The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP 6 ). In this study, we demonstrated that InsP 6 is not simply an allosteric cofactor, but rather binding of InsP 6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP 6 -bound unprocessed form of CPD was determined and revealed the scissile bond Leu 3428 -Ala 3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP 6 , but was reactivated for high affinity binding of InsP 6 by cooperative binding of both a new substrate and InsP 6 . Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3 toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1). The best characterized MARTX is the Ͼ450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2, 3). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4, 5), the Rho-inactivating domain (RID) that disables small Rho GTPases (6), an ␣␤ hydrolase of unknown function (1), and an autoprocessing cysteine protease domain (CPD) (7,8).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7,8). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP 6 ) (7), a molecule ubiquitously present in eukaryotic cell cytosol (9 -11), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys 3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8).While it is clear that InsP 6 activates the CPD and that auto...

A novel homologue of insect defensin designated lucifensin (Lucilia defensin) was purified from the extracts of various tissues (gut, salivary glands, fat body, haemolymph) of green bottle fly (Lucilia sericata) larvae and from their excretions/secretions. The primary sequence of this peptide of 40 residues and three intramolecular disulfide bridges was determined by ESI-QTOF mass spectrometry and Edman degradation and is very similar to that of sapecin and other dipteran defensins. We assume that lucifensin is the key antimicrobial component that protects the maggots when they are exposed to the highly infectious environment of a wound during the medicinal process known as maggot therapy. We also believe that lucifensin is that long-sought larger molecular weight antimicrobial factor of the Lucilia sericata excretions/secretions believed to be effective against pathogenic elements of the wound microbial flora.

Two novel antimicrobial peptides, named halictines, were isolated from the venom of the eusocial bee Halictus sexcinctus. Their primary sequences were established by ESI-QTOF mass spectrometry, Edman degradation and enzymatic digestion as Gly-Met-Trp-Ser-Lys-Ile-Leu-Gly-His-Leu-Ile-Arg-NH2 (HAL-1), and Gly-Lys-Trp-Met-Ser-Leu-Leu-Lys-His-Ile-Leu-Lys-NH2 (HAL-2). Both peptides exhibited potent antimicrobial activity against Gram-positive and Gram-negative bacteria but also noticeable hemolytic activity. The CD spectra of HAL-1 and HAL-2 measured in the presence of trifluoroethanol or SDS showed ability to form an amphipathic alpha-helical secondary structure in an anisotropic environment such as bacterial cell membrane. NMR spectra of HAL-1 and HAL-2 measured in trifluoroethanol/water confirmed formation of helical conformation in both peptides with a slightly higher helical propensity in HAL-1. Altogether, we prepared 51 of HAL-1 and HAL-2 analogs to study the effect of such structural parameters as cationicity, hydrophobicity, alpha-helicity, amphipathicity, and truncation on antimicrobial and hemolytic activities. The potentially most promising analogs in both series are those with increased net positive charge, in which the suitable amino acid residues were replaced by Lys. This improvement basically relates to the increase of antimicrobial activity against pathogenic Pseudomonas aeruginosa and to the mitigation of hemolytic activity.

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

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Two propart peptides of aspartic proteinases. the propart peptide of chicken pepsin and human cathepsin D. respectively. were investigated from the point of view of their inhibitory activity for a set of aspartic proteinases. These peptides display a very broad Inhibitory spectrum. The strongest inhibition was observed for pepsin A-like proteinases where propart peptides can be used as titrants of active enzymes.

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