Anticancer mechanisms of temporin-1CEa,an amphipathic α-helical antimicrobial peptide,in Bcap-37 human breast cancer cells

AimsTemporin-1CEa, a 17-residue antimicrobial peptide, is known to exert broad-spectrum anticancer activity that acts preferentially on cancer cells instead of normal cells. However, the mechanism of cancer cell death induced by temporin-1CEa is weakly understood.Main methodsHere, we investigated the cytotoxic and membrane-disrupting effects of temporin-1CEa on human breast cancer cell line Bcap-37, using MTT assay, electronic microscope observation, fluorescence imaging and flow cytometry analysis.Key findingsThe MTT assay indicated that one-hour temporin-1CEa treatment led to rapid cell death in either caspase-dependent or -independent manner. The electronic microscope observation suggested that temporin-1CEa exposure resulted in profound morphological changes in Bcap-37 cells. The fluorescence imaging and flow cytometry analysis demonstrated that temporin-1CEa exhibited membrane-disrupting property characterized by induction of cell-surface phosphatidylserine exposure, elevation of plasma membrane permeability, and rapid transmembrane potential depolarization. Moreover, temporin-1CEa might also induce rapid cell death through mitochondria-involved mechanisms, including rapid intracellular Ca^2 + leakage, collapse of mitochondrial membrane potential (Δφm) and over-generation of reactive oxygen species (ROS).SignificanceIn summary, the present study indicates that temporin-1CEa triggers a rapid cytotoxicity in Bcap-37 cells through membrane-destruction and intracellular mechanisms involving mitochondria. These intracellular mechanisms and direct membrane-destruction effect were evaluated helping to understand the detail action of antimicrobial peptides in mammalian cancer cells.     

Rapid Cytotoxicity of Antimicrobial Peptide Tempoprin-1CEa in Breast Cancer Cells through Membrane Destruction and Intracellular Calcium Mechanism

Temporin-1CEa is an antimicrobial peptide isolated from the skin secretions of the Chinese brown frog (Rana chensinensis). We have previously reported the rapid and broad-spectrum anticancer activity of temporin-1CEa in vitro. However, the detailed mechanisms for temporin-1CEa-induced cancer cell death are still weakly understood. In the present study, the mechanisms of temporin-1CEa-induced rapid cytotoxicity on two human breast cancer cell lines, MDA-MB-231 and MCF-7, were investigated. The MTT assay and the LDH leakage assay indicated that one-hour of incubation with temporin-1CEa led to cytotoxicity in a dose-dependent manner. The morphological observation using electronic microscopes suggested that one-hour exposure of temporin-1CEa resulted in profound morphological changes in both MDA-MB-231 and MCF-7 cells. The membrane-disrupting property of temporin-1CEa was further characterized by induction of cell-surface exposure of phosphatidylserine, elevation of plasma membrane permeability and rapid depolarization of transmembrane potential. Moreover, temporin-1CEa evoked intracellular calcium ion and reactive oxygen species (ROS) elevations as well as collapse of mitochondrial membrane potential (Δφm). In summary, the present study indicates that temporin-1CEa triggers rapid cell death in breast cancer cells. This rapid cytotoxic activity might be mediated by both membrane destruction and intracellular calcium mechanism.     

Purification, molecular cloning, and antimicrobial activity of peptides from the skin secretion of the black-spotted frog, Rana nigromaculata

