Functional Characterization of Temporin-SHe, a New Broad-Spectrum Antibacterial and Leishmanicidal Temporin-SH Paralog from the Sahara Frog (Pelophylax saharicus).

Amphibian skin is a promising natural resource for antimicrobial peptides (AMPs), key effectors of innate immunity with attractive therapeutic potential to fight antibiotic-resistant pathogens. Our previous studies showed that the skin of the Sahara Frog (Pelophylax saharicus) contains broad-spectrum AMPs of the temporin family, named temporins-SH. Here, we focused our study on temporin-SHe, a temporin-SHd paralog that we have previously identified in this frog but was never structurally and functionally characterized. We synthesized and determined the structure of temporin-SHe. This non-amphipathic α-helical peptide was demonstrated to strongly destabilize the lipid chain packing of anionic multilamellar vesicles mimicking bacterial membranes. Investigation of the antimicrobial activity revealed that temporin-SHe targets Gram-negative and Gram-positive bacteria, including clinical isolates of multi-resistant Staphylococcus aureus strains. Temporin-SHe exhibited also antiparasitic activity toward different Leishmania species responsible for visceral leishmaniasis, as well as cutaneous and mucocutaneous forms. Functional assays revealed that temporin-SHe exerts bactericidal effects with membrane depolarization and permeabilization, via a membranolytic mechanism observed by scanning electron microscopy. Temporin-SHe represents a new member of the very limited group of antiparasitic temporins/AMPs. Despite its cytotoxicity, it is nevertheless an interesting tool to study the AMP antiparasitic mechanism and design new antibacterial/antiparasitic agents.

The effects of curcumin, mangiferin, resveratrol and other natural plant products on aminopeptidase B activity.

Aminopeptidase B (Ap-B) is a Zn2+-aminopeptidase of the M1 family which is implicated, in conjunction with the nardilysin endoprotease, in the generation of miniglucagon, a peptide involved in the maintenance of glucose homeostasis. Other in vivo physiological roles have been established for this vertebrate enzyme, such as the processing of Arg-extended forms of human insulin and cholecystokinin 9 and the degradation of viral epitopes in the cytoplasm. Among M1 family members, Ap-B is phylogenetically close to leukotriene A4 hydrolase (LTA4H), a bi-functional aminopeptidase also able to transform LTA4 in LTB4 (a lipid mediator of inflammation). As the activities of LTA4H are reported to be inhibited by resveratrol, a polyphenolic molecule from red wine, the effect of this molecule was investigated on the Ap-B activity. Several other active phenolic compounds produced in plants were also tested. Among them, curcumin and mangiferin are the most effective inhibitors. Dixon analysis indicates that curcumin is a non-competitive inhibitor with a Ki value of 46 µmol.L-1. Dixon and Lineweaver-Burk representations with mangiferin show a mixed non-competitive inhibition with Ki' and Ki values of 194 µmol.L-1 and 105 µmol.L-1, respectively. At 200 µmol.L-1, no significant effect was observed with caffeic, chlorogenic, ferulic, salicylic and sinapic acids as well as with resveratrol. Analyses on the 3D-structure of LTA4H with resveratrol (pdb: 3FTS) and the Ap-B 3D-model allow hypothesis to explain theses results.

Engineering of Antimicrobial Surfaces by Using Temporin Analogs to Tune the Biocidal/antiadhesive Effect.

Proliferation of resistant bacteria on biomaterials is a major problem leading to nosocomial infections. Due to their broad-spectrum activity and their ability to disrupt bacterial membranes through a rapid membranolytic mechanism, antimicrobial peptides (AMPs) are less susceptible to the development of bacterial resistance and therefore represent good candidates for surface coating strategies to prevent biofilm formation. In this study, we report on the covalent immobilization of temporin-SHa, a small hydrophobic and low cationic antimicrobial peptide exhibiting broad-spectrum activity, and (SHa) analogs on modified gold surfaces. Several analogs derived from SHa with either a carboxamidated ([K³]SHa, d-[K³]SHa) or a carboxylated C-terminus ([K³]SHa-COOH) were used to achieve peptide grafting on gold surfaces modified by a thiolated self-assembled monolayer (SAM). Surface functionalization was characterized by polarization modulation infrared reflection absorption spectroscopy (PM-RAIRS) and X-ray photoemission spectroscopy (XPS). The antibacterial properties of the temporin-functionalized surfaces were tested against the Gram-positive Listeria ivanovii. Direct visualization of the peptide effects on the bacterial membrane was investigated by scanning electron microscopy equipped with a field emission gun (SEM-FEG). All active temporin analogs were successfully grafted and display significant antibacterial activity (from 80 to 90% killing efficiency) in addition to a 2-fold decrease of bacterial adhesion when all d-SHa analogs were used.

ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

CC chemokine receptor type 2 (CCR2) is a key molecule in inflammatory diseases and is an obvious drug target for the treatment of inflammation. A number of nonpeptidic, competitive CCR2 antagonists have been developed, but none has yet been approved for clinical use. Our aim was to identify a short peptide that showed allosteric antagonism against human and mouse CCR2. On the basis of sequence analysis and 3-dimensional modeling, we identified an original 7-d-amino acid peptidic CCR2 inhibitor that we have called extracellular loop 1 inverso (ECL1i), d(LGTFLKC). In vitro, ECL1i selectively and potently inhibits CC chemokine ligand type 2 (CCL2)-triggered chemotaxis (IC50, 2 µM) but no other conventional CCL2-associated events. We used the classic competitive CCR2 antagonist, BMS22 {2-[(isopropylaminocarbonyl)amino]-N-[2-[[cis-2-[[4-(methylthio)benzoyl]amino]cyclohexyl]amino]-2-oxoethyl]-5-(trifluoromethyl)benzamide}, as positive control and inhibited CCL2-dependent chemotaxis with an IC50 of 18 nM. As negative control, we used a peptide with the same composition as ECL1i, but in a different sequence, d(FKLTLCG). In vivo, ECL1i (4 mg/kg) interfered with CCR2-positive cell recruitment and attenuated disease progression in experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. This study establishes ECL1i as the first allosteric inhibitor of CCR2 with functional selectivity. ECL1i is a promising new agent in therapeutic development, and it may, by its selective effect, increase our understanding of CCR2 signaling pathways and functions.-Auvynet, C., Baudesson de Chanville, C., Hermand, P., Dorgham, K., Piesse, C., Pouchy, C., Carlier, L., Poupel, L., Barthélémy, S., Felouzis, V., Lacombe, C., Sagan, S., Salomon, B., Deterre, P., Sennlaub, F., Combadière, C. ECL1i, d(LGTFLKC), a novel, small peptide that specifically inhibits CCL2-dependent migration.

The sequence Pro295-Thr311 of the hinge region of oestrogen receptor α is involved in ERK1/2 activation via GPR30 in leiomyoma cells.

The ERα (oestrogen receptor α)-derived peptide ERα17p activates rapid signalling events in breast carcinoma cells under steroid-deprived conditions. In the present study, we investigated its effects in ELT3 leiomyoma cells under similar conditions. We show that it activates ERK1/2 (extracellular-signal-regulated kinase 1/2), the Gαi protein, the trans-activation of EGFR (epidermal growth factor receptor) and, finally, cell proliferation. It is partially internalized in cells and induces membrane translocation of ß-arrestins. The activation of ERK1/2 is abolished by the GPR30 (G-protein-coupled receptor 30) antagonist G15 and GPR30 siRNA. When ERα is down-regulated by prolonged treatment with E2 (oestradiol) or specific ERα siRNA, the peptide response is blunted. Thus the simultaneous presence of GPR30 and ERα is required for the action of ERα17p. In addition, its PLM sequence, which interferes with the formation of the ERα-calmodulin complex, appears to be requisite for the phosphorylation of ERK1/2 and cell proliferation. Hence ERα17p is, to our knowledge, the first known peptide targeting ERα-GPR30 membrane cross-talk and the subsequent receptor-mediated biological effects.

Effects of residue 5-point mutation and N-terminus hydrophobic residues on temporin-SHc physicochemical and biological properties.

