The lethal toxin of Bacillus anthracis consists of two proteins, PA and LF, which together induce lethal effects in animals and cause macrophage lysis. LF is a zinc-endopeptidase which cleaves two mitogen-activated proten kinase kinases (MAPKKs), Mek1 and Mek2, within the cytosol. Here, we show that also MKK3, another dual-specificity kinase that phosphorylates and activates p38 MAP kinase, is cleaved by LF in macrophages. No direct correlation between LF-induced cell death and cleavage of these MAPKKs was found in macrophage cell lines and primary peritoneal cells exhibiting different sensitivity to LF. However, we present the first evidence that sublytic doses of LF cleave Meks and cause a substantial reduction in the production of NO and tumour necrosis factor-K K induced by lipopolysaccharide/interferonQ Q. We suggest that this effect of LF is relevant during the first stages of B. anthracis infection, when a reduction of the inflammatory response would permit growth and diffusion of the bacterium.z 1999 Federation of European Biochemical Societies.

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The clostridial neurotoxins responsible for tetanus and botulism are proteins consisting of three domains endowed with di¡erent functions: neurospeci¢c binding, membrane translocation and proteolysis for speci¢c components of the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular junction, is internalized and transported retroaxonally to the spinal cord. The spastic paralysis induced by the toxin is due to the blockade of neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven serotypes of botulinum neurotoxins (BoNTs) act at the periphery by inducing a £accid paralysis due to the inhibition of acetylcholine release at the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G cleave speci¢cally at single but di¡erent peptide bonds, of the vesicle associated membrane protein (VAMP) synaptobrevin, a membrane protein of small synaptic vesicles (SSVs). BoNT types A, C and E cleave SNAP-25 at di¡erent sites located within the carboxyl-terminus, while BoNT type C additionally cleaves syntaxin. The remarkable speci¢city of BoNTs is exploited in the treatment of human diseases characterized by a hyperfunction of cholinergic terminals.

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by sitedirected mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.Tetanus neurotoxin (TeNT) 1 and botulinum neurotoxins (BoNTs, seven types from A to G) are three-domain protein toxins that bind selectively to the neuronal presynaptic membrane. They are internalized inside intracellular compartments from which the amino-terminal 50-kDa domain (termed L chain) enters into the cytosol (1-4). The L chains of TeNT and BoNTs are zinc-endopeptidases that cleave specifically three proteins of the neuroexocytosis apparatus, thereby blocking neurotransmitter release (4 -7). TeNT and BoNT/B, BoNT/D, BoNT/F, and BoNT/G recognize and cleave specifically a synaptic vesicle-associated membrane protein (VAMP, also referred to as synaptobrevin) at different single peptide bonds (4, 8 -12). BoNT/A and BoNT/E specifically recognize and cut SNAP-25 (synaptosomal-associated protein of 25 kDa) at two different peptide bonds near the COOH terminus (10, 13, 14), whereas BoNT/C cleaves syntaxin (15, 16) and . VAMP, SNAP-25, and syntaxin are collectively termed SNARE proteins, because they act as receptors of soluble Nethylmaleimide-sensitive factor accessory proteins, involved in vesicle-membrane fusion (5-7).Sequence comparison of the L chains of the eight clostridial neurotoxins show strong similarities (20), which are even more extensive at the level of predicted secondary structure (21). These similarities suggest that they derive from a common ancestral metalloproteinase. On this basis, to account for their different substrate specificity, we considered the possibility that the three SNAREs contain a common neurotoxin recognition site in addition to the cleavage sites specific for each neurotoxin type. We identified a nine-residue-long motif (SNARE motif) present in eukaryotes only in the three proteins known to be proteolytic substrates of the neurotoxins (22). The SNARE motif is included within regions...

The lethal toxin (LeTx) of Bacillus anthracis is the major virulence factor responsible for the death of infected animals and for cytolysis of cultured macrophages. Its catalytic component, LF, contains the characteristic zinc-binding motif of metalloproteases, it binds zinc and indirect evidence suggests that this hydrolytic activity is essential for LeTx cytotoxicity ( Limpel et al. 1994 ; Kochi et al. 1994 ). To identify substrates of LF, we have used the yeast two-hybrid system, employing an LF inactive mutant as bait. This approach has led to the identification of the MAP kinase kinases (MAPKKs) Mek1 and Mek2 as proteins capable of specific interaction with LF. LF cleaves Mek1 and Mek2 within their N-terminus in vitro and in vivo, hydrolysing a Pro8-Ile9 and a Pro10-Arg11 peptide bond in Mek1 and Mek2, respectively ( Vitale et al. 1998 ), similarly to that found with a different approach by Duesbery et al. (1998) . The removal of the amino terminus of MAPKKs eliminates the ‘docking site’ involved in the specific interaction with MAPKs and interferes with the phospho-activation of the MAPKs ERK1 and ERK2, which become phosphorylated in cultured macrophages following toxin challenge. We are currently investigating the relevance of MAPKKs cleavage for LeTx cytotoxicity and the consequences for the activity of the MAP pathway

Botulinum neurotoxins type A and E (BoNT/A and BoNT/E) are metalloproteases with a unique specificity for SNAP-25 (synaptosome-associated protein of 25 kDa), an essential protein component of the neuroexocytotic machinery. It has been suggested that this specificity is directed through the recognition of a nine residue sequence, termed SNARE motif, that is common to the other two SNARE proteins: VAMP (vesicle-associated membrane protein) and syntaxin, the only known substrates of the other six clostridial neurotoxins. Here we analyse the involvement of the four copies of the SNARE motif present in SNAP-25 in its interaction with BoNT/A and BoNT/E by following the kinetics of proteolysis of SNAP-25 mutants deleted of SNARE motifs. We show that a single copy of the motif is sufficient for BoNT/A and BoNT/E to recognise SNAP-25. While the copy of the motif proximal to the cleavage site is clearly involved in recognition, in its absence, other more distant copies of the motif are able to support proteolysis. Also, a non-neuronal isoform of SNAP-25, Syndet, is shown to be sensitive to BoNT/E, but not BoNT/A, whilst the SNAP-25 isoforms from Torpedo marmorata and Drosophila melanogaster were demonstrated not to be substrates of these metalloproteases.

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Botulinum neurotoxins type D and F are zincendopeptidases with a unique specificity for VAMP/synaptobrevin, an essential component of the exocytosis apparatus. VAMP contains two copies of a nine residue motif, termed VI and V2, which are determinants of the interaction with tetanus and botulinum B and G neurotoxins. Here, we show that VI plays a major role in VAMP recognition by botulinum neurotoxins D and F and that V2 is also involved in F binding. Site-directed mutagenesis of VI and V2 indicates that different residues are the determinants of the VAMP interaction with the two endopeptidases. The study of the VAMP-neurotoxins interaction suggest a pairing of the VI and V2 segments.