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Biomedical subjects

C Montecucco

Publications and source records attributed to C Montecucco.

At least 163 records · Page 9Linked to original sources
PubMed1993

Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.

SNAP-25, a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln197-Arg198 peptide bond, while serotype E cleaves the Arg180-Ile181 peptide lineage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.

Amino Acid Sequence↗
PubMed1993

Identification of the nerve terminal targets of botulinum neurotoxin serotypes A, D, and E.

Botulinum neurotoxins are metalloproteins with one zinc atom bound to the zinc binding motif of zinc endopeptidases. Here we show that botulinum neurotoxin serotypes A, D, and E are zinc endoproteases specific for components of the synaptic vesicle docking and fusion complex. Serotypes A and E cleave SNAP-25, a 25-kDa protein of the synaptic terminal, while serotype D is specific for VAMP/synaptobrevin, a membrane protein of synaptic vesicles. Both rat brain VAMP isoforms are cleaved at a single Lys-Leu peptide bond. The proteolytic activity of these neurotoxins is inhibited by EDTA and captopril.

Amino Acid Sequence↗
PubMed1993

Cell vacuolization induced by Helicobacter pylori: inhibition by bafilomycins A1, B1, C1 and D.

All available bafilomycins (A1, B1, C1 and D) inhibit and revert macroscopic vacuolization induced by Helicobacter pylori cell-free extracts. Bafilomycin A1 displays the highest activity, followed by bafilomycin B1, C1 and D. The different potency of bafilomycins correlates with their ability to inhibit the vacuolar-type ATPase (V-ATPase) and to dissipate the membrane pH gradient of intracellular acidic organelles. These results suggest that bafilomycins should be considered as possible therapeutic agents in the treatment of gastritis.

Adenosine Triphosphatases↗
PubMed1993

Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synaptobrevin.

Botulinum neurotoxin serotype F contains the zinc binding motif of zinc endopeptidases. Atomic adsorption analysis of highly purified toxin preparation revealed the presence of one atom of zinc per molecule of toxin, which could be removed with EDTA or o-phenanthroline. The light chain of the neurotoxin was shown to have a zinc-dependent protease activity specific for VAMP/synaptobrevin, an integral membrane protein of synaptic vesicles. Both isoforms of rat VAMP were cleaved at the same site corresponding to the single Gln-Lys peptide bond present in their sequences. This proteolytic activity was inhibited by EDTA, o-phenanthroline, and captopril as well as by VAMP peptides spanning the cleavage site.

Amino Acid Sequence↗
PubMed1993

Cell penetration of diphtheria toxin. Reduction of the interchain disulfide bridge is the rate-limiting step of translocation in the cytosol.

The pathway of cell penetration of diphtheria toxin (DT) was studied in Vero cells by following the kinetics of uptake, reduction, degradation, and sub-cellular distribution of 125I-DT in the absence or presence of bafilomycin A1 (baf-A1), a powerful inhibitor of the endosomal H(+)-ATPase. After a lag phase of 4 min, DT, bound to Vero cells, reached an acidic intracellular compartment, where about one-third of it underwent a transition to a state competent for subsequent reduction and membrane translocation. After further 4 min, this DT fraction was reduced in a baf-A1-insensitive reaction and DT-A, the intracellularly active protomer of DT, was immediately released in the cytosol. The present data indicate that cell-mediated reduction of the toxin is the rate-determining step of the DT cell intoxication process. Subcellular fractionation showed that DT underwent the low pH-driven conformational change in an early endosome, distinct from the subsequent endosomal compartment where reduction took place. DT-B remained endosome-bound and was proteolyzed at low pH as well as the portion of DT which was not reduced after the exposure to low pH in early endosomes.

Animals↗
PubMed1993

The sensitivity of cystic fibrosis cells to diphtheria toxin.

Cystic fibrosis (CF) cells have been reported to have a defective acidification and it has been suggested that they may be less sensitive than normal cells to diphtheria toxin (DT). A comparative analysis of DT toxicity in CF and normal cells, both in terms of kinetics of cell intoxication and of cell survival, shows very little difference, which makes it unlikely that diphtheria epidemics contribute to accounting for the large diffusion of the CF trait. DT penetrates cells from endosomal compartments different from those defective in CF cells.

Cell Division↗
PubMed1993

Novel targets and catalytic activities of bacterial protein toxins.

Among bacterial protein toxins with intracellular targets, tetanus and botulinum toxins form a group with unique properties. They are absolutely neurospecific and act in the cytosol of neurons. Recent evidence indicates that they are zinc proteases specific for proteins of the neuroexocytosis apparatus.