Antimicrobial peptides from a wide range of amphibian species, especially frogs of the genus Rana, have been characterised and are potential therapeutic agents. Here we describe the isolation, purification, and structural and biological characterisation of three novel antimicrobial peptides from the skin secretions of the black spotted frog, Rana nigromaculata, from Northeastern China. The peptides were identified as belonging to two known families: the temporin, which was first identified in R. nigromaculata from China, and the brevinin-2. Temporin-1RNa and temporin-1RNb both containing three positive charges and have a high potency against microorganisms (MIC: 3.13–8.3 μM against Gram-positive bacteria, 12.5–25.0 μM against Gram-negative bacteria, and 6.25–12.5 μM against Candida albicans) and a high haemolytic activity against human erythrocytes (HC₅₀: 100–150 μM). Brevinin-2RNa contains a single intra-disulphide bridge at the C-terminus that is active towards the tested Gram-positive bacteria but is not active against E. coli and P. aeruginosa. The cDNAs encoding three novel peptide precursors were also subsequently cloned from an R. nigromaculata skin cDNA library and sequenced. The precursors contain 58–72 amino acid residues, which include a conserved signal peptide, acidic propeptide, and the mature temporin-1RNa, temporin-1RNb and brevinin-2RNa. The CD spectra of temporin-1RNa and temporin-1RNb in water, 30 mM SDS and 50 % trifluoroethanol (TFE) indicated that both peptides adopted an aperiodic structure in water and an organised structure with an α-helical conformation in TFE and SDS solution. The conformational transition induced by TFE or SDS reflects the potential ability of temporin-1RNa and temporin-1RNb to interact with anionic membranes.     

中国林蛙皮肤抗菌肽基因的cDNA克隆及抗菌、抗癌和溶血活性的测定

采用RT-PCR和3￠RACE方法, 从中国林蛙皮肤总RNA中克隆出了6条编码不同抗菌肽前体的cDNA序列, 分别命名为: preprotemporin-1CEa、preprotemporin-1CEb、preprotemporin-1CEc、preprobrevinin-1CEa、preprobrevinin-1CEb和preprochensinin-1。cDNA碱基序列全长为289~315 bp, 编码59~65个氨基酸。6个抗菌肽前体由3部分结构域组成: 22个氨基酸残基组成的信号肽、多个酸性氨基酸残基组成的中间序列、高度变异的成熟肽。preprotemporin-1CEa、preprotemporin-1CEb和preprotemporin-1CEc属于temporin-1家族抗菌肽前体, 具有肽链短, C-端酰胺化的特点; preprobrevinin-1CEb和preprobrevinin-1CEa属于brevinin-1家族抗菌肽前体, 在肽链的C-端含有RANA盒结构, 可在2个半胱氨酸残基间形成二硫键, 组成7肽环; preprochensinin-1在已知多种数据库中没有发现序列同源的多肽, 被鉴定为新抗菌肽。人工合成temporin-1CEa、temporin-1CEb、brevinin-1CEa和chensinin-1四种中国林蛙皮肤抗菌肽, 活性检测结果表明: 它们对金黄葡萄球菌等3种革兰氏阳性细菌的生长具有明显抑制作用, 同时抑制乳腺癌MCF-7细胞和宫颈癌HeLa细胞生长。     

Antimicrobial peptides and protease inhibitors in the skin secretions of the crawfish frog, Rana areolata

The dorsal skin of the crawfish frog, Rana areolata, is associated with numerous prominent granular glands. Proteomic analysis of electrically stimulated skin secretions from these glands enabled the identification and characterization of eight peptides with antimicrobial and hemolytic activity belonging to the previously identified brevinin-1, temporin-1, palustrin-2, palustrin-3, esculentin-1 (two peptides), and ranatuerin-2 (two peptides) families. The primary structures of the peptides were consistent with a close phylogenetic relationship between R. areolata and the pickerel frog, Rana palustris. Three structurally related cationic, cysteine-containing peptides were identified that show sequence similarity to peptide Leucine–Arginine, a peptide with immunomodulatory and histamine-releasing properties from the skin of the northern leopard frog, Rana pipiens. The skin secretions contained a 61-amino-acid-residue peptide that inhibited porcine trypsin and possessed a 10-cysteine-residue motif that is characteristic of a protease inhibitor previously isolated from the parasitic nematode, Ascaris suum. A 48-amino-acid-residue protein containing eight cysteine residues in the whey acidic protein (WAP) motif, characteristic of elafin (skin-derived antileukoproteinase) and secretory leukocyte protease inhibitor, was also isolated. The data suggest that protease inhibitors in skin secretions may play a role complementary to cationic, amphipathic α-helical peptides in protecting anurans from invasions by microorganisms.     

