Zinc endopeptidases

The most ingenious exocytosis toxins, however, come from the anaerobic bacteria Clostridium botulinum and Clostridium tetani. The former produces the seven botulinum neurotoxins (BoNTs) A-G the latter produces tetanus neurotoxin (TeNT). All eight toxins consist of a heavy (H) chain and a light (L) chain that are associated by an interchain S-S bond. The L-chains enter the cytosol of axon terminals. Importantly, BoNT L-chains mainly enter peripheral cholinergic terminals, whereas the TeNT L-chain mainly enters cerebral and spinal cord GABAergic and glycinergic terminals. The L-chains are the active domains of the toxins. They are zinc-endopeptidases and specifically split the three core proteins of exocytosis, i.e. the SNAREs (Fig. 1 inset). Each ofthe eight toxins splits a... 

Schiavo, G., Shone, C.C., Rossetto, O., Alexander, F.C. and Montecucco, C., Botulinum neurotoxin serotype 1 is a zinc endopeptidase specific for VAMP/ synaptobrevin, J. Biol. Chern., 268, 11516-11519, 1993. 

Botulinus toxin from Clostridium botuli-num is the most potent poison known. The estimated lethal dose for 50% of an exposed human population is 1 / 10 9 g/kg (i.e., about 75 nanograms for an adult individual). The toxin, a zinc endopeptidase, blocks exo-cytosis of ACh in motor (and also parasympathetic) nerve endings. Death is caused by paralysis of respiratory muscles. 

Another ATP-dependent protease identified among heat shock proteins of E. coli is known as HslV-HslU or (ClpQ-ClpY). It has a threonine protease active site and is even more closely remeniscient of eukaryotic proteasomes. Also active in E. coli is another ring-like protease, a membrane-bormd zinc endopeptidase FtsH (or HflB). " Similar eukaryotic proteases also exist. ... 

The most conserved segment of the L chain of CNTs is a central region that contains a His-Glu-Xaa-Xaa-His zinc-binding motif characteristic of zinc-endopeptidases, thus suggesting that TeTx and the BoNTs may inhibit neuroexocytosis through a zinc-endopeptidase activity. This hypothesis was confirmed with two experimental approaches in Aply-sia neurons. First, the lack of toxicity of the apo-TeTx L chain demonstrated the essential role of the metal atom in toxin activity (Schiavo et ai, 1992 a, b). Second, phosphoramidon, a very specific inhibitor of zinc-endopeptidases, was shown to inhibit TeTx-induced blockade of ACh release (Schiavo ef ai, 1992 b). These results were the first clear evidence that the L chain of TeTx was acting via a metallo-protease activity. 

TeTx and BoNTs are zinc-endopeptidases highly specific for their substrates and do not cleave short peptides spanning the respective cleavage sites. As a consequence, a simple spectrophotometric or fluorometric assay of their activity is not yet available. Their zinc-dependent activity must be assayed with the target protein or a substantial portion of it. The target proteins can be obtained by sub-... 

The clostridial neurotoxins responsible for tetanus and botulism form a new group of zinc-endopeptidases endowed with peculiar properties. They are produced as inactive precursors which are activated by specific proteolysis, followed by intracellular reduction of a single di-... 

Schiavo G, Poulain B, Rossetto O, Benfenati F, Tauc L, Montecucco C (1992 b) Tetanus toxin is a zinc protein and its inhibition of neurotrasmitter release and protease activity depend on zinc. In EMBOJ. 11 3577-83 Schiavo G, Rossetto O, Santucci A, DasGupta BR, Montecucco C (1992 c) Botulinum neurotoxins are zinc proteins. In J. Biol. Chem. 267 23479-83 Schiavo G, Rossetto O, Catsicas S, Polverino de Laureto P, DasGupta BR, Benfenati F, Montecucco C (1993 a) Identification of the nerve-terminal targets of botulinum neurotoxins serotypes A, D and E. In J. Biol. Chem. 268 23784-7 Schiavo G, Santucci A, DasGupta BR, Metha PP, Jontes J, Benfenati F, Wilson MC, Montecucco C (1993 b) Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds. In FEBS Lett. 335 99-103 Schiavo G, Shone CC, Rossetto O, Alexandre FCG, Montecucco C (1993 c) Botulinum neurotoxin serotype F is a zinc endopeptidase specific for VAMP/synOp-tobrevin. In J. Biol. Chem. 268 11516-9... 

Schmid MF, Robinson JP, DasGupta BR (1993) Direct visualization of bofulinum neurotoxin-induced channels in phospholipid vesicles. In Nature 364 827-30 Shone CC, Roberts AK (1994) Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin. In Eur. J. Biochem. 225 263-70... 

Bartlett and Otake experimentally verified the applicability of tripeptide analogues with the Cbz-Gly-xP[(Z)-CF=CH]-Leu-Xaa structure as ground-state analogue inhibitors of the zinc endopeptidase thermolysin (see Figure 10.4) [19]. These fluoro-olefin tripeptide... 

Stocker, W. and Bode, W. (1995) Structural features of a superfamily of zinc-endopeptidases the metzincins. Curr. Opin. Struct. Biol., 5(3), 383-390. 

Merino, L, Thompson, J.D., Millard, C.B., Schmidt, J.J., and Pang, Y.P. 2006. Bis-imidazoles as molecular probes for peripheral sites of the zinc endopeptidase of botulinum neurotoxin serotype A. Bioorg. Med. Chem. 14 3583-3591. 

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