Amino Acid Sequence↗
PubMed1993

Tetanus and botulism neurotoxins: a new group of zinc proteases.

The active forms of tetanus and botulinum neurotoxins, released from the precursor molecule by specific proteolysis and reduction, block the release of neurotransmitters via a Zn(2+)-dependent protease activity. VAMP/synaptobrevin, an integral membrane protein of the synaptic vesicles, is cleaved at a single site by tetanus and botulinum B, D and F neurotoxins. The unique sequence, mechanism of activation and site of activity of clostridial neurotoxins mark them out as an independent group of Zn(2+)-endopeptidases.

Animals↗
PubMed1993

Is Tolosa-Hunt syndrome a limited form of Wegener's granulomatosis? Report of two cases with anti-neutrophil cytoplasmic antibodies.

Two patients with recurrent painful ophthalmoplegia due to granulomatous involvement of the intracavernous sinus (Tolosa-Hunt syndrome) had circulating anti-neutrophil cytoplasmic antibodies with a granular cytoplasmic immunofluorescence pattern during a flare-up of the disease. The presence of these antibodies suggests that Tolosa-Hunt syndrome might be regarded as a localized form of Wegener's granulomatosis.

Adult↗
PubMed1993

Bafilomycin A1 inhibits Helicobacter pylori-induced vacuolization of HeLa cells.

Bafilomycin A1, a specific inhibitor of the vacuolar-type H(+)-ATPase, responsible for acidification of intracellular compartments, prevents the vacuolization of Hela cells induced by H. pylori, with an inhibitory concentration giving 50% of maximal (ID50) of 4 nM. Bafilomycin A1 is also very efficient in restoring vacuolated cells to a normal appearance. The vacuolating activity of Helicobacter pylori is not inhibited by a series of specific inhibitors of vacuolar H(+)-ATPases. These findings indicate that a transmembrane pH gradient is needed for the formation and growth of vacuoles caused by the bacterium and that this pH gradient is due to the activity of a vacuolar ATPase proton pump of HeLa cells.

Anti-Bacterial Agents↗
PubMed1993

Antibodies against rat brain vesicle-associated membrane protein (synaptobrevin) prevent inhibition of acetylcholine release by tetanus toxin or botulinum neurotoxin type B.

Tetanus and botulinum B neurotoxins are zinc endopeptidases that cleave vesicle-associated membrane protein (VAMP or synaptobrevin) at a single peptide bond. To test the possibility that in vivo also the toxin-induced blockade of neurotransmission is due to cleavage of VAMP, rat brain VAMP-specific antibodies were raised in rabbits. IgGs purified from one antiserum, which bind specifically to rat brain VAMP, also specifically recognize proteins from Aplysia californica in immunoblotting. When injected into neurons in the buccal ganglion of Aplysia, these IgGs did not affect the release of acetylcholine but effectively prevented the inhibitory action of both toxins on neurotransmitter release, thus indicating that the block of neurotransmission by these neurotoxins is consequent to the cleavage of VAMP or specific interaction with VAMP.

Acetylcholine↗
PubMed1993

Botulinum A like type B and tetanus toxins fulfils criteria for being a zinc-dependent protease.

Although botulinum neurotoxin (BoNT) types A and B and tetanus toxin (TeTx) are specific inhibitors of transmitter release whose light chains contain a zinc-binding motif characteristic of metalloendoproteases, only the latter two proteolyse synaptobrevin. Chelation of zinc or its readdition at high concentration hindered blockade of neuromuscular transmission by BoNT/A and B, indicating that type A also acts via a zinc-dependent mechanism. Such treatments prevented proteolysis of synaptobrevin II in rat brain synaptic vesicles by BoNT/B and TeTx but only the activity of the latter was antagonised appreciably by ASQFETS, a peptide spanning their cleavage site. The toxin's neuroparalytic activities were attenuated by phosphoramidon or captopril, inhibitors of certain zinc requiring proteases. However, these agents were ineffective in reducing the toxins' degradation of synaptobrevin except that a high concentration of captopril partially blocked the activity of TeTx but not BoNT/B, as also found for these drugs when tested on synaptosomal noradrenaline release. These various criteria establish that a zinc-dependent protease activity underlies the neurotoxicity of BoNT/A, a finding confirmed at motor nerve endings for type B and TeTx. Moreover, the low potencies of captopril and phosphoramidon in counteracting the toxins' effects necessitate the design of improved inhibitors for possible use in the clinical treatment of tetanus or botulism.

Acetylcholine↗
PubMed1992

Botulinum neurotoxins are zinc proteins.