Biophysical characterization and antitumor activity of synthetic Pantinin peptides from scorpion's venom

Antimicrobial peptides have been extensively described as bioactive agents, mainly considering their selective toxicity towards bacteria but not to healthy mammalian cells. In past years, this class of compounds has been classified as an attractive and novel family of anticancer agents. Pantinin peptides isolated from scorpion Pandinus imperator presented antimicrobial activity. In this study, we have explored the in vitro antitumor activity of antimicrobial pantinin peptides against the tumor cell lines MDA-MB-231 (breast adenocarcinoma) and DU − 145 (prostate adenocarcinoma) and healthy fibroblasts HGF − 1. To further improve our mechanistic understanding for this class of compounds, we have also performed a biophysical characterization of these peptides in lipid model membranes. Cell viability assays revealed that all peptides were more effective on tumor cells when compared to fibroblasts, indicating selectivity towards cancer cells. Furthermore, flow cytometry analysis revealed that all peptides induced apoptosis in cancer cells in a different way from fibroblasts. Circular dichroism spectroscopy showed that all peptides adopted an α-helical structure and an evaluation of the binding constant indicates a higher affinity of the peptides to negatively charged phospholipids. Additionally, permeabilization assays showed that POPG and POPS anionic vesicles were more susceptible to peptide-induced lysis than POPC:Chol and POPC:POPE vesicles. Moreover, we have observed that increasing concentrations of cholesterol inhibits peptide binding process. Therefore, our findings suggest that Pantinin peptides may have chemotherapeutic potential for cancer treatment.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris.

Six peptides with antimicrobial activity were isolated from an extract of freeze-dried skin of the Japanese mountain brown frog Rana ornativentris. Two structurally related peptides (brevinin-20a GLFNVFKGALKTAGKHVAGSLLNQLKCKVSGGC, 11 nmol/g dried tissue, and brevinin-20b GIFNVFKGALKTAGKHVAGSLLNQLKCKVSGEC, 170 nmol/g) belong to the brevinin-2 family, previously identified in Asian and European, but not North American, Ranid frogs. Four peptides (temporin-10a FLPLLASLFSRLL.NH2, 13 nmol/g; temporin-10b FLPLIGKILGTI L.NH2, 350 nmol/g; temporin-10c FLPLLASLFSRLF.NH2, 14 nmol/g; and temporin-10d FLPLLASLFSGLF.NH2, 8 nmol/g) are members of the temporin family first identified in the European common frog Rana temporaria but also found in the skins of North American Ranids. The brevinin-2 peptides showed broad-spectrum activity against the gram-positive bacterium, Staphylococcus aureus, the gram-negative bacterium, Escherichia coli and the yeast Candida albicans, whereas the temporins showed potent activity only against S. aureus. The brevinins and temporins belong to the class of cationic antimicrobial peptides that adopt an amphipathic alpha-helical conformation but it is significant that temporin-10d, which lacks a basic amino acid residue, is still active against S. aureus (minimum inhibitory concentration=13 microM compared with 2 microM for temporin-10a). This suggests that strong electrostatic interaction between the peptide and the negatively charged phospholipids of the cell membrane is not an absolute prerequisite for antimicrobial activity.     

Strategies for transformation of naturally-occurring amphibian antimicrobial peptides into therapeutically valuable anti-infective agents

The emergence of strains of pathogenic microorganisms with resistance to commonly used antibiotics has necessitated a search for novel types of antimicrobial agents. Many frog species produce amphipathic alpha-helical peptides with broad spectrum antimicrobial activity in the skin but their therapeutic potential is limited by varying degrees of cytolytic activity towards eukaryotic cells. Methods for development of such peptides into anti-infective drugs are illustrated by the example of temporin-1DRa (HFLGTLVNLAK KIL.NH(2)). Studies with model alpha-helical peptides have shown that increase in cationicity promotes antimicrobial activity whereas increases in hydrophobicity, helicity and amphipathicity promote hemolytic activity and loss of selectivity for microorganisms. Analogs of temporin-1DRa in which each amino acid is replaced by L-lysine and D-lysine were synthesized and their cytolytic activities tested against a range of microorganisms and human erythrocytes. Small changes in structure produced marked changes in conformation, as determined by retention time on reversed-phase HPLC, and in biological activity. However, peptides containing the substitutions (Val(7) -->L-Lys), (Thr(5)-->D-Lys) and (Asn(8)-->D-Lys) retained the high solubility and potent, broad spectrum antimicrobial activity of the naturally occurring peptide but were appreciably (up to 10-fold) less hemolytic. In contrast, analogs in which Leu(9) and Ile(13) were replaced by the more hydrophobic cyclohexylglycine residue showed slightly increased antimicrobial potencies (up to 2-fold) but a 4-fold increase in hemolytic activity. The data suggest a strategy of selective increases in cationicity concomitant with decreases in helicity and hydrophobicity in the transformation of naturally-occurring antimicrobial peptides into non-toxic therapeutic agents.     

