Structural and Functional Characteristics of the Proline-Rich Antimicrobial Peptide Minibactenecin from Leukocytes of Domestic Goat Capra hircus.

We performed structural and functional studies of minibactenecin mini-ChBac7.5Nα, a natural proline-rich cathelicidin from domestic goat Capra hircus. To identify the key residues important for the biological action of the peptide, a panel of its alanine-substituted analogues was produced. The development of E. coli resistance to the natural minibactenecin, as well as to its analogues carrying substitutions for hydrophobic amino acids in the C-terminal residues was studied. The data obtained indicate the possibility of rapid development of the resistance to this class of peptides. The main factors in the formation of the antibiotic resistance are various mutations leading to inactivation of the SbmA transporter.

Analysis of Antibacterial Action of Mammalian Host-Defense Cathelicidins and Induction of Resistance to Them in MßL-Producing Pseudomonas aeruginosa.

Recombinant analogs of a number of natural host-defense mammalian cathelicidins were obtained and predominant mechanism of their antibacterial action was studied. The ability of cathelicidins to suppress the growth of Pseudomonas aeruginosa producing metallo-ß-lactamases (MßL) was studied, and the possibility of appearance of cathelicidin-resistant bacteria was evaluated. Among peptides with different structures and mechanisms of action, only the strains resistant to ChMAP-28 were not obtained, which indicated minimum risk of the development of natural resistance to this cathelicidin. High antibacterial activity, wide spectrum of action, and the absence of cross-resistance effects allow considering ChMAP-28 peptide as a candidate to be developed further as a therapeutic agent against MßL-producing bacteria.

New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2.

Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the "bottom" entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the "bottom" entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation.

Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria.

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

Pediocin-Like Antimicrobial Peptides of Bacteria.

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

In Vitro Study of Antitumor Effect of Antimicrobial Peptide Tachyplesin I in Combination with Cisplatin.

We studied combined effect of ß-hairpin antimicrobial peptide tachyplesin I and cytotoxic agent cisplatin on tumor and normal human cell lines. MTT assay and flow cytometry showed that tachyplesin I selectively sensitized cancer cells to cisplatin in specified concentration ratios. In vitro experiments demonstrated that combined use of tachyplesin I and cisplatin allows decreasing the effective dose of the cytostatic thus reducing nonspecific toxicity.

The First Recombinant Viper Three-Finger Toxins: Inhibition of Muscle and Neuronal Nicotinic Acetylcholine Receptors.

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.

Analysis of Synergistic Effects of Antimicrobial Peptide Arenicin-1 and Conventional Antibiotics.

We studied combined effects of antimicrobial peptide arenicin-1 from lugworm Arenicola marina and some conventional antibiotics. A number of drug combinations with pronounced synergistic effects were revealed. The influence of antibacterial activity assessment conditions was determined and the methodology excluding false-positive test results was developed.

Effect of N- and C-Terminal Modifications on Cytotoxic Properties of Antimicrobial Peptide Tachyplesin I.

We analyze the effects of N-terminal acetylation and C-terminal amidation on the cytotoxic properties of ß-hairpin antimicrobial peptide tachyplesin I. MTT-assay showed that modified tachyplesin I exhibited increased cytotoxicity toward both tumor and normal human cells. Hemolytic activity of modified tachyplesin I was also higher than that of the initial molecule. In contrast to non-modified tachyplesin I, the peptide with C- and N-terminal modifications is resistant to proteolytic degradation in fresh human serum. C- and N-terminal modifications make tachyplesin I more attractive prototype of anticancer drug due to its more potent cytotoxic effect and better pharmacokinetic properties.

Minibactenecins ChBac7.Nα and ChBac7. Nß - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nß), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nß exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 µM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nß have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

Lipid Transfer Proteins As Components of the Plant Innate Immune System: Structure, Functions, and Applications.

Among a variety of molecular factors of the plant innate immune system, small proteins that transfer lipids and exhibit a broad spectrum of biological activities are of particular interest. These are lipid transfer proteins (LTPs). LTPs are interesting to researchers for three main features. The first feature is the ability of plant LTPs to bind and transfer lipids, whereby these proteins got their name and were combined into one class. The second feature is that LTPs are defense proteins that are components of plant innate immunity. The third feature is that LTPs constitute one of the most clinically important classes of plant allergens. In this review, we summarize the available data on the plant LTP structure, biological properties, diversity of functions, mechanisms of action, and practical applications, emphasizing their role in plant physiology and their significance in human life.

Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity.

[Synthesis of Oligocaccharides Containing Internal and Terminal Fragment Galß1-3GlcNAc].

Synthesis of oligocaccharides Galß1-3GlcNAcß-sp, GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-4GlcNAcß-sp, Galß1-3GlcNAcß1-6Galß1-4GlcNAcß-sp (sp = O(CH2)3NH2 or O(CH2)2NH2) was carried out using glycosylation by N-Troc-protected derivatives of glycosamine or disaccharide Galß1-3GlcN.

