Proteolysis of synthetic peptides by type A botulinum neurotoxin

Type A botulinum neurotoxin catalyzed the hydrolysis of synthetic peptides based on the sequence of the 25-kD synaptosomal protein SNAP-25. In each peptide, the toxin cleaved at a single glutaminyl-arginine bond corresponding to residues 197 and 198 of SNAP-25, confirming earlier reports on the enzymatic specificity of the toxin in synaptosomal preparations. Metal chelators inhibited catalysis, consistent with a metalloprotease activity. In contrast to tetanus toxin and other botulinum toxin serotypes, type A toxin hydrolyzed relatively short, 17-to 20-residue peptides. In the substrates, SNAP-25 residue 202 and one or more of residues 187–191 were required for efficient hydrolysis, but residues 167–186 and 203–206 were not. The highest rates of hydrolysis were found when the C-terminal residues of the peptides were amidated.     

Redox properties of the disulfide bond of human Cu,Zn superoxide dismutase and the effects of human glutaredoxin 1

Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.     

Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons

We examined effects of botulinum neurotoxin A (BoNTA) on sympathetic constrictions of the vena cava and uterine artery from guinea pigs to test the role of soluble NSF attachment protein receptor (SNARE) proteins in release of the cotransmitters norepinephrine (NE) and neuropeptide Y (NPY). Protein extracts of venae cavae and uterine arteries showed partial cleavage of synaptosomal associated protein of 25 kDa (SNAP-25) after treatment in vitro with BoNTA (50-100 nM). The rising phase of isometric contractions of isolated venae cavae to field stimulation at 20 Hz, mediated by NE acting on alpha-adrenoceptors, was reduced significantly by 100 nM BoNTA. However, sustained sympathetic contractions mediated by NPY were not affected by BoNTA. In uterine arteries, noradrenergic contractions to 1-Hz stimulation were almost abolished by BoNTA, and contractions at 10 Hz were reduced by 50-60%. We conclude that SNARE proteins are involved in exocytosis of NE from synaptic vesicles at low frequencies of stimulation but may not be essential for exocytosis of NPY and NE from large vesicles at high stimulation frequencies.     

Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.     

Retention of cleaved synaptosome-associated protein of 25 kDa (SNAP-25) in neuromuscular junctions: a new hypothesis to explain persistence of botulinum A poisoning

Botulinum neurotoxins can block neurotransmitter release for several months. The molecular mechanism of these toxins' action is known, but the persistence of neuromuscular paralysis that they cause is unexplained. At frog neuromuscular junctions, application of botulinum toxin type A caused paralysis and reduced the C-terminus immunoreactivity of SNAP-25, but not that of the remaining N-terminus fragment. Botulinum toxin type C caused paralysis and reduced syntaxin immunoreactivity without affecting that of SNAP-25. Co-application of botulinum A and C reduced syntaxin immunoreactivity, and that of both C and N termini of SNAP-25. Application of hydroxylamine to de-palmitoylate SNAP-25 resulted in a slight reduction of the immunoreactivity of SNAP-25 N terminus, while it had no effect on immunoreactivity of botulinum A cleaved SNAP-25. In contrast, application of hydroxylamine to nerve terminals where syntaxin had been cleaved by botulinum C caused a considerable reduction in SNAP-25 N-terminus immunoreactivity. Hence the retention of immunoreactive SNAP-25 at the neuromuscular junction depends on its interactions with syntaxin and plasma membrane. Persistence of cleaved SNAP-25 in nerve terminals may prevent insertion of new SNAP-25 molecules, thereby contributing to the longevity of botulinum A effects.     

Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons

The role of the soluble NSF attachment protein receptor (SNARE) protein complex in release of multiple cotransmitters from autonomic vasodilator neurons was examined in isolated segments of guinea pig uterine arteries treated with botulinum neurotoxin A (BoNTA; 50 nM). Western blotting of protein extracts from uterine arteries demonstrated partial cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) to a NH2-terminal fragment of approximately 24 kDa by BoNTA. BoNTA reduced the amplitude (by 70-80%) of isometric contractions of arteries in response to repeated electrical stimulation of sympathetic axons at 1 or 10 Hz. The amplitude of neurogenic relaxations mediated by neuronal nitric oxide (NO) was not affected by BoNTA, whereas the duration of peptide-mediated neurogenic relaxations to stimulation at 10 Hz was reduced (67% reduction in integrated responses). In contrast, presynaptic cholinergic inhibition of neurogenic relaxations was abolished by BoNTA. These results demonstrate that the SNARE complex has differential involvement in release of cotransmitters from the same autonomic neurons: NO release is not dependent on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependent on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.     

