Biomedical subjects

Igor Zelezetsky

Publications and source records attributed to Igor Zelezetsky.

10 recordsLinked to original sources

PubMed2006

Evolution of the primate cathelicidin. Correlation between structural variations and antimicrobial activity.

Cathelicidin genes homologous to the human CAMP gene, coding for the host defense peptide LL-37, have been sequenced and analyzed in 20 primate species, including Great Apes, hylobatidae, cercopithecidae, callithricidae, and cebidae. The region corresponding to the putative mature antimicrobial peptide is subject to a strong selective pressure for variation, with evidence for positive selection throughout the phylogenetic tree relating the peptides, which favors alterations in the charge while little affecting overall hydrophobicity or amphipathicity. Selected peptides were chemically synthesized and characterized, and two distinct types of behavior were observed. Macaque and leaf-eating monkey RL-37 peptides, like other helical antimicrobial peptides found in insect, frog, and mammalian species, were unstructured in bulk solution and had a potent, salt and medium independent antimicrobial activity in vitro, which may be the principal function also in vivo. Human LL-37 and the orangutan, hylobates, and callithrix homologues instead showed a salt-dependent structuring and likely aggregation in bulk solution that affected antimicrobial activity and its medium dependence. The two types of peptides differ also in their interaction with host cells. The evolution of these peptides has thus resulted in distinct mechanisms of action that affect the direct antimicrobial activity and may also modulate accessory antimicrobial functions due to interactions with host cells.

Amino Acid Sequence↗

PubMed2006

Alpha-helical antimicrobial peptides--using a sequence template to guide structure-activity relationship studies.

An important class of cytolytic antimicrobial peptides (AMPs) assumes an amphipathic, alpha-helical conformation that permits efficient interaction with biological membranes. Host defence peptides of this type are widespread in nature, and numerous synthetic model AMPs have been derived from these or designed de novo based on their characteristics. In this review we provide an overview of the 'sequence template' approach which we have used to design potent artificial helical AMPs, to guide structure-activity relationship studies aimed at their optimization, and to help identify novel natural AMP sequences. Combining this approach with the rational use of natural and non-proteinogenic amino acid building blocks has allowed us to probe the individual effects on the peptides' activity of structural and physico-chemical parameters such as the size, propensity for helical structuring, amphipathic hydrophobicity, cationicity, and hydrophobic or polar sector characteristics. These studies furthermore provided useful insights into alternative modes of action for natural membrane-active helical peptides.

Amino Acid Substitution↗

PubMed2005

Controlled alteration of the shape and conformational stability of alpha-helical cell-lytic peptides: effect on mode of action and cell specificity.

A novel method, based on the rational and systematic modulation of macroscopic structural characteristics on a template originating from a large number of natural, cell-lytic, amphipathic alpha-helical peptides, was used to probe how the depths and shapes of hydrophobic and polar faces and the conformational stability affect antimicrobial activity and selectivity with respect to eukaryotic cells. A plausible mode of action explaining the peptides' behaviour in model membranes, bacteria and host cells is proposed. Cytotoxic activity, in general, correlated strongly with the hydrophobic sector depth, and required a majority of aliphatic residue side chains having more than two carbon atoms. It also correlated significantly with the size of polar sector residues, which determines the penetration depth of the peptide via the so-called snorkel effect. Both an oblique gradient of long to short aliphatic residues along the hydrophobic face and a stabilized helical structure increased activity against host cells but not against bacteria, as revealed by haemolysis, flow cytofluorimetric studies on lymphocytes and surface plasmon resonance studies with model phosphatidylcholine/cholesterol membranes. The mode of interaction changes radically for a peptide with a stable, preformed helical conformation compared with others that form a structure only on membrane binding. The close correlation between effects observed in biological and model systems suggests that the 'carpet model' correctly represents the type of peptides that are bacteria-selective, whereas the behaviour of those that lyse host cells is more complex.

Amino Acid Sequence↗

PubMed2005

Tuning the biological properties of amphipathic alpha-helical antimicrobial peptides: rational use of minimal amino acid substitutions.

In nature, alpha-helical antimicrobial peptides present the small and flexible residue glycine at positions 7 or 14 with a significant frequency. Based on the sequence of the non-proteinogenic alpha-helical model peptide P1(Aib7), with a potent, broad spectrum antimicrobial activity, six peptides were designed by effecting a single amino acid substitution to investigate how tuning the structural characteristics at position 7 could lead to optimization of selectivity without affecting antimicrobial activity against a broad panel of multidrug resistant bacterial and yeast indicator strains. The relationship between structural features (size/hydrophobicity of the side chain as well as conformation and flexibility) and biological activity, in terms of minimum inhibitory concentration, membrane permeabilization kinetics and lysis of red blood cells are discussed. On conversion of the peptide to proteinogenic residues, these principles allowed development of a potent antimicrobial peptide with a reduced cytotoxicity. However, while results suggest that both hydrophobicity of residue 7 and chain flexibility at this position can be modulated to improve selectivity, position 14 is less tolerant of substitutions.

Amino Acid Substitution↗

PubMed2005

Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin.

