Amphiphilic Α-helical Peptides Research Articles

A novel antimicrobial peptide screened by a Bacillus subtilis expression system, derived from Larimichthys crocea Ferritin H, exerting bactericidal and parasiticidal activities.

Antimicrobial peptides (AMPs) may be the most promising substitute for antibiotics due to their effective antimicrobial activities and multiple function mechanisms against pathogenic microorganisms. In this study, a novel AMP containing 51 amino acids, named Lc1687, was screened from the large yellow croaker (Larimichthys crocea) via a B. subtilis system. Bioinformatics and circular dichroism (CD) analyses showed that Lc1687 is a novel anionic amphiphilic α-helical peptide, which was derived from the C-terminal of a Ferritin heavy subunit. The recombinant Lc1687 (named rLc1687) purified from Escherichia coli exhibited strong activities against Gram-positive (Gram+) bacterium Staphylococcus aureus, Gram-negative (Gram-) bacteria Vibrio vulnificus, V. parahaemolyticus, and Scuticociliatida. Scanning electron microscope (SEM) and transmission electron microscopy (TEM) revealed the possible function mechanisms of this peptide, which is to target and disrupt the bacterial cell membranes, including pore-forming, loss of fimbriae, and cytoplasm overflow, whereas gel retardation assay revealed that peptide Lc1687 cannot bind bacterial DNA. The peptide stability analysis showed that rLc1687 acts as a stable antimicrobial agent against Gram+ and Gram- bacteria at temperatures ranging from 25 to 100°C, pH 3-12, and UV radiation time ranging from 15 to 60 min. A hemolytic activity assay confirmed that this peptide may serve as a potential source for clinical medicine development. Taken together, Lc1687 is a novel AMP as it is a firstly confirmed Ferritin fragment with antimicrobial activity. It is also a promising agent for the development of peptide-based antibacterial and anti-parasitic therapy.

Heterogeneous Structural Disturbance of Cell Membrane by Peptides with Modulated Hydrophobic Properties.

Extensive effort has been devoted to developing new clinical therapies based on membrane-active peptides (MAPs). Previous models on the membrane action mechanisms of these peptides mostly focused on the MAP-membrane interactions in a local region, while the influence of the spatial heterogeneity of the MAP distribution on the membrane was much ignored. Herein, three types of natural peptide variants, AS4-1, AS4-5, and AS4-9, with similar amphiphilic α-helical structures but distinct hydrophobic degrees (AS4-1 < AS4-5 < AS4-9) and net charges (+9 vs. +7 vs. +5), were used to interact with a mixed phosphatidylcholine (PC) and phosphatidylglycerol (PG) membrane. A combination of giant unilamellar vesicle (GUV) leakage assays, atomic force microscopy (AFM) characterizations, and molecular dynamics (MD) simulations demonstrated the coexistence of multiple action mechanisms of peptides on a membrane, probably due to the spatially heterogeneous distribution of peptides on the membrane surface. Specifically, the most hydrophobic peptide (i.e., AS4-9) had the strongest membrane binding, perturbation, and permeabilization effects, leading to the formation of large peptide-lipid aggregates (10 ± 5 nm in height and 150 ± 50 nm in size), as well as continuous fragments and ridges on the supported membrane surface. The AS4-5 peptides, with a half-hydrophilic and half-hydrophobic structure, induced membrane lysis in addition to reconstruction. The most hydrophilic peptide AS4-1 only exhibited unstable binding on the supported membrane surface. These results demonstrate the heterogeneous structural disturbance of model cell membranes by amphiphilic α-helical peptides, which could be significantly strengthened by increasing the degree of hydrophobicity and/or local number density of peptides. This work provides support for the modulation of the membrane activity of MAPs by adjusting their hydrophobicity and local concentration.

Structure-Activity Relationship of New Chimeric Analogs of Mastoparan from the Wasp Venom Paravespula lewisii.