A discontinuous SNAP-25 C-terminal coil supports exocytosis

Membrane fusion requires the formation of four-helical bundles comprised of the SNARE proteins syntaxin, vesicle-associated membrane protein (VAMP), and the synaptosomal-associated protein of 25 kDa (SNAP-25). Botulinum neurotoxin E cleaves the C-terminal coil of SNAP-25, inhibiting exocytosis of norepinephrine from permeabilized PC12 cells. Addition of a 26-mer peptide comprising the C terminus of SNAP-25 that is cleaved by the toxin restores exocytosis, demonstrating that continuity of the SNAP-25 C-terminal helix is not critical for its function. By contrast, vesicle-associated membrane protein peptides could not rescue botulinum neurotoxin D-treated cells, suggesting that helix continuity is critical for VAMP function. Much higher concentrations of the SNAP-25 C-terminal peptide are required for rescuing exocytosis (K(assembly) = approximately 460 microm) than for binding to other SNAREs in vitro (Kd < 5 microm). Each residue of the peptide was mutated to alanine to assess its functional importance. Whereas most mutants rescue exocytosis with lower efficiency than the wild type peptide, D186A rescues with higher efficiency, and kinetic analysis suggests this is because of higher affinity for the cellular binding site. This is consistent with Asp-186 contributing to negative regulation of the fusion process.     

Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins

In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum. Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin. Type A C. botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs). Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes. BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively. Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions. Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs. We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.     

Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol.

Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes. The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract. We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25). While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form. Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment. Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy. BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.     

Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.     

Inhibition of calcium-dependent release of noradrenaline from PC12 cells by botulinum type-A neurotoxin. Long-term effects of the neurotoxin on intact cells.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line

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.     

Enhanced exocytotic-like insertion of Orai1 into the plasma membrane upon intracellular Ca2+ store depletion

Ca+ release-activated Ca2+ (CRAC) channels are activated when free Ca2+ concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+ entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+ store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+ concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+ chelator that prevented TG-evoked cytosolic free Ca2+ concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+ entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+ entry.     

A dileucine in the protease of botulinum toxin A underlies its long-lived neuroparalysis: transfer of longevity to a novel potential therapeutic

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT(A)) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT(A) was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC(A)) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6-7 N terminus residues diminished BoNT(A) activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC(E) to a BoNT(A) enzymically inactive mutant (BoTIM(A)) yielded a novel LC(E)-BoTIM(A) protein that targets neurons, and the BoTIM(A) moiety also delivers and stabilizes the inhibitory LC(E), giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT(E). LC(E-)BoTIM(A)(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT(E), reaffirming that these residues are critical for the persistent action of LC(E)-BoTIM(A) as well as BoNT(A). LC(E)-BoTIM(A) inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.     

Antagonism of botulinum toxin type A-induced cleavage of SNAP-25 in rat cerebral synaptosome by toosendanin

Toosendanin (TSN), a triterpenoid derivative extracted from Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study is designed to explore its antibotulismic mechanism by Western blotting. The results showed that TSN incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes, but made the synaptosomes completely resistant to botulinum neurotoxin A (BoNT/A)-mediated cleavage of SNAP-25. After binding of BoNT/A to synaptosomes, TSN still partially antagonized the toxin-mediated cleavage of SNAP-25. However, TSN-incubated synaptosomal membrane fraction did not resist the cleavage of SNAP-25 by the light chain of BoNT/A. It is suggested that the antibotulismic effect of TSN results from blocking the toxin's approach to its enzymatic substrate.     

Botulinum neurotoxin a impairs neurotransmission following retrograde transynaptic transport

The widely used botulinum neurotoxin A (BoNT/A) blocks neurotransmission via cleavage of the synaptic protein SNAP-25 (synaptosomal-associated protein of 25 kDa). Recent evidence demonstrating long-distance propagation of SNAP-25 proteolysis has challenged the idea that BoNT/A remains localized to the injection site. However, the extent to which distant neuronal networks are impacted by BoNT/A retrograde trafficking remains unknown. Importantly, no studies have addressed whether SNAP-25 cleavage translates into structural and functional changes in distant intoxicated synapses. Here we show that the BoNT/A injections into the adult rat optic tectum result in SNAP-25 cleavage in retinal neurons two synapses away from the injection site, such as rod bipolar cells and photoreceptors. Retinal endings displaying cleaved SNAP-25 were enlarged and contained an abnormally high number of synaptic vesicles, indicating impaired exocytosis. Tectal injection of BoNT/A in rat pups resulted in appearance of truncated-SNAP-25 in cholinergic amacrine cells. Functional imaging with calcium indicators showed a clear reduction in cholinergic-driven wave activity, demonstrating impairments in neurotransmission. These data provide the first evidence for functional effects of the retrograde trafficking of BoNT/A, and open the possibility of using BoNT/A fragments as drug delivery vehicles targeting the central nervous system.     

