Tetanus and botulinum neurotoxins: turning bad guys into good by research

Rossetto, Ornella; Seveso, Michela; Caccin, Paola; Schiavo, Giampietro; Montecucco, Cesare

doi:10.1016/s0041-0101(00)00163-x

Cited by 154 publications

(103 citation statements)

References 138 publications

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“…Effects of clostridial toxins that cleave SNARE proteins in neuronal cells include inhibition of neurotransmitter release (25). In exocrine cells, VAMP2 and VAMP8, described in separate ZG populations, may have different roles but share unique participation in transport and exocytotic events (42).…”

Section: Discussionmentioning

confidence: 99%

“…The activity of this protease is dependent on divalent ions, in agreement with the zinc-binding motif present in the sequence. Before this report, proteolytic cleavage of SNARE family components was known to be associated only with large microbial proteins from the genus Clostridium that require intracellular cleavage before release of smaller active proteolytic enzymes (23)(24)(25)(26). Here we introduce a proteolytic enzyme in venom from the New World scorpion T. serrulatus, which cleaves v-SNAREs.…”

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confidence: 99%

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Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Fletcher

Weninger

et al. 2010

Journal of Biological Chemistry

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We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction , an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction that cleaves VAMP2, and report its amino acid sequence.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Fletcher

Weninger

et al. 2010

Journal of Biological Chemistry

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“…Cleavage of VAMP-1 disrupts neurotransmitter release through disruption of the vesicle docking complex, thus inhibiting neurotransmission. 13,14 However, inadvertent release of LC should not pose a threat since this fragment is not capable of neuronal binding and uptake. The brainstem contains densely packed structures responsible for a variety of critical activities including the vegetative functions of the reticular activating system necessary to maintain a wakeful state and defensive reactions including freezing.…”

Section: Discussionmentioning

confidence: 99%

“…9 In other words, inactivation of the dSC/DpMe only blocks potentiated startle without interfering the other behaviors that are mediated by the adjacent areas such as freezing and spontaneous locomotor activity. Freezing and locomotor activity are mediated by the central gray [10][11][12] and the mesencephalic locomotor region (MLR), 1,13,14 respectively, which are located in close proximity to the dSC/DpMe. A control study demonstrates that inaction of the central gray has little effect on fear-potentiated startle but tend to reduce freezing.…”

Section: Introductionmentioning

confidence: 99%

Anatomically discrete functional effects of adenoviral clostridial light chain gene-based synaptic inhibition in the midbrain

et al. 2006

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The gene for the Light Chain fragment of Tetanus Toxin (LC) induces synaptic inhibition by preventing the release of synaptic vesicles. The present experiment applied this approach within the rat midbrain in order to demonstrate that LC gene expression can achieve functionally and anatomically discrete effects within a sensitive brain structure. The deep layers of the superior colliculus/deep mesencephalic nucleus (dSC/DpMe) that are located in the rostral midbrain has been implicated in fear-induced increase of the acoustic startle reflex (fear potentiated startle) but exists in close proximity to neural structures important for a variety of critical functions. The dSC/DpMe of adult rats was injected bilaterally with adenoviral vectors for LC, green fluorescent protein, or vehicle. Synaptobrevin was depleted in brain regions of adenoviral LC expression. LC gene expression in the dSC/DpMe inhibited the increase in startle amplitude seen with the control viral infection, and blocked context-dependent potentiation of startle induced by fear conditioning. Although LC gene expression reduced the absolute amount of cue-specific fear potentiated startle, it did not decrease percent potentiated startle to a cue, nor did it reduce fear-induced contextual freezing, nonspecific locomotor activity, or general health, indicating that its effects were functionally and anatomically specific. Thus, vector-driven LC expression inhibits the function of deep brain nuclei without altering the function of surrounding structures supporting its application to therapeutic neuromodulation. Gene Therapy (2006) 13, 942-952.

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“…BoNTs have also emerged as important medical tools to treat muscle dysfunction, inflammation, and chronic pain (2)(3)(4)(5). There are seven related toxins (BoNT͞A-G).…”

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confidence: 99%

Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells

Dong

Tepp

Johnson

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

118

111

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Botulinum neurotoxins (BoNTs) act as zinc-dependent endopeptidases that cleave proteins required for neurotransmitter release. To detect toxin activity, fragments of the toxin substrate proteins, synaptobrevin (Syb) or synaptosome-associated protein of 25 kDa (SNAP-25), were used to link cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP). Cleavage of these fusion proteins by BoNTs abolished fluorescence resonance energy transfer between the CFP and YFP, providing a sensitive means to detect toxin activity in real-time in vitro. Furthermore, using full-length SNAP-25 and Syb as the linkers, we report two fluorescent biosensors that can detect toxin activity within living cells. Cleavage of the SNAP-25 fusion protein abolished fluorescence resonance energy transfer between CFP and YFP, and cleavage of Syb resulted in spatial redistribution of YFP fluorescence in cells. This approach provides a means to carry out cell-based screening of toxin inhibitors and to study toxin activity in situ. By using these biosensors, we found that the subcellular localizations of SNAP-25 and Syb are critical for efficient cleavage by BoNT/A and B, respectively. B otulinum neurotoxins (BoNT) are the most lethal substances known and thus pose a bioterrorism threat (1). BoNTs have also emerged as important medical tools to treat muscle dysfunction, inflammation, and chronic pain (2-5). There are seven related toxins (BoNT͞A-G). Each toxin is composed of a heavy chain, which mediates the entry of toxin into neurons, and a light chain, which functions as a zinc-dependent endoprotease inside cells (6). BoNT͞A, -E, and -C cleave the peripheral plasma membrane protein SNAP-25 (synaptosome-associated protein of 25 kDa); BoNT͞C also cleaves the integral plasma membrane protein syntaxin; and BoNT͞B, -D, -F, and -G cleave the secretory vesicle membrane protein synaptobrevin (Syb) (see ref.6 for a review). Cleavage of these substrates inhibits neuronal exocytosis (6).Characterizing BoNT activity traditionally relies on functional assays to monitor the effect of the toxin (e.g., toxicity in mice, blockade of exocytosis) or biochemical assays (e.g., immunoblot͞ immunoassays, chromatography) to detect cleaved substrate molecules (7). Several methods, based on intramolecular quenching of fluorigenic amino acid derivatives, have been recently explored and used to screen toxin inhibitors in vitro (8-10). However, this approach is limited to a subset of toxins and cannot be used to monitor the action of the toxins in live cells. At present, there are no available methods that are amenable to cell-based high-throughput screening of BoNT inhibitors.Here we report a recently developed method based on monitoring fluorescence resonance energy transfer (FRET) changes between cyan (CFP) and yellow (YFP) fluorescent protein pairs, to detect toxin activity in real time in vitro. Furthermore, using CFP͞YFP pairs, we are able to detect BoNT activity in living cells, which enabled us to study toxin substrate cleavage in situ. Materials and Methodsc...

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Tetanus and botulinum neurotoxins: turning bad guys into good by research

Cited by 154 publications

References 138 publications

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus

Anatomically discrete functional effects of adenoviral clostridial light chain gene-based synaptic inhibition in the midbrain

Using fluorescent sensors to detect botulinum neurotoxin activity in vitro and in living cells

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