A template based on positional residue frequencies in the N-terminal stretch of natural alpha-helical antimicrobial peptides was used to prepare sequence patterns and to scan the Swiss-Prot Database, using the ScanProsite tool. This search identified a segment in pilosulin 1, a cytotoxic peptide from the venom of the jumper ant Myrmecia pilosula, as a potential novel antimicrobial peptide sequence. This segment, corresponding to the 20 N-terminal residues, was synthesized and its structural properties and biological activities were investigated. It showed a potent and broad spectrum antimicrobial activity including standard and multi-drug resistant gram-positive and gram-negative bacteria and Candida albicans, confirming the validity of the search method. A rational redesign approach resulting in four amino acid substitutions yielded a variant with improved antibacterial and significantly reduced hemolytic activity.

Amino Acid Sequence↗

PubMed2005

Structural aspects and biological properties of the cathelicidin PMAP-36.

PMAP-36 is a cathelicidin-derived host defence peptide originally deduced by a transcript from pig bone marrow RNA. The expression of the propeptide in leukocytes, and the structure, antimicrobial activity, and mechanism of action of the mature peptide were investigated. The proform is stored as a dimeric precursor of 38 kDa formed by a dimerization site at its C-terminal cysteine residue; it is likely that the mature peptide is dimeric when released. Monomeric and dimeric forms of PMAP-36 were chemically synthesized and their activity compared. Both forms assumed an amphipathic alpha-helical conformation and exhibited a potent and rapid microbicidal activity against a wide spectrum of microorganisms, mediated by their ability to permeabilize the microbial membranes rapidly. A shortened fragment localized the helical region to the N terminus, but showed a significantly lower potency and slower permeabilization kinetics, indicating an important role of the nonhelical C-terminal hydrophobic portion of this molecule. Dimerization modulated the effectiveness of the peptide in terms of killing and permeabilization kinetics, and reduced medium dependence. It allows the molecule to achieve an impressive charge density (+28 in 70 residues), although the significance of this feature with respect to biological activity has yet to be determined.

Amino Acid Sequence↗

PubMed2005

Primate beta-defensins--structure, function and evolution.

Host defense peptides (HDPs) are endogenous antibiotics that play a multifunctional role in the innate immunity of mammals. Among these, beta-defensins contribute to mucosal and epithelial defense, also acting as signal molecules for cellular components of innate and adaptive immunity. Numerous members of this family have been identified in mammalian and avian species, and genomic studies in human and mouse indicate a considerable complexity in their gene organization. Recent reports on the evolution of primate and rodent members of this family indicate quite a complex pattern of variation. In this review we briefly discuss the evolution of mammalian beta-defensins in relation to other types of defensins, and then concentrate on the evolution of beta-defensins 1 to 4 in primates. In particular, the surprisingly varied patterns of evolution, which range from neutral or weakly purifying, to positive selection to a high level of conservation are analyzed in terms of possible genetics, structural or functional implications, as well as to observed variations on the antimicrobial activity in vitro. The role of polymorphisms in the genes encoding for these host defense peptides in determining susceptibility to human diseases are also briefly considered.

Amino Acid Sequence↗

PubMed2004

Effects of positively selected sequence variations in human and Macaca fascicularis beta-defensins 2 on antimicrobial activity.

The evolution of orthologous genes coding for beta-defensin 2 (BD2) in primates has been subject to positive selection during the divergence of the platyrrhines from the catarrhines and of the Cercopithecidae from the Hylobatidae, great apes, and humans. Three peptides have been selected for a functional analysis of the effects of sequence variations on the direct antimicrobial activity: human BD2 (hBD2), Macaca fascicularis BD2 (mfaBD2), and a variant of the human peptide lacking Asp(4), (-D)hBD2, which is characteristic only of the human/great ape peptides. hBD2 and mfaBD2 showed a significant difference in specificity, the former being more active towards Escherichia coli and the later towards Staphylococcus aureus and Candida albicans. Asp(4) in the human peptide appears to be important, as (-D)hBD2 was less structured and had a markedly lower antimicrobial activity. The evolution of beta-defensin 2 in primates may thus have been driven, at least in part, by different environmental pressures so as to modulate antimicrobial activity.

Amino Acid Sequence↗

PubMed2003

A study of host defence peptide beta-defensin 3 in primates.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.

Amino Acid Sequence↗

PubMed2002

Solid-phase synthesis of fullerene-peptides.

The solid-phase synthesis of peptides (SPPS) containing [60]fullerene-functionalized amino acids is reported. A new amino acid, fulleropyrrolidino-glutamic acid (Fgu), is used for the SPPS of a series of analogues of different length based on the natural Leu(5)-Enkephalin and on cationic antimicrobial peptides. These fullero-peptides were prepared on different solid supports to analyze the influence of the resin on the synthesis. Optimized protocols for the coupling and deprotection procedures were determined allowing the synthesis of highly pure peptides in sufficient quantities for evaluation of biological activities. In particular, to avoid side reactions of the fullerene moiety with bases and nucleophiles, the removal of the protecting groups was performed under inert conditions (nitrogen or argon in the dark). We have encountered serious problems with the recovery of the crude compounds, especially when Fgu was inserted in the proximity of the resin core as fullero-peptides tend to remain embedded inside the resin. Eventually, all of the fullero-peptides were easily purified, and the cationic peptides were tested for their antimicrobial activities. They displayed a specific activity against the Gram-positive bacterium S. aureus and also lysed erythrocytes. The availability of a fullero-amino acid easily useable in the SPPS of fullero-peptides may thus open the way to the synthesis of new types of biologically active oligomers.

Amino Acids↗