Mastoparan (MP) is an antimicrobial cationic tetradecapeptide with the primary structure INLKALAALAKKIL-NH2. This amphiphilic α-helical peptide was originally isolated from the venom of the wasp Paravespula lewisii. MP shows a variety of biological activities, such as inhibition of the growth of Gram-positive and Gram-negative bacteria, as well as hemolytic activity and activation of mast cell degranulation. Although MP appears to be toxic, studies have shown that its analogs have a potential therapeutic application as antimicrobial, antiviral and antitumor agents. In the present study we have designed and synthesized several new chimeric mastoparan analogs composed of MP and other biologically active peptides such as galanin, RNA III inhibiting peptide (RIP) or carrying benzimidazole derivatives attached to the ε-amino side group of Lys residue. Next, we compared their antimicrobial activity against three reference bacterial strains and conformational changes induced by membrane-mimic environments using circular dichroism (CD) spectroscopy. A comparative analysis of the relationship between the activity of peptides and the structure, as well as the calculated physicochemical parameters was also carried out. As a result of our structure–activity study, we have found two analogs of MP, MP-RIP and RIP-MP, with interesting properties. These two analogs exhibited a relatively high antibacterial activity against S. aureus compared to the other MP analogs, making them a potentially attractive target for further studies. Moreover, a comparative analysis of the relationship between peptide activity and structure, as well as the calculated physicochemical parameters, may provide information that may be useful in the design of new MP analogs.

Proton transfer through tetrameric charge zipper TisB peptide in membrane

The formation of persister cells are among the most important strategies employed by the bacterial population to get rid of antibiotic treatment and external stress. E. coli overproduces amphiphilic α-helical peptide TisB(toxic protein) in harmful conditions, which is localized into the inner membrane. Whenever bacteria are under stress, the persister cells help to equilibrate PH gradient, thereby Proton Motive Force (PMF) is eliminated across the membrane. This phenomenon leads to the depletion of ATP levels in the membrane, which results in the formation of biofilm and induces the state of temporary dormancy.

Designed Antitumor Peptide for Targeted siRNA Delivery into Cancer Spheroids.

Antimicrobial/anticancer peptides (AMPs/ACPs) have shown promising results as new therapeutic agents in cancer thearpy. Among them, the designed amphiphilic α-helical peptide G(IIKK)3I-NH2 (G3) displayed great affinity and specificity in targeting cancer cells. Here, we report new insights on how G3 penetrates cancer cells. G3 showed high specificity to HCT-116 colon cancer cells compared to the HDFs (human neonatal primary dermal fibroblasts) control. With high concentrations of peptide, a clear cancer cell membrane disruption was observed through SEM. Gene knockdown of the endocytic pathways demonstrated that an energy-dependent endocytic pathway is required for the uptake of the peptide. In addition, G3 can protect and selectively deliver siRNAs into cancer cells and successfully modulated their gene expression. Gene delivery was also tested in 3D cancer spheroids and showed deep penetration delivery into the cancer spheroids. Finally, the in vivo toxicity of G3 was evaluated on zebrafish embryos, showing an increasing toxicity effect with concentration. However, the toxicity of the peptide was attenuated when complexed with siRNA. In addition, negligible toxicity was observed at the concentration range for efficient gene delivery. The current results demonstrate that G3 is promising as an excellent agent for cancer therapy.

Tetrameric Charge-Zipper Assembly of the TisB Peptide in Membranes—Computer Simulation and Experiment

TisB is a short amphiphilic α-helical peptide from Escherichia coli that induces a breakdown of the pH gradient across the inner membrane when the bacteria are under stress and require to form persister cells to turn into a biofilm. A computational-experimental approach combining all-atom and coarse-grained molecular dynamics simulation with circular dichroism spectroscopy and gel electrophoresis was used to reveal its structure and oligomeric assembly in a phospholipid bilayer. TisB is found to be inserted upright in the membrane as a tetrameric bundle with a left-handed sense of supercoiling, best described as an antiparallel dimer-of-dimers. The tetramer is stabilized by means of a regular but dynamically interchanging pattern of salt bridges and hydrogen bonds, in accordance with the recently proposed "charge-zipper" motif.

Cell-selectivity of tryptophan and tyrosine in amphiphilic α-helical antimicrobial peptides against drug-resistant bacteria

The increasing emergence of drug-resistant bacteria creates a requirement for new antibiotics and various types of antibiotic materials such as proteins, peptides, polymers, and chemical compounds. Among these, antimicrobial peptides (AMPs) are considered to be promising antibiotic candidates for clinical treatments. In this study, we have designed a novel series of peptides with repeated sequences of minimum membrane-active motif, ‘XWZX’ basic sequence (X: lysine or arginine, Z: leucine, tyrosine, valine, or glycine), and an α-helical secondary structure. Some peptides displayed a potent antibacterial activity via membranolytic action and high therapeutic index (toxic dose/minimum inhibitory concentration) in vitro. Furthermore, in vivo experiments using bacterial ear-skin infection models verified that these peptides have the potential to be powerful and safe antibiotics. The present study provides a lead sequence for designing peptide antibiotics against bacterial membranes and information for cell-selectivity of hydrophobic amino acids with aromatic side chains such as Trp and Tyr.