Rescue of exocytosis in botulinum toxin A-poisoned chromaffin cells by expression of cleavage-resistant SNAP-25. Identification of the minimal essential C-terminal residues

Botulinum neurotoxin (BoNT) types A and B selectively block exocytosis by cleavage of SNAP-25 and synaptobrevin, respectively; in humans, many months are required for full recovery from the resultant neuromuscular paralysis. To decipher the molecular basis for such prolonged poisoning, intoxication in adreno-chromaffin cells was monitored over 2 months. Exocytosis from BoNT/B-treated cells resumed after 56 days because of the appearance of intact synaptobrevin. However, inhibition continued in BoNT/A-treated cells, throughout the same interval, with a continued predominance of cleaved SNAP-25-(1-197) over the intact protein. When recovery from poisoning was attempted by transfection of the latter cells with the gene encoding full-length SNAP-25-(1-206), no restoration of exocytosis ensued even after 3 weeks. To ascertain if this failure was because of the persistence of the toxin's protease activity, the cells were transfected with BoNT/A-resistant SNAP-25 constructs; importantly, exocytosis was rescued. C-terminal truncation of the toxin-insensitive SNAP-25 revealed that residues 1-201, 1-202, 1-203 afforded a significant return of exocytosis, unlike shorter forms 1-197, -198, -199, or -200; accordingly, mutants M202A or L203A of full-length SNAP-25 rescued secretion. These findings give insights into the C-terminal functional domain of SNAP-25, demonstrate the longevity of BoNT/A protease, and provide the prospect of a therapy for botulism.     

Targeted delivery into motor nerve terminals of inhibitors for SNARE-cleaving proteases via liposomes coupled to an atoxic botulinum neurotoxin

A targeted drug carrier (TDC) is described for transferring functional proteins or peptides into motor nerve terminals, a pivotal locus for therapeutics to treat neuromuscular disorders. It exploits the pronounced selectivity of botulinum neurotoxin type B (BoNT/B) for interacting with acceptors on these cholinergic nerve endings and becoming internalized. The gene encoding an innocuous BoNT/B protease-inactive mutant (BoTIM) was fused to that for core streptavidin, expressed in Escherichia coli and the purified protein was conjugated to surface-biotinylated liposomes. Such decorated liposomes, loaded with fluorescein as traceable cargo, acquired pronounced specificity for motor nerve terminals in isolated mouse hemidiaphragms and facilitated the intraneuronal transfer of the fluor, as revealed by confocal microscopy. Delivery of the protease light chain of botulinum neurotoxin type A (BoNT/A) via this TDC accelerated the onset of neuromuscular paralysis, indicative of improved translocation of this enzyme into the presynaptic cytosol with subsequent proteolytic inactivation of synaptosomal-associated protein of molecular mass 25 kDa (SNAP-25), an exocytotic soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) essential for neurotransmitter release. BoTIM-coupled liposomes, loaded with peptide inhibitors of proteases, yielded considerable attenuation of the neuroparalytic effects of BoNT/A or BoNT/F as a result of their cytosolic transfer, the first in situ demonstration of the ability of designer antiproteases to suppress the symptoms of botulism ex vivo. Delivery of the BoNT/A inhibitor by liposomes targeted with the full-length BoTIM proved more effective than that mediated by its C-terminal neuroacceptor-binding domain. This demonstrated versatility of TDC for nonviral cargo transfer into cholinergic nerve endings has unveiled its potential for direct delivery of functional targets into motor nerve endings.     

Evaluation of the therapeutic usefulness of botulinum neurotoxin B,C1, E,and F compared with the long lasting type A. Basis for distinct durations of inhibition of exocytosis in central neurons

Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they selectively proteolyze SNAP-25 (BoNT/A, BoNT/C1, and BoNT/E), syntaxin1 (BoNT/C1), and synaptobrevin (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), SNARE proteins responsible for transmitter release, to cause neuromuscular paralysis but of different durations. BoNT/A paralysis lasts longest (4-6 months) in humans, hence its widespread clinical use for the treatment of dystonias. Molecular mechanisms underlying these distinct inhibitory patterns were deciphered in rat cerebellar neurons by quantifying the half-life of the effect of each toxin, the speed of replenishment of their substrates, and the degradation of the cleaved products, experiments not readily feasible at motor nerve endings. Correlation of target cleavage with blockade of transmitter release yielded half-lives of inhibition for BoNT/A, BoNT/C1, BoNT/B, BoNT/F, and BoNT/E (31, 25, approximately 10, approximately 2, and approximately 0.8 days, respectively), equivalent to the neuromuscular paralysis times found in mice, with recovery of release coinciding with reappearance of the intact SNAREs. A limiting factor for the short neuroparalytic durations of BoNT/F and BoNT/E is the replenishment of synaptobrevin or SNAP-25, whereas pulse labeling revealed that extended inhibition by BoNT/A, BoNT/B, or BoNT/C1 results from longevity of each protease. These novel findings could aid development of new toxin therapies for patients resistant to BoNT/A and effective treatments for human botulism.     

The 25-kDa synaptosome-associated protein (SNAP-25) binds and inhibits delayed rectifier potassium channels in secretory cells

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.