Optimizing the synthesis of dimeric peptides: influence of the reaction medium and effects that modulate kinetics and reaction yield.

Peptides are remarkably interesting alternatives to several applications. In particular, antimicrobial sequences have raised major interest of the scientific community due to the resistance acquired by commonly used antibiotics. Amongst these, some dimeric peptides have shown very promising characteristics as strong biological activities and resistance against degradation by peptidases. However, despite such promising characteristics, a relatively small number of studies address dimeric peptides, mainly due to the synthesis-related obstacles in their production, whereas the well-implemented routines of solid phase peptide synthesis-which includes the possibility of automation-makes life significantly easier. Here, we present kinetic investigations of the dimerization of a cysteine-containing sequence to obtain the homodimeric antimicrobial peptide homotarsinin. Based on the structural and membrane interaction data already available for the dimer and its monomeric chain, we have proposed distinct dimerization protocols in selected environments, namely, aqueous buffer, TFE:H2O and micellar solutions. The experimental results were adjusted by a theoretical model. Both the kinetic profiles and the reaction yields are dependent on the reaction medium, clearly indicating that aggregation, peptide structure, and peptide-membrane interactions play major roles in the formation of the disulfide bond. Finally, the rationalization of the different aspects addressed here is expected to contribute to research and applications that demand the obtainment of dimeric peptides.

Nicotiana benthamiana is a suitable transient system for high-level expression of an active inhibitor of cotton boll weevil α-amylase.

BACKGROUND: Insect resistance in crops represents a main challenge for agriculture. Transgenic approaches based on proteins displaying insect resistance properties are widely used as efficient breeding strategies. To extend the spectrum of targeted pathogens and overtake the development of resistance, molecular evolution strategies have been used on genes encoding these proteins to generate thousands of variants with new or improved functions. The cotton boll weevil (Anthonomus grandis) is one of the major pests of cotton in the Americas. An α-amylase inhibitor (α-AIC3) variant previously developed via molecular evolution strategy showed inhibitory activity against A. grandis α-amylase (AGA). RESULTS: We produced in a few days considerable amounts of α-AIC3 using an optimised transient heterologous expression system in Nicotiana benthamiana. This high α-AIC3 accumulation allowed its structural and functional characterizations. We demonstrated via MALDI-TOF MS/MS technique that the protein was processed as expected. It could inhibit up to 100% of AGA biological activity whereas it did not act on α-amylase of two non-pathogenic insects. These data confirmed that N. benthamiana is a suitable and simple system for high-level production of biologically active α-AIC3. Based on other benefits such as economic, health and environmental that need to be considerate, our data suggested that α-AIC3 could be a very promising candidate for the production of transgenic crops resistant to cotton boll weevil without lethal effect on at least two non-pathogenic insects. CONCLUSIONS: We propose this expression system can be complementary to molecular evolution strategies to identify the most promising variants before starting long-lasting stable transgenic programs.

Synthesis, biophysical and functional studies of two BP100 analogues modified by a hydrophobic chain and a cyclic peptide.

Antimicrobial peptides (AMPs) work as a primary defense against pathogenic microorganisms. BP100, (KKLFKKILKYL-NH2), a rationally designed short, highly cationic AMP, acts against many bacteria, displaying low toxicity to eukaryotic cells. Previously we found that its mechanism of action depends on membrane surface charge and on peptide-to-lipid ratio. Here we present the synthesis of two BP100 analogs: BP100­alanyl­hexadecyl­1­amine (BP100-Ala-NH-C16H33) and cyclo(1­4)­d­Cys1, Ile2, Leu3, Cys4-BP100 (Cyclo(1­4)­cILC-BP100). We examined their binding to large unilamellar vesicles (LUV), conformational and functional properties, and compared with those of BP100. The analogs bound to membranes with higher affinity and a lesser dependence on electrostatic forces than BP100. In the presence of LUV, BP100 and BP100-Ala-NH-C16H33 acquired α-helical conformation, while Cyclo(1­4)­cILC-BP100) was partly α-helical and partly ß-turn. Taking in conjunction: 1. particle sizes and zeta potential, 2. effects on lipid flip-flop, 3. leakage of LUVs internal contents, and 4. optical microscopy of giant unilamellar vesicles, we concluded that at high concentrations, all three peptides acted by a carpet mechanism, while at low concentrations the peptides acted by disorganizing the lipid bilayer, probably causing membrane thinning. The higher activity and lesser membrane surface charge dependence of the analogs was probably due to their greater hydrophobicity. The MIC values of both analogs towards Gram-positive and Gram-negative bacteria were similar to those of BP100 but both analogues were more hemolytic. Confocal microscopy showed Gram-positive B. subtilis killing with concomitant extensive membrane damage suggestive of lipid clustering, or peptide-lipid aggregation. These results were in agreement with those found in model membranes.