Lipid Nanodisc Formation using Pxt-5 Peptide Isolated from Amphibian (Xenopus tropicalis) Skin, and its Altered Form, Modify-Pxt-5.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that accumulate around the circumference of a lipid bilayer. In this study, Pxt-5, which is an antimicrobial peptide isolated from the skin of Xenopus tropicalis, and its modified peptide (Modify-Pxt-5) were synthesized by solid-phase peptide synthesis (SPPS).Their surface properties, which are an important factor in inducing nanodisc formation, were investigated by circular dichroism (CD) spectroscopy, surface tension measurement, phospholipid vesicle clearance assay, and negative-staining transmission electron microscopy (NS-TEM). The α-helicity of Pxt-5 (8.4%) improved drastically to 45.6% by four amino-acid substitutions (Modify-Pxt-5). Both the peptides, having hydrophobic and hydrophilic faces, behaved like general surfactants, and the surface activity of Modify-Pxt-5 (CAC: 9.5×10-5 M, γCAC: 30.3 mN·m-1) was much higher than that of Pxt-5 (CAC: 7.9×10-5 M, γCAC: 38.1 mN·m-1). A turbid L-α-dimyristoylphosphatidylcholine (DMPC) vesicle solution (T = 0.3%) quickly turned transparent upon addition of Pxt-5 or Modify-Pxt-5. After twelve hours, the transmittance of vesicle solution with Modify-Pxt-5 (T = 96.2%) was found to be higher than that of vesicle solution with Pxt-5 (T = 83.5%), and then the micro-solubilized solutions were observed by NS-TEM. Interestingly, nanodisc structures were found in the vicinity of DMPC vesicles in both the images, and the average diameter of the nanodiscs was 11.2 ± 6.0 nm for those containing Pxt-5 and 10.8 ± 5.8 nm for those containing Modify-Pxt-5. It was also found that Modify-Pxt-5 effectively self-assembles into nanodiscs compared to Pxt-5 without any substitutions.

The structure and configuration changes of multifunctional peptide vectors enhance gene delivery efficiency.

We designed a series of peptide vectors that contain functional fragments with the goal of enhancing cellular internalization and gene transfection efficiency. The functional fragments included a cell-penetrating peptide (R9), a cationic amphiphilic α-helical peptide [(LLKK)3-H6 or (LLHH)3], a stearyl moiety, and cysteine residues. Vectors were also synthesized with D-type amino acids to improve their proteolytic stability. The conformations, particle sizes, and zeta potentials for complexes of these peptides with pGL3 plasmid DNA were characterized by circular dichroism and dynamic light scattering. In addition, cellular uptake of the peptide/DNA complexes and gene transfection efficiency were investigated with fluorescence-activated cell sorting and confocal laser-scanning microscopy. Greater transfection efficiency was achieved with the vectors containing the R9 segment, and the efficiency was greater than Lipo2000. In addition, the D-type C18-c(llkk)3ch6-r9 had about 7 times and 5.5 times the transfection efficiency of Lipo2000 in 293T cells and NIH-3T3 cells at the N/P ratio of 6, respectively. Overall, the multifunctional peptide gene vectors containing the R9 segment exhibited enhanced cellular internalization, a high gene transfection efficiency, and low cytotoxicity.