Temporin-SHc (FLSHIAGFLSNLFamide) first isolated from skin extraction of the Tunisian frog Pelophylax saharica, which shows potent antimicrobial activity against Gram-positive bacteria and is highly active against yeasts and fungi without hemolytic activity at antimicrobial concentrations. The peptide adopts well-defined α-helical conformation when bound to SDS micelles. In this study, we explored the effects of residue at position 5 and the N-terminus hydrophobic character on the hydrophilic/polar face of temp-SHc, on its biological activities (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity and helicity). Antibacterial and hemolytic properties of temporin-SHc derivatives depend strongly on physicochemical properties. Therefore, slight decreasing amphipathicity together with hydrophobicity and helicity by the substitution Ile(5) → Leu decreased antimicrobial potency approximately twofold without changing of hemolytic activity. It is noteworthy that a conservative amino acid substitution decreases the antimicrobial activity, underlining the differences between Leu/Ile side chains insertion into the lipid bilayer. While the modification of N-terminal hydrophobic character by four residue inversion decreased amphipathicity (twofold) of (4-1)L5temp-SHc and resulted in an increase in antibacterial activity against E. coli, E. faecalis and C. parapsilosis of at least fourfold, its therapeutic potential is limited by its drastic increase of hemolysis (LC50 = 2 µM). We found that the percentage of helicity of temp-SHc analog is directly correlated to its hemolytic activity. Last, the hydrophobic N-terminal character is an important determinant of antimicrobial activity.

Cyclodextrin-based supramolecular nanogels decorated with mannose for short peptide encapsulation.

Nanogels are aqueous dispersions of hydrogel particles formed by physically or chemically cross-linked polymer networks of nanoscale size. Herein, we devised a straightforward technique to fabricate a novel class of physically cross-linked nanogels via a self-assembly process in water involving α-cyclodextrin and a mannose molecule that was hydrophobically modified using an alkyl chain. The alkyl chain-modified mannose was synthesized in five steps, starting with D-mannose. Subsequently, nanogels were formed by subjecting α-cyclodextrin and the hydrophobically modified mannose to magnetic stirring in water. By adjusting the mole ratio between the hydrophobically modified mannose and α-cyclodextrin, nanogels with an average 100-150 nm diameter were obtained. Physicochemical and structural analyses by 1H NMR and X-ray diffraction unveiled a supramolecular and hierarchical mechanism underlying the creation of these nanogels. The proposed mechanism of nanogel formation involves two distinct steps: initial interaction of hydrophobically modified mannose with α-cyclodextrin resulting in the formation of inclusion complexes, followed by supramolecular interactions among these complexes, ultimately leading to nanogel formation after 72 h of stirring. We demonstrated the nanogels' ability to encapsulate a short peptide ([p-tBuF2, R5]SHf) as a water-soluble drug model. This discovery holds promise for potentially utilizing these nanogels in drug delivery applications.

Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor.

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

Development and Characterization of Innovative Multidrug Nanoformulation for Cardiac Therapy.

For several decades, various peptides have been under investigation to prevent ischemia/reperfusion (I/R) injury, including cyclosporin A (CsA) and Elamipretide. Therapeutic peptides are currently gaining momentum as they have many advantages over small molecules, such as better selectivity and lower toxicity. However, their rapid degradation in the bloodstream is a major drawback that limits their clinical use, due to their low concentration at the site of action. To overcome these limitations, we have developed new bioconjugates of Elamipretide by covalent coupling with polyisoprenoid lipids, such as squalenic acid or solanesol, embedding self-assembling ability. The resulting bioconjugates were co-nanoprecipitated with CsA squalene bioconjugate to form Elamipretide decorated nanoparticles (NPs). The subsequent composite NPs were characterized with respect to mean diameter, zeta potential, and surface composition by Dynamic Light Scattering (DLS), Cryogenic Transmission Electron Microscopy (CryoTEM) and X-ray Photoelectron Spectrometry (XPS). Further, these multidrug NPs were found to have less than 20% cytotoxicity on two cardiac cell lines even at high concentrations, while maintaining an antioxidant capacity. These multidrug NPs could be considered for further investigations as an approach to target two important pathways involved in the development of cardiac I/R lesions.

Improving the selectivity of antimicrobial peptides from anuran skin.