Structure and Biological Functions of ß-Hairpin Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are evolutionarily ancient factors of the innate immune system that serve as a crucial first line of defense for humans, animals, and plants against infection. This review focuses on the structural organization, biosynthesis, and biological functions of AMPs that possess a ß-hairpin spatial structure. Representatives of this class of AMPs are among the most active antibiotic molecules of animal origin. Due to their wide spectrum of activity and resistance to internal environmental factors, natural ß-hairpin AMPbased compounds might become the most promising drug candidates.

[Interaction of Arenicin-1 with C1q Protein].

The interaction between arenicin-1, that is an antimicrobial peptide from polychaeta Arenicola marina, and human complement system protein C1q was studied using enzyme-linked receptor sorbent assay and ELISA. We revealed that arenicin-1 and C1q form complex that is stable in high ionic strength condition 0.5 M NaCl. The ability of C1q to interact with arenicin-1 is comparable with the binding activity of C1q towards another antimicrobial peptide, porcine cathelicidin protegrin-1, which has a similar spatial arrangement with arenicin-1. Namely, both arenicin-1 and protegrin-1 form cystine-stabilized antiparallel ß-hairpin structure.

Lipid-protein nanodiscs offer new perspectives for structural and functional studies of water-soluble membrane-active peptides.

Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.

Acipensins - Novel Antimicrobial Peptides from Leukocytes of the Russian Sturgeon Acipenser gueldenstaedtii.

Antimicrobial peptides (AMPs) play an important role in the innate defense mechanisms in humans and animals. We have isolated and studied a set of antimicrobial peptides from leukocytes of the Russian sturgeon Acipenser gueldenstaedtii belonging to a subclass of chondrosteans, an ancient group of bony fish. Structural analysis of the isolated peptides, designated as acipensins (Ac), revealed in leukocytes of the Russian sturgeon six novel peptides with molecular masses of 5336.2 Da, 3803.0 Da, 5173.0 Da, 4777.5 Da, 5449.4 Da, and 2740.2 Da, designated as Ac1-Ac6, respectively. Complete primary structures of all the isolated peptides were determined, and the biological activities of three major components - Ac1, Ac2, and Ac6 - were examined. The peptides Ac1, Ac2, Ac3, Ac4, and Ac5 were found to be the N-terminal acetylated fragments 1-0, 1-5, 1-9, 1-4, and 1-1 of the histone H2A, respectively, while Ac6 was shown to be the 62-5 fragment of the histone H2A. The peptides Ac1 and Ac2 displayed potent antimicrobial activity towards Gram-negative and Gram-positive bacteria (Escherichia coli ML35p, Listeria monocytogenes EGD, MRSA ATCC 33591) and the fungus Candida albicans 820, while Ac6 proved effective only against Gram-negative bacteria. The efficacy of Ac 1 and Ac2 towards the fungus and MRSA was reduced upon an increase in the ionic strength of the solution. Ac1, Ac2, and Ac6, at concentrations close to their minimum inhibitory concentrations, enhanced the permeability of the E.coli ML35p outer membrane to the chromogenic marker, but they did not affect appreciably the permeability of the bacterial inner membrane in comparison with a potent pore-forming peptide, protegrin 1. Ac1, Ac2, and Ac6 revealed no hemolytic activity against human erythrocytes at concentrations of 1 to 40 µM and had no cytotoxic effect (1 to 20 µM) on K-562 and U-937 cells in vitro. Our findings suggest that histone-derived peptides serve as important anti-infective host defense molecules.

Lentil (Lens culinaris) lipid transfer protein Len c 3: a novel legume allergen.

BACKGROUND: Lentils are increasingly consumed in many parts of the world.Two allergens, Len c 1 and 2, have been reported previously. Recently, peanut and green bean lipid transfer proteins (LTPs) have been identified as the first two members of an important group of allergens that might be associated with severe food allergies. OBJECTIVE: To investigate lentil LTP as a potential new allergen. METHODS: Efficacy of LTP extraction was monitored at different acidic pH values, using immunoblotting with cross-reactive anti-peach LTP antiserum. Natural LTP was purified from lentil extract and expressed as recombinant allergen in Escherichia coli. Sera from 10 lentil-allergic and/or -sensitized patients (Spain: 6, Italy: 1 and the Netherlands: 3) were used to further characterize lentil LTP. RESULTS: Natural lentil LTP, purified from the homogenized germinated seeds and optimally extracted at pH 3, was identified and designated as allergen Len c 3. By CAP, 9/10 sera showed specific IgE to Len c 3. Recombinant (r) Len c 3 was successfully purified. The natural (n) Len c 3 CAP was completely inhibited by rLen c 3/rPru p 3. IgE binding to lentil pH 3 extract blot was completely inhibited by rLen c 3. CONCLUSION: The availability of immunochemically active nLen/rLen c 3 as a novel legume allergen facilitates further development and implementation of a third (next to peanut and green bean) legume LTP in component-resolved diagnosis strategies and contributes to evaluate the clinical importance of legume LTPs. Preferential extraction of Len c 3 (pH 3) will affect the production of sensitive extract-based diagnostic tests.

Lipid-protein nanodiscs: possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides.

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). (15)N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and (31)P-NMR spectroscopy. The 2D 1H-(15)N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased (1)H(N) line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. (15)N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (approximately 40 nsec at 45 degrees C) is indicative of additional peptide motions within the Aam-I/LPN complex.