Molecular mechanism of synergy between the antimicrobial peptides PGLa and magainin 2

PGLa and magainin 2 (MAG2) are amphiphilic α-helical membranolytic peptides from frog skin with known synergistic antimicrobial activity. By systematically mutating residues in the two peptides it was possible to identify the ones crucial for the synergy, as monitored by biological assays, fluorescence vesicle leakage, and solid-state 15N-NMR. Electrostatic interactions between anionic groups in MAG2 and cationic residues in PGLa enhance synergy but are not necessary for the synergistic effect. Instead, two Gly residues (7 and 11) in a so-called GxxxG motif in PGLa are necessary for synergy. Replacing either of them with Ala or another hydrophobic residue completely abolishes synergy according to all three methods used. The designer-made peptide MSI-103, which has a similar sequence as PGLa, shows no synergy with MAG2, but by introducing two Gly mutations it was possible to make it synergistic. A molecular model is proposed for the functionally active PGLa-MAG2 complex, consisting of a membrane-spanning antiparallel PGLa dimer that is stabilized by intimate Gly-Gly contacts, and where each PGLa monomer is in contact with one MAG2 molecule at its C-terminus.

Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes.

PGLa and magainin 2 (MAG2) are amphiphilic α-helical frog peptides with synergistic antimicrobial activity. In vesicle leakage assays we observed the strongest synergy for equimolar mixtures of PGLa and MAG2. This result was consistent with solid-state (15)N-NMR data on the helix alignment in model membranes. The Hill coefficients determined from the vesicle leakage data showed that the heterodimeric (PGLa-MAG2) interactions were stronger than the homodimeric (PGLa-PGLa and MAG2-MAG2) interactions. This result was also reflected in the free energy of dimerization determined from oriented circular dichroism and quantitative solid-state (19)F-NMR analysis.

Peptide amphiphiles with multifunctional fragments promoting cellular uptake and endosomal escape as efficient gene vectors.

To overcome barriers associated with gene delivery, a series of peptides consisting of multifunctional fragments, including a cationic amphiphilic α-helical antimicrobial peptide (AMP), a cell penetrating peptide (CPP), TAT, a stearyl moiety, and cysteine residues, were designed and synthesized for evaluation as non-viral gene vectors. TAT and AMP segments were utilized to mediate cellular uptake and endosomal escape, respectively. Stearyl moieties provide an intramolecular hydrophobic environment to promote AMPs to form an α-helical conformation in PBS, and this is beneficial for DNA binding, cellular uptake, and endosomal escape. The α-helical content of the peptides, as well as the particle size, zeta potential, and morphology of the peptide/DNA complexes, was characterized. Fluorescence activated cell sorting (FACS) and confocal microscopy data showed that the peptides were able to efficiently translocate a pGL3 control plasmid across the plasma membrane via endocytosis, and then they successfully evaded endosomal entrapment and possible metabolic degradation. Moreover, one of the peptide vectors exhibited a high transfection efficiency similar to that of Lipofectamine 2000, concomitant with lower cytotoxicity. Overall, a combination of the four functional segments tested was used to generate a non-viral gene vector that synergistically promoted cellular uptake, endosomal escape, and gene expression.

Asymmetric triplex metallohelices with high and selective activity against cancer cells.

Small cationic amphiphilic α-helical peptides are emerging as agents for the treatment of cancer and infection, but they are costly and display unfavourable pharmacokinetics. Helical coordination complexes may offer a three-dimensional scaffold for the synthesis of mimetic architectures. However, the high symmetry and modest functionality of current systems offer little scope to tailor the structure to interact with specific biomolecular targets, or to create libraries for phenotypic screens. Here, we report the highly stereoselective asymmetric self-assembly of very stable, functionalized metallohelices. Their anti-parallel head-to-head-to-tail 'triplex' strand arrangement creates an amphipathic functional topology akin to that of the active sub-units of, for example, host-defence peptides and p53. The metallohelices display high, structure-dependent toxicity to the human colon carcinoma cell-line HCT116 p53(++), causing dramatic changes in the cell cycle without DNA damage. They have lower toxicity to human breast adenocarcinoma cells (MDA-MB-468) and, most remarkably, they show no significant toxicity to the bacteria methicillin-resistant Staphylococcus aureus and Escherichia coli.