The available amino acid sequences of 150-kDa botulinum and tetanus neurotoxins show the presence of a closely homologous segment in the middle of the light chain (NH2-terminal 50 kDa), which is the intracellularly active portion of the toxin. This segment contains the zinc binding motif of metalloendopeptidases, HEXXH. Atomic adsorption analysis of botulinum neurotoxins (serotypes A, B, and E) made on the basis of this observation demonstrated the presence of one zinc atom/molecule of 150-kDa neurotoxin. Conditions were found for the removal of the zinc ion with chelating agents and for the restoration of the normal metal content. The conserved segment, which includes the zinc binding motif, was synthesized and shown to bind [65Zn]2+. Chemical modification experiments indicated that two histidines and no cysteines are involved in Zn2+ coordination in agreement with a probable catalytic role for the zinc ion. The present findings suggest the possibility that botulinum neurotoxins are zinc proteases.

Amino Acid Sequence↗
PubMed1992

Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin.

Clostridial neurotoxins, including tetanus toxin and the seven serotypes of botulinum toxin (A-G), are produced as single chains and cleaved to generate toxins with two chains joined by a single disulphide bond (Fig. 1). The heavy chain (M(r) 100,000 (100K)) is responsible for specific binding to neuronal cells and cell penetration of the light chain (50K), which blocks neurotransmitter release. Several lines of evidence have recently suggested that clostridial neurotoxins could be zinc endopeptidases. Here we show that tetanus and botulinum toxins serotype B are zinc endopeptidases, the activation of which requires reduction of the interchain disulphide bond. The protease activity is localized on the light chain and is specific for synaptobrevin, an integral membrane protein of small synaptic vesicles. The rat synaptobrevin-2 isoform is cleaved by both neurotoxins at the same single site, the peptide bond Gln 76-Phe 77, but the isoform synaptobrevin-1, which has a valine at the corresponding position, is not cleaved. The blocking of neurotransmitter release of Aplysia neurons injected with tetanus toxin or botulinum toxins serotype B is substantially delayed by peptides containing the synaptobrevin-2 cleavage site. These results indicate that tetanus and botulinum B neurotoxins block neurotransmitter release by cleaving synaptobrevin-2, a protein that, on the basis of our results, seems to play a key part in neurotransmitter release.

Amino Acid Sequence↗
PubMed1992

Lipid interaction of tetanus neurotoxin. A calorimetric and fluorescence spectroscopy study.

The interaction of Tetanus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl-phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.

Calorimetry, Differential Scanning↗
PubMed1992

Surface topography of histidine residues of tetanus toxin probed by immobilized-metal-ion affinity chromatography.

Tetanus toxin contains 14 histidine residues: six of them are localized in the light chain (L), one is present in the N-terminal half of the heavy chain (HN) and the remaining seven histidines are localized in the C-terminal half of the heavy chain (Hc). Using immobilized-metal-ion affinity chromatography with Chelating Superose-Zn(II), we show that histidines of Hc are exposed to the protein surface and are responsible for the binding of tetanus toxin and of Hc to the immobilized metal. The histidines of the L chain are not available for co-ordination of matrix-bound Zn2+; however, two of them and three of the histidines of fragment Hc are accessible to diethyl pyrocarbonate. Chromatography on Superose-Zn(II) is also shown to be a simple and efficient method for the rapid isolation of tetanus toxin and of its Hc fragment, which can be extended to the botulinum neurotoxins.

Cations, Divalent↗
PubMed1992

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus and botulinum neurotoxins are the most potent toxins known. They bind to nerve cells, penetrate the cytosol and block neurotransmitter release. Comparison of their predicted amino acid sequences reveals a highly conserved segment that contains the HexxH zinc binding motif of metalloendopeptidases. The metal content of tetanus toxin was then measured and it was found that one atom of zinc is bound to the light chain of tetanus toxin. Zinc could be reversibly removed by incubation with heavy metal chelators. Zn2+ is coordinated by two histidines with no involvement in cysteines, suggesting that it plays a catalytic rather than a structural role. Bound Zn2+ was found to be essential for the tetanus toxin inhibition of neurotransmitter release in Aplysia neurons injected with the light chain. The intracellular activity of the toxin was blocked by phosphoramidon, a very specific inhibitor of zinc endopeptidases. Purified preparations of light chain showed a highly specific proteolytic activity against synaptobrevin, an integral membrane protein of small synaptic vesicles. The present findings indicate that tetanus toxin, and possibly also the botulinum neurotoxins, are metalloproteases and that they block neurotransmitter release via this protease activity.

Acetylcholine↗