Anuran skin is known to be a rich source of antimicrobial peptides although their therapeutic potential is often limited due to their toxicity against mammalian cells. The analysis of structure-activity relationships among anuran antimicrobial peptides provided the parameters to construct the "Mutator" tool for improving their selectivity for bacterial cells, by suggesting appropriate point substitutions. Double substitution analogues [K2, K16] of the Xenopus tropicalis peptide XT-7 and [I2, K19] of the Ascaphus truei peptide ascaphin-8 were predicted by this tool to have an increased 'therapeutic index' (TI = HC(50)/MIC for erythrocytes with respect to bacteria) > 80. The mutated peptides were synthesized and respectively found to have experimental TI values > 130 for S. aureus or E. coli, a considerable improvement with respect to TI < 37 for the parent compounds. Circular dichroism studies of the mutated peptides suggested this may in part be due to variations in the α-helical structure. For P. aeruginosa, which is more resistant to XT-7, the TI increased in the mutated peptide from 5 to >270, also due to a significant improvement in minimal inhibitory concentration. We have shown that the Mutator tool is capable of suggesting limited variations in natural anuran peptides capable of increasing peptide selectivity, by decreasing toxicity against mammalian erythrocytes, in general without compromising antibacterial activity. The tool is freely available on the Mutator Web server at http://split4.pmfst.hr/mutator/.

Temporin-SHf, a new type of phe-rich and hydrophobic ultrashort antimicrobial peptide.

Because issues of cost and bioavailability have hampered the development of gene-encoded antimicrobial peptides to combat infectious diseases, short linear peptides with high microbial cell selectivity have been recently considered as antibiotic substitutes. A new type of short antimicrobial peptide, designated temporin-SHf, was isolated and cloned from the skin of the frog Pelophylax saharica. Temporin-SHf has a highly hydrophobic sequence (FFFLSRIFa) and possesses the highest percentage of Phe residues of any known peptide or protein. Moreover, it is the smallest natural linear antimicrobial peptide found to date, with only eight residues. Despite its small size and hydrophobicity, temporin-SHf has broad-spectrum microbicidal activity against Gram-positive and Gram-negative bacteria and yeasts, with no hemolytic activity. CD and NMR spectroscopy combined with restrained molecular dynamics calculations showed that the peptide adopts a well defined non-amphipathic alpha-helical structure from residue 3 to 8, when bound to zwitterionic dodecyl phosphocholine or anionic SDS micelles. Relaxation enhancement caused by paramagnetic probes showed that the peptide adopts nearly parallel orientations to the micelle surface and that the helical structure is stabilized by a compact hydrophobic core on one face that penetrates into the micelle interior. Differential scanning calorimetry on multilamellar vesicles combined with membrane permeabilization assays on bacterial cells indicated that temporin-SHf disrupts the acyl chain packing of anionic lipid bilayers, thereby triggering local cracks and microbial membrane disintegration through a detergent-like effect probably via the carpet mechanism. The short length, compositional simplicity, and broad-spectrum activity of temporin-SHf make it an attractive candidate to develop new antibiotic agents.

Reverse Immunology Approach to Define a New HIV-gp41-Neutralizing Epitope.

The design of immunogens susceptible to elicit potent and broadly neutralizing antibodies against the human immunodeficiency virus type 1 (HIV-1) remains a veritable challenge in the course of vaccine development. Viral envelope proteins adopt different conformational states during the entry process, allowing the presentation of transient neutralizing epitopes. We focused on the highly conserved 3S motif of gp41, which is exposed to the surface envelope in its trimeric prefusion state. Vaccination with a W614A-modified 3S peptide induces in animals neutralizing anti-HIV-1 antibodies among which we selected clone F8. We used F8 as bait to select for W614A-3S phage-peptide mimics. Binding and molecular docking studies revealed that F8 interacts similarly with W614A-3S and a Mim_F8-1 mimotope, despite their lack of sequence homology, suggesting structural mimicry. Finally, vaccination of mice with the purified Mim_F8-1 phage elicited HIV-1-neutralizing antibodies that bound to the cognate W614A-3S motif. Collectively, our findings provide new insights into the molecular design of immunogens to elicit antibodies with neutralizing properties.

Solution structure and model membrane interactions of temporins-SH, antimicrobial peptides from amphibian skin. A NMR spectroscopy and differential scanning calorimetry study.