A Computational Study of the Interaction of Amphiphilic α-Helical Cell-Penetrating Peptides with Heparan Sulfate

Abstract Recent studies have suggested that extracellular glycosaminoglycans (GAGs), such as heparan sulfate (HS), can bind cell-penetrating peptides (CPPs) and cluster during CPP binding, thereby affecting the efficiency of a wide range of CPPs. Recently, we experimentally investigated the HS interactions of four peptides, which were chosen from our amphiphilic α-helical CPP library and had only one or two amino acid(s) differences (Arg/Glu and Ala/Phe). Here, we describe the computational methods and molecular dynamics (MD) simulations used to reveal the detailed binding and clustering mechanisms of these four peptides with HS. The order of the HS-binding affinities and HS-clustering abilities of the four CPPs, which were obtained from the binding free energies calculated in our MD simulations, almost agreed with our previous experimental data. Using free energy decomposition analyses, Arg-containing peptides were found to contribute more electrostatic energy to the binding enthalpy in comparison with Glu-containing peptides, whereas Phe-containing peptides benefited more from nonelectrostatic energy in comparison with Ala-containing peptides. Furthermore, although electrostatic interactions were revealed as the initial driving force in HS–CPP binding, nonelectrostatic interactions might be more favorable during the HS–CPP clustering process because of their tolerance to local charge mismatch. Consequently, CPPs that adopt a nonelectrostatic-dominant HS-binding mechanism could be internalized by cells more efficiently. Therefore, the present study provides important insights into the design of useful CPPs.

Surfactant-like properties of an amphiphilic α-helical peptide leading to lipid nanodisc formation.

Nanodiscs are self-assembled discoidal nanoparticles composed of amphiphilic α-helical scaffold proteins or peptides that wrap themselves around the circumference of a lipid bilayer in a beltlike manner. In this study, an amphiphilic helical peptide that mimics helix 10 of human apoA-I was newly synthesized by solid phase peptide synthesis using Fmoc chemistry, and its physicochemical properties, including surface tension, self-association, and solubilization abilities, were evaluated and related directly to nanodisc formation. The synthesized peptide having hydrophobic and hydrophilic faces behaves like a general surfactant, affording a critical association concentration (CAC) of 2.7 × 10(-5) M and a γCAC of 51.2 mN m(-1) in aqueous solution. Interestingly, only a peptide solution above its CAC was able to microsolubilize L-α-dimyristoylphosphatidylcholine (DMPC) vesicles, and lipid nanodiscs with an average diameter of 9.5 ± 2.7 nm were observed by dynamic light scattering and negative stain transmission electron microscopy. Moreover, the ζ potentials of the lipid nanodiscs were measured for the first time as a function of pH, and the values changed from positive (20 mV) to negative (-30 mV). In particular, nanodisc solutions at acidic pH 4 (20 mV) or basic pH 9 (-20 mV) were found to be stable for more than 6 months as a result of the electrostatic repulsion between the particles.

Minimum amino acid residues of an α-helical peptide leading to lipid nanodisc formation.

Nanodiscs are a relatively new class of nanoparticles composed of amphiphilic α-helical scaffold peptides and a phospholipid bilayer, and find potential applications in various fields. In order to identify the minimum number of amino acid residues of an amphiphilic α-helical peptide that leads to nanodisc formation, seven peptides differing in lengths (22-, 18-, 14-, 12-, 10-, 8-, and 6-mers) that mimic and modify the C-terminal domain of apoA-I (residues 220-241) were synthesized. At a concentration of 0.3 mM, the 6- and 8-mer peptides did not present any surface activity. In case of the 10-mer peptide, the aqueous surface tension initially decreased and reached a constant value of 51.9 mN/m with the 14-, 18-, and 22-mer peptides. Moreover, upon mixing the surface-active peptides (14-, 18-, and 22-mers) with dipalmitoylphosphatidylcholine (DMPC) liposomes (2.5:1, peptide : DMPC), the turbid DMPC liposome solution rapidly became transparent. Further analysis of this solution by negative-stain transmission electron microscopy (NS-TEM) indicated the presence of disk-like nanostructures. The average diameter of the nanodiscs formed was 9.5 ± 2.7 nm for the 22-mer, 8.1 ± 2.7 nm for the 18-mer, and 25.5 ± 8.5 nm for the 14-mer peptides. These results clearly demonstrate that the surface properties of peptides play a critical role in nanodisc formation. Furthermore, the minimum length of an amphiphilic peptide from the C-terminal of apoA-I protein that can lead to nanodisc formation is 14 amino acid residues.

Interaction of amphiphilic α-helical cell-penetrating peptides with heparan sulfate.