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria

BackgroundSome cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some β-sheets.MethodsThe software Kamal was used to scan the human reference proteome for short (7–11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of β-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli.ResultsThousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a β-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. Conclusions: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained.General SignificanceThis work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.     

Anticancer peptide NK-2 targets cell surface sulphated glycans rather than sialic acids

Abstract Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.     

TAT Modification of Alpha-Helical Anticancer Peptides to Improve Specificity and Efficacy

HPRP-A1 is an amphipathic α-helical anticancer peptide (ACP) derived from the N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In our previously study, HPRP-A1 has been reported that induced HeLa cell apoptosis in a caspase-dependent approach and involved both by the death receptor ‘extrinsic’ pathway and the mitochondria ‘intrinsic’ pathway. Here we report the construction of a new hybrid peptide, HPRP-A1-TAT, comprising the cell-permeating peptide TAT linked to the C-terminus of HPRP-A1. This peptide exhibits higher anticancer activity against HeLa cells with lower toxicity against human RBC than HPRP-A1. Two FITC-labeled peptides, FITC-HPRP-A1 and FITC-HPRP-A1-TAT, were used to investigate and compare the cellular uptake mechanism using fluorescence spectra and flow cytometry. Compared with HPRP-A1, HPRP-A1-TAT quickly crossed cell, entered the cytoplasm via endocytosis, and disrupted the cell membrane integrity. HPRP-A1-TAT exhibited stronger anticancer activity than HPRP-A1 at the same concentration by increasing early apoptosis of HeLa cells and inducing caspase activity. Notably, after 24 h, the cellular concentration of HPRP-A1-TAT was higher than that of HPRP-A1. This result suggests that TAT protects HPRP-A1 against degradation, likely due to its high number of positively charged amino acids or the further release of peptides into cancer cells from endocytotic vesicles. We believe that this TAT modification approach may provide an effective new strategy for improving the therapeutic index and anticancer activity of ACPs for clinical use.     

Antimicrobial peptides from the skin of the Japanese mountain brown frog Rana ornativentris: evidence for polymorphism among preprotemporin mRNAs

A previous study led to the isolation of antimicrobial peptides belonging to the temporin and brevinin-2 families from a pooled extract of the skin of adult specimens of the Japanese mountain brown frog Rana ornativentris Werner 1903. In order to ascertain whether individual frogs expressed the full complement of temporin genes, we individually cloned cDNAs encoding the temporin precursors from total RNA extracted from the skins of 12 frogs by RT-PCR using a set of preprotemporin-specific primers. All the specimens examined contained mRNAs directing the synthesis of the novel, but inactive, temporin-1Oe (ILPLLGNLLNGLL x NH2). Nucleotide sequence analysis revealed marked polymorphism among individual frogs. Twenty-seven distinct preprotemporin-1Oe mRNAs were identified that contained synonymous substitutions in the antimicrobial peptide region and both synonymous and non-synonymous substitutions in the signal peptide and intervening sequence regions. Up to eight preprotemporin-1Oe mRNA variants were found within a single frog. In addition, several cDNAs encoding preprotemporin-1Oa and -1Ob and a single cDNA encoding preprotemporin-1Oc were characterized. Peptidomic analysis of norepinephrine-stimulated skin secretions revealed the presence of temporin-1Oe, temporin-1Of (SLILKGLASIAKLF x NH2), temporin-1Og (FLSSLLSKVVSLFT x NH2), four members of the ranatuerin-2 family and one member of the palustrin-2 family in addition to previously characterized temporin and brevinin-2 peptides.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

Venom peptides from solitary hunting wasps induce feeding disorder in lepidopteran larvae

