to the II and III intracellular loop of the alpha1C subunit of the L(Ca). In this study, using patch-clamp methods on primary pancreatic beta-cells and insulinoma HIT-T15 cells, we examined the functional interactions of the botulinum neurotoxin A (BoNT/A) cleavage products of SNAP-25, including NH(2)-terminal (1-197 amino acids) and COOH-terminal (amino acid 198-206) domains, on the L(Ca), particularly at the Lc(753-893) domain. Intracellular application of SNAP-25(1-206) in primary beta-cells decreased L(Ca) currents by approximately 15%. The reduction in L(Ca) currents was counteracted by coapplication of Lc(753-893). Overexpression or injection of wild-type SNAP-25 in HIT cells reduced L(Ca) currents by approximately 30%, and this inhibition was also blocked by the recombinant Lc(753-893) peptide. Expression of BoNT/A surprisingly caused an even greater reduction of L(Ca) currents (by 41%), suggesting that the BoNT/A cleavage products of SNAP-25 might possess distinct inhibitory and positive regulatory domains. Indeed, expression of SNAP-25(1-197) increased L(Ca) currents (by 19% at 10 mV), and these effects were blocked by the Lc(753-893) peptide. In contrast, injection of SNAP-25(198-206) peptide into untransfected cells inhibited L(Ca) currents (by 47%), and more remarkably, these inhibitory effects dominated over the stimulatory effects of SNAP-25(1-197) overexpression (by 34%). Therefore, the SNARE protein SNAP-25 possesses distinct inhibitory and stimulatory domains that act on the L(Ca). The COOH-terminal 197-206 domain of SNAP-25, whose inhibitory actions dominate over the opposing stimulatory NH(2)-terminal domain, likely confers the inhibitory actions of SNAP-25 on the L(Ca). We postulate that the eventual accelerated proteolysis of SNAP-25 brought about by BoNT/A cleavage allows the relatively intact NH(2)-terminal SNAP-25 domain to assert its stimulatory action on the L(Ca) to increase Ca(2+) influx, and this could in part explain the observed weak or inconsistent inhibitory effects of BoNT/A on insulin secretion. The present study suggests that distinct domains within SNAP-25 modulate L(C) subtype Ca(2+) channel activity in both primary beta-cells and insulinoma HIT-T15 cells.
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PMID:Modulation of L-type Ca(2+) channels by distinct domains within SNAP-25. 1197 39

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

Clostridium botulinum neurotoxins (BoNTs) are effective therapeutics for a variety of neurological disorders, such as strabismus, blepharospam, hemificial spasm, and cervical dystonia, because of the toxin's tropism for neurons and specific cleavage of neuronal soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptors (SNARE) proteins. Modifying BoNT to bind nonneuronal cells has been attempted to extend therapeutic applications. However, prerequisite to develop nonneuronal therapies requires the retargeting the catalytic activity of BoNTs to nonneuronal SNARE isoforms. Here, we reported the engineering of a BoNT derivative that cleaves SNAP23, a nonneuronal SNARE protein. SNAP23 mediates vesicle-plasma membrane fusion processes, including secretion of airway mucus, antibody, insulin, gastric acids, and ions. This mutated BoNT/E light chain LC/E(K(224)D) showed extended substrate specificity to cleave SNAP23, and the natural substrate, SNAP25, but not SNAP29 or SNAP47. Upon direct protein delivery into cultured human epithelial cells, LC/E(K(224)D) cleaved endogenous SNAP23, which inhibited secretion of mucin and IL-8. These studies show the feasibility of genetically modifying LCs to target a nonneuronal SNARE protein that extends therapeutic potential for treatment of human hypersecretion diseases.
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PMID:Engineering botulinum neurotoxin to extend therapeutic intervention. 1948 72

Syntaxin (stx)-1 is an integral plasma membrane protein that is crucial for two distinct steps of regulated exocytosis, docking of secretory granules at the plasma membrane and membrane fusion. During docking, stx1 clusters at the granule docking site, together with the S/M protein munc18. Here we determined features of stx1 that contribute to its clustering at granules. In live insulin-secreting cells, stx1 and stx3 (but not stx4 or stx11) accumulated at docked granules, and stx1 (but not stx4) rescued docking in cells expressing botulinum neurotoxin-C. Using a series of stx1 deletion mutants and stx1/4 chimeras, we found that all four helical domains (Ha, Hb, Hc, SNARE) and the short N-terminal peptide contribute to recruitment to granules. However, only the Hc domain confers specificity, and it must be derived from stx1 for recruitment to occur. Point mutations in the Hc or the N-terminal peptide designed to interfere with binding to munc18-1 prevent stx1 from clustering at granules, and a mutant munc18 deficient in binding to stx1 does not cluster at granules. We conclude that stx1 is recruited to the docking site in a munc18-1-bound conformation, providing a rationale for the requirement for both proteins for granule docking.
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PMID:Syntaxin clusters at secretory granules in a munc18-bound conformation. 3015 74


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