Cell-penetrating peptides (CPPs) are able to be taken up by cells and can deliver macromolecular cargos. However, the mechanism of this internalization is not yet fully understood. Recent theories suggest that the binding of cationic CPPs to negatively charged extracellular glycosaminoglycans, such as heparan sulfate (HS), is a possible mechanism of cellular uptake (CU). Our group has screened the CU activities of 54 systematically designed amphiphilic α-helical peptides in HeLa cells. Notably, a mutation in even a single residue significantly alters the CU ability of a peptide. To determine the structure-CU activity relationship of CPPs, four peptides, which contain a difference in one or two amino acids (i.e., Arg/Glu and Ala/Phe), were chosen from our CPP library to examine their interactions with HS. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering analysis indicated that the HS-binding affinities and HS-clustering abilities of the four CPPs correlated well with their CU activities in HeLa and A549 cells. The heat capacities of the CPPs, determined using ITC and binding free energy decomposition analyses in molecular dynamics simulations, revealed that electrostatic interactions were more dominant in the HS-binding processes of Arg-containing peptides in comparison to Glu-containing peptides, whereas hydrophobic contributions were the primary mode of interaction of Phe-containing peptides in comparison to Ala-containing peptides. Furthermore, it was implied that hydrophobic interactions may be more favourable than electrostatic interactions during the CU process.

Five Different Piscidins from Nile Tilapia, Oreochromis niloticus: Analysis of Their Expressions and Biological Functions

Piscidins are antimicrobial peptides (AMPs) that play important roles in helping fish resist pathogenic infections. Through comparisons of tilapia EST clones, the coding sequences of five piscidin-like AMPs (named TP1∼5) of Nile tilapia, Oreochromis niloticus, were determined. The complete piscidin coding sequences of TP1, -2, -3, -4, and -5 were respectively composed of 207, 234, 231, 270, and 195 bases, and each contained a translated region of 68, 77, 76, 89, and 64 amino acids. The tissue-specific, Vibrio vulnificus stimulation-specific, and Streptococcus agalactiae stimulation-specific expressions of TP2, -3, and -4 mRNA were determined by a comparative RT-PCR. Results of the tissue distribution analysis revealed high expression levels of TP2 mRNA in the skin, head kidneys, liver, and spleen. To study bacterial stimulation, S. agalactiae (SA47) was injected, and the TP4 transcript was upregulated by >13-fold (compared to the wild-type (WT) control, without injection) and was 60-fold upregulated (compared to the WT control, without injection) 24 h after the S. agalactiae (SA47) injection in the spleen and gills. Synthesized TP3 and TP4 peptides showed antimicrobial activities against several bacteria in this study, while the synthesized TP1, -2, and -5 peptides did not. The synthesized TP2, -3, and -4 peptides showed hemolytic activities and synthesized TP3 and TP4 peptides inhibited tilapia ovary cell proliferation with a dose-dependent effect. In summary, the amphiphilic α-helical cationic peptides of TP3 and TP4 may represent novel and potential antimicrobial agents for further peptide drug development.

Lipid shape is a key factor for membrane interactions of amphipathic helical peptides

The membrane alignment of the amphiphilic α-helical model peptide MSI-103 (sequence [KIAGKIA]3-NH2) was examined by solid state 2H-NMR in different lipid systems by systematically varying the acyl chain length and degree of saturation, the lipid head group type, and the peptide-to-lipid molar ratio. In liquid crystalline phosphatidylcholine (PC) lipids with saturated chains, the amphiphilic helix changes its orientation from a surface-bound “S-state” to a tilted “T-state” with increasing peptide concentration. In PC lipids with unsaturated chains, on the other hand, the S-state is found throughout all concentrations. Using phosphatidylethanolamine lipids with a small head group or by addition of lyso-lipids with only one acyl chain, the spontaneous curvature of the bilayer was purposefully changed. In the first case with a negative curvature only the S-state was found, whereas in systems with a positive curvature the peptide preferred the obliquely immersed T-state at high concentration. The orientation of MSI-103 thus correlates very well with the shape of the lipid molecules constituting the membrane. Lipid charge, on the other hand, was found to affect only the initial electrostatic attraction to the membrane surface but not the alignment preferences. In bilayers that are “sealed” with 20% cholesterol, MSI-103 cannot bind in a well-oriented manner and forms immobilized aggregates instead. We conclude that the curvature properties of a membrane are a key factor in the interactions of amphiphilic helical peptides in general, whose re-alignment and immersion preferences may thus be inferred in a straightforward manner from the lipid-shape concept.

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis)

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH2, and exhibits 50–56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.