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Chapter 19 : Proteases

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Proteases, Page 1 of 2

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Abstract:

This chapter focuses on bacterial protein toxins that target eukaryotic cells acting as zinc-metalloproteases. Additional understanding of the mode of action of proteases that contribute to pathogenicity could lead to the development of inhibitors that could prevent or interrupt the disease process. The seven botulinum neurotoxins (BoNTs, types A to G) and the single tetanus neurotoxin (TeNT) are responsible for the clinical manifestations of botulism and tetanus, respectively, and constitute a group of metalloproteases endowed with unique properties. These bacterial metalloproteases do not act on the cell surface but exert their enzymatic action in the cell cytosol on selected proteins that form the core of the neuroexocytosis machinery. The SNARE complex is insensitive to clostridial neurotoxins (CNT) proteolysis, as expected on the basis that proteases are known to attack predominantly unstructured exposed loops. toxin (BFT) is the first bacterial toxin known to remodel the intestinal epithelial cytoskeleton and F-actin architecture via cleavage of a cell surface molecule and represents the prototype of a novel class of bacterial toxins that act without cell internalization and covalent modification of intracellular substrates. In analogy with tetanus, botulism, and anthrax, one is tempted to suggest that BFT is an essential virulence factor.

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19

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Bacterial Proteins
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Figures

Image of Figure 1
Figure 1

Three-domain structure of CNTs. (A) These neurotoxins consist of three domains of similar size (50 kDa). The N-terminal domain (L) is a zincendopeptidase containing the His-Glu-Xaa-Xaa-His zinc-binding motif of zincendopeptidases. Its intracellular activity is expressed after reduction of the interchain disulfide bond. H, the central domain, is responsible for the membrane translocation of the L chain into the neuronal cytosol. The Cterminal H domain consists of two equally sized subdomains. The N-terminal subdomain (H) has a structure similar to that of sugar-binding proteins. The C-terminal subdomain (H) folds similarly to proteins known to be involved in protein-protein binding functions such as the K+ channel-specific dendrotoxin. Such structure is consistent with the toxin binding to the presynaptic membrane via a double interaction, most likely with two different molecules of the nerve terminal. (B) Crystallographic structure of BoNT/A oriented as in the top panel: the zinc atom and the -helix containing the HEXXH motif are shown in black. Notice the unusual 10-nm-long pair of -helices present in the Hdomain (from reference with permission).

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19
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Image of Figure 2
Figure 2

A mechanism for substrate proteolysis by CNTs and by anthrax LF. The active site of the CNTs and of LF is centered around a structural zinc cation coordinated by a strictly conserved HEXXH+E motif. In both cases zinc is directly coordinated by the two histidines of the motif, by a glutamic acid (Glu-268 in BoNT/B and Glu-735 in LF) and by a water molecule that forms a strong hydrogen-bonding interaction with the Glu of the motif. A conserved tyrosine of the active site (Tyr-373 in BoNT/B and Tyr-728 in LF) likely functions as a general acid to protonate the amine leaving group in the transition state, assisting the hydrolysis reaction to yield the cleaved products. A conserved Arg present in the active site of CNTs but not in the one of LF probably has a role in stabilizing the transition state in combination with the zinc ion.

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19
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Image of Figure 3
Figure 3

Scheme of a presynaptic cell. The figure shows the normal process of the neurotransmitter (NT) release (left) and the mechanism of action of TeNT and BoNTs (right). Vesicles containing the NT have a transmembrane protein called VAMP that binds specifically two proteins of the cell membrane (syntaxin and SNAP25). The three proteins make a complex that forces the vesicle and cellular membranes to come in close contact and finally to fuse, thus releasing the NT into the intersynaptic space. TeNT and BoNTs are zincendopeptidases that cleave VAMP, syntaxin, and SNAP25, thus preventing the NT release.

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19
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Image of Figure 4
Figure 4

Stereo ribbon representation of LF, which reveals the presence of four distinct domains: the aminoterminal PA-binding domain (1, in black), the second domain resembling the ADPribosylating toxin VIP2 from (2, in light gray), the third domain inserted between the second and third helices of the second domain (3, in gray), and the fourth Cterminal catalytic domain (4, in dark gray). Zinc ion in the active site of the catalytic domain is shown as a black sphere. Domains 2, 3, and 4 together create a long deep groove that holds the Nterminal tail of the MAPKK prior to cleavage (adapted from reference ).

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19
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Image of Figure 5
Figure 5

Schematic structure of fragilysin. The predicted amino acid sequences of the , , and genes suggest that the encoded proteins are preproprotein toxins. Cleavage between Arg and Ala residues appears to release the mature BFT protein. Each protein contains a HEXXHXXGXXH motif (and a conserved methionine near the C terminus), suggesting that they belong to the metzincins group of metalloproteases.

Citation: Rossetto O, Tonello F, Montecucco C. 2003. Proteases, p 271-282. In Burns D, Barbieri J, Iglewski B, Rappuoli R (ed), Bacterial Protein Toxins. ASM Press, Washington, DC. doi: 10.1128/9781555817893.ch19
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References

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