Temporin-SHa and temporin-SHc are 13 residue long antimicrobial peptides from frog skin that have similar sequences but differ markedly in their membrane-damaging properties. Temporin-SHa contains a single basic lysine residue and has a unique antimicrobial spectrum of action among temporins, being very potent against Gram-positive and Gram-negative bacteria, yeasts, fungi, and protozoa. Temporin-SHc, which contains a single basic histidine residue, is inactive against Gram-negative bacteria, has a reduced efficacy against Gram-positive bacteria, but is still active against yeasts and fungi. Temporin-SHb, with no basic residue, has no antimicrobial activity. The three-dimensional structures of the peptides bound to SDS micelles were analyzed by CD and NMR spectroscopy combined with restrained molecular dynamics calculations. The peptides adopt well-defined amphipathic alpha-helical structures extending from residue 3 to residue 12, when bound to SDS micelles. The structures are stabilized by extensive interactions between aliphatic and aromatic side chains on the nonpolar face. Relaxation enhancements caused by paramagnetic probes showed that the peptides adopt nearly parallel orientations to the micelle surface and do not deeply penetrate into the micelle. The interaction of the peptides with model membranes was investigated by differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles and membrane-permeabilization assays on calcein-loaded large unilamellar vesicles. Calorimetric data indicated that both temporin-SHa and -SHc reside at the hydrocarbon core-water interface of the anionic lipid bilayer but interact with anionic bilayers in a very different manner. This suggests that the charge-induced activity of temporins-SH for bacterial cells is due to changes in the membrane-disturbing mechanism of the bound peptides.

Peptidoglycan potentiates the membrane disrupting effect of the carboxyamidated form of DMS-DA6, a Gram-positive selective antimicrobial peptide isolated from Pachymedusa dacnicolor skin.

The occurrence of nosocomial infections has been on the rise for the past twenty years. Notably, infections caused by the Gram-positive bacteria Staphylococcus aureus represent a major clinical problem, as an increase in antibiotic multi-resistant strains has accompanied this rise. There is thus a crucial need to find and characterize new antibiotics against Gram-positive bacteria, and against antibiotic-resistant strains in general. We identified a new dermaseptin, DMS-DA6, produced by the skin of the Mexican frog Pachymedusa dacnicolor, with specific antibacterial activity against Gram-positive bacteria. This peptide is particularly effective against two multiple drug-resistant strains Enterococcus faecium BM4147 and Staphylococcus aureus DAR5829, and has no hemolytic activity. DMS-DA6 is naturally produced with the C-terminal carboxyl group in either the free or amide forms. By using Gram-positive model membranes and different experimental approaches, we showed that both forms of the peptide adopt an α-helical fold and have the same ability to insert into, and to disorganize a membrane composed of anionic lipids. However, the bactericidal capacity of DMS-DA6-NH2 was consistently more potent than that of DMS-DA6-OH. Remarkably, rather than resulting from the interaction with the negatively charged lipids of the membrane, or from a more stable conformation towards proteolysis, the increased capacity to permeabilize the membrane of Gram-positive bacteria of the carboxyamidated form of DMS-DA6 was found to result from its enhanced ability to interact with peptidoglycan.

Identification of the Single Immunodominant Region of the Native Human CC Chemokine Receptor 6 Recognized by Mouse Monoclonal Antibodies.

Chemokines and their receptors play an important role in cell trafficking and recruitment. The CCR6 chemokine receptor, selectively expressed on leukocyte populations, has been shown to play a deleterious role in the pathogenesis of various chronic inflammatory diseases and, as such, may constitute a prime target in the development of immunotherapeutic treatment. However, to date no neutralizing mouse monoclonal antibodies (mAbs) specific for this chemokine receptor have been reported, whereas information on small molecules capable of interfering with the interaction of CCR6 and its ligands is scant. Here, we report the failure to generate neutralizing mouse mAbs specific for human (hu)CCR6. Immunization of mice with peptides mimicking extracellular domains, potentially involved in CCR6 function, failed to induce Abs reactive with the native receptor. Although the use of NIH-3T3 cells expressing huCCR6 resulted in the isolation of mAbs specific for this receptor, they were not able to block the interaction between huCCR6 and huCCL20. Investigation of the anti-huCCR6 mAbs generated in the present study, as well as those commercially available, show that all mAbs invariably recognize a unique, non-neutralizing, immunodominant region in the first part of its N-terminal domain. Together, these results indicate that the generation of potential neutralizing anti-huCCR6 mAbs in the mouse is unlikely to succeed and that alternative techniques, such as the use of other animal species for immunization, might constitute a better approach to generate such a potentially therapeutic tool for the treatment of inflammatory disease.