The cell lytic activity and toxicity against lepidopteran larvae of 13 venom peptides (4 OdVPs and 9 EpVPs) from two solitary hunting wasps, Orancistrocerus drewseni and Eumenes pomiformis, were examined with mastoparan as a reference peptide. Of the 13 peptides, 7 were predicted to have α-helical structures that exhibit the typical character of amphipathic α-helical antimicrobial peptides. The remaining peptides exhibited coil structures; among these, EpVP5 possesses two Cys residues that form an internal disulfide bridge. All the helical peptides including mastoparan showed antimicrobial and insect cell lytic activities, whereas only two of them were hemolytic against human erythrocytes. The helical peptides induced a feeding disorder when injected into the vicinity of the head and thorax of Spodoptera exigua larvae, perhaps because their non-specific neurotoxic or myotoxic action induced cell lysis. At low concentrations, however, these helical peptides increased cell permeability without inducing cell lysis. These findings suggest that the helical venom peptides may function as non-specific neurotoxins or myotoxins and venom-spreading factors at low concentrations, as well as preservatives for long-term storage of the prey via antimicrobial, particularly antifungal, activities.     

Anticancer activity of CopA3 dimer peptide in human gastric cancer cells

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic. [BMB Reports 2015; 48(6): 324-329]     

The effect of d-amino acid substitution on the selectivity of temporin L towards target cells: Identification of a potent anti-Candida peptide

The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro³]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6–13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro³]TL with the corresponding d isomers, known as helix breakers. Structure–activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro³]TL, without giving a toxic effect towards human cells.     

Purification and characterization of antimicrobial and vasorelaxant peptides from skin extracts and skin secretions of the North American pig frog Rana grylio

Eight peptides with differential growth–inhibitory activity against the gram-positive bacterium Staphylococcus aureus, the gram-negative bacterium Escherichia coli and the yeast, Candida albicans were isolated from an extract of the skin of the North American pig frog Rana grylio. The primary structures of these antimicrobial peptides were different from previously characterized antimicrobial peptides from Ranid frogs but on the basis of sequence similarities, the peptides may be classified as belonged to four previously characterized peptide families: the ranatuerin-1, ranatuerin-2 and ranalexin families, first identified in the North American bullfrog, Rana catesbeiana, and the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of other species of Ranid frogs, were not identified in the extracts. The ranatuerin-1 and ranalexin peptides showed broadest spectrum of antimicrobial activity whereas the temporins were active only against S. aureus. Synthetic replicates of temporin-1Gb (SILPTIVSFLSKFL.NH₂) and temporin-1Gd (FILPLIASFLSKFL.NH₂) produced concentration-dependent relaxation of preconstricted vascular rings from the rat thoracic aorta (EC₅₀=2.4±0.1 μM for temporin-1Gb and 2.3±0.2 μM for temporin-1Gd). The antimicrobial peptides that were isolated in extracts of the skin R. grylio were present in the same molecular forms in electrically-stimulated skin secretions of the animal demonstrating that the peptides are stored in the granular glands of the skin in their fully processed forms.     

The effects of Leu or Val residues on cell selectivity of α-helical peptides

In this study, the peptides were designed to compare the effect of multiple Leu or Val residues as the hydrophobic side of an α-helical model on their structure, function, and interaction with model membranes. The Leu-rich peptides displayed 4- to 16-fold stronger antimicrobial activity than Val-rich peptides, while Val-containing peptides showed no haemolysis and weak cytotoxicity. The peptides LR and VR showed an α-helical-rich structure under a membranemimicking environment. Different cell selectivity for Leu- or Val-containing peptides correlated with the targeted cell membranes. The Leu-rich peptide LR(W) and Val-rich peptide VR(W) interacted preferentially with negatively charged phospholipids over zwitterionic phospholipids. VR(W) displayed no interaction with zwitterionic phospholipids, which was consistent with its lack of haemolytic activity. The ability of LR to depolarize bacterial cells was much greater than that of VR. Val- and Leu-rich peptides appeared to kill bacteria in a membrane-targeted fashion, with different modes of action. Leu-rich peptides appeared to be active via a membrane-disrupting mode, while Val-rich peptides were active via the formation of small channels.