Pachymodulin, a new functional formyl peptide receptor 2 peptidic ligand isolated from frog skin has Janus-like immunomodulatory capacities.

Recruitment of leukocytes is essential to fight infections or to heal injuries; however, excessive and/or prolonged responses favor the development of major inflammatory pathologies, such as cardiovascular or neurodegenerative diseases. Thus, it is of great interest to seek novel compounds that can regulate leukocyte recruitment depending on the degree of inflammation. We have isolated and characterized, by different chromatographic techniques, mass spectrometry, and Edman sequencing, a new hexapeptide (SSLSKL) from the Mexican frog Pachymedusa dacnicolor, which we named pachymodulin. In vitro, pachymodulin promotes the migration of leukocytes through the binding and activation of the human and mouse N-formyl peptide receptor 2 (huFPR2). In vivo, it exhibits opposite biological activities: under homeostatic conditions, pachymodulin induces the recruitment of leukocytes, whereas under inflammatory conditions, it inhibits this process. Therefore, pachymodulin represents an interesting template in the quest to design new immunomodulatory drugs in the therapy of immune-related diseases.

Structure-Activity Relationship-based Optimization of Small Temporin-SHf Analogs with Potent Antibacterial Activity.

Short antimicrobial peptides represent attractive compounds for the development of new antibiotic agents. Previously, we identified an ultrashort hydrophobic and phenylalanine-rich peptide, called temporin-SHf, representing the smallest natural amphibian antimicrobial peptide known to date. Here, we report on the first structure-activity relationship study of this peptide. A series of temporin-SHf derivatives containing insertion of a basic arginine residue as well as residues containing neutral hydrophilic (serine and α-hydroxymethylserine) and hydrophobic (α-methyl phenylalanine and p-(t)butyl phenylalanine) groups were designed to improve the antimicrobial activity, and their α-helical structure was investigated by circular dichroism and nuclear magnetic resonance spectroscopy. Three compounds were found to display higher antimicrobial activity with the ability to disrupt (permeabilization/depolarization) the bacterial membrane while retaining the nontoxic character of the parent peptide toward rat erythrocytes and human cells (THP-1 derived macrophages and HEK-293). Antimicrobial assays were carried out to explore the influence of serum and physiological salt concentration on peptide activity. Analogs containing d-amino acid residues were also tested. Our study revealed that [p-(t)BuF(2), R(5)]SHf is an attractive ultrashort candidate that is highly potent (bactericidal) against Gram-positive bacteria (including multidrug resistant S. aureus) and against a wider range of clinically interesting Gram-negative bacteria than temporin-SHf, and also active at physiological salt concentrations and in 30% serum.

Human aminopeptidase B (rnpep) on chromosome 1q32.2: complementary DNA, genomic structure and expression.

Aminopeptidase B (APB) is a Zn(2+)-metalloexopeptidase, which selectively removes Arg and/or Lys residues from the N-terminus of several peptide substrates. Several data strongly support the hypothesis that this enzyme could participate in the final stages of precursor processing mechanisms and/or in particular inflammatory processes and tumor developments. Therefore, we have cloned the complementary DNA encoding the human APB, a 658-residues protein, containing the canonical "HEXXH(X(18))E", a signature allowing its classification in the M1 family of metallopeptidases. The genomic structure of the human APB gene (rnpep; 1q32.1-q32.2) was also determined. rnpep is bracketed by pre-protein translocase of the inner mitochondrial membrane gene and ETS family transcription factor ELF3 gene. It spans more than 24 kbp and contains 11 exons ranging from 109 to 574 bp. Finally, expression of the human APB messenger RNA (mRNA) was investigated using a pre-made dot-blot. This mRNA seems to be ubiquitous although its expression level varies depending of the cells or tissues considered.

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.

Structure, antimicrobial activities and mode of interaction with membranes of novel [corrected] phylloseptins from the painted-belly leaf frog, Phyllomedusa sauvagii.

Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6) and also of phylloseptin-1 (PSN-1, here renamed PLS-S1), the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79-95% amino acid sequence identity; net charge  = +2) were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge  = +1) and -S5 (net charge  = +2) were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of phylloseptins.