Characterization of multiple antilisterial peptides produced by sakacin P-producing Lactobacillus sakei subsp. sakei 2a.

Antimicrobial compounds produced by lactic acid bacteria can be explored as natural food biopreservatives. In a previous report, the main antimicrobial compounds produced by the Brazilian meat isolate Lactobacillus sakei subsp. sakei 2a, i.e., bacteriocin sakacin P and two ribosomal peptides (P2 and P3) active against Listeria monocytogenes, were described. In this study, we report the spectrum of activity, molecular mass, structural identity and mechanism of action of additional six antilisterial peptides produced by Lb. sakei 2a, detected in a 24 h-culture in MRS broth submitted to acid treatment (pH 1.5) and proper fractionation and purification steps for obtention of free and cell-bound proteins. The six peptides presented similarity to different ribosomal proteins of Lb. sakei subsp sakei 23K and the molecular masses varied from 4.6 to 11.0 kDa. All peptides were capable to increase the efflux of ATP and decrease the membrane potential in Listeria monocytogenes. The activity of a pool of the obtained antilisterial compounds [enriched active fraction (EAF)] against Listeria monocytogenes in a food model (meat gravy) during refrigerated storage (4 °C) for 10 days was also tested and results indicated that the populations of L. monocytogenes in the food model containing the acid extract remained lower than those at time 0-day, evidencing that the acid extract of a culture of Lb. sakei 2a is a good technological alternative for the control of growth of L. monocytogenes in foods.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH2). Two glycotriazole-peptides, namely [p-Glc-trz-G1]HSP1-NH2 and [p-GlcNAc-trz-G1]HSP1-NH2, were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G1]HSP1-NH2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH2 presents relatively low and [trz-G1]HSP1-NH2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

BACKGROUND: Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. METHODS: Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. RESULTS: Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. CONCLUSIONS: These findings revealed that vivax-infected patients with anaemia have increased levels of IgG auto-antibodies against nRBCs and that their deposition on the surface of non-infected erythrocytes decreases their deformability, which, in turn, may enhance nRBC clearance by phagocytes, contributing to the anaemic outcome. These data provide insights into the immune mechanisms associated with vivax malaria anaemia and may be important to the development of new therapy and vaccine strategies.

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies.

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3-11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events - large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.

A voltammetric peptide biosensor for Cu2+ metal ion quantification in coffee seeds.

A prion-derived copper(II)-binding peptide was assembled onto a gold electrode for the building of a voltammetric biosensor for measuring the Cu2+ metal ion in biological samples. The chosen sequence was H-CVNITKQHTVTTTT-NH2, with an appended cysteine residue for binding to the gold surface as a self-assembled monolayer and a histidine residue as the anchorage point for copper(II) complexation. The biosensor showed a linear range of 10-7 to 10-6 M with an 8.0 × 10-8 M detection limit and a 1.0 × 10-7 M quantification limit, with good precision, trueness, and absence of matrix effect. The quantification of Cu2+ was performed in the presence of other transition metal ions, such as Zn2+, Cd2+, Fe2+, or Ni2+, which indicates the excellent selectivity of the biosensor. When the modified electrode was applied for measuring copper(II) in calcined coffee seeds, a difference in copper amount was observed between two Coffea arabica cultivars that were submitted to a treatment with a copper-based antifungal, showing the applicability of the biosensor in the agricultural field.

Non-Cryoprecipitation Separation of Coagulation FVIII and Prothrombin Complex Proteins by Pseudoaffinity Calcium Elution Chromatography Using Anion Exchange Resin.

Hemophilia A is treated with human plasma coagulation factor VIII (FVIII) replacement therapy and Hemophilia B with coagulation factor IX, which is purified from prothrombin complex concentrate (PCC). In this paper we evaluated the separation of FVIII and PCC by directly loading raw thawed plasma to an anion exchange resin (AEX). Under this relatively high ionic strength, most of the plasma proteins such as albumin, immunoglobulins and others were not adsorbed. Five resins commonly used in protein purification (plasma fractionation) were tested. With all resins, PCC was eluted by pseudoaffinity in a calcium gradient step. Afterwards, FVIII could be recovered with a good yield and high purification factor in the salt gradient step with 400-500 mM NaCl. Using ANX Sepharose FF and Q Sepharose FF, the CaCl2 elution step was introduced after the intermediate wash with 200 mM NaCl, whereas using DEAE Sepharose FF, Fractogel EMD TMAE and Fractogel EMD DEAD, PCC eluted after the wash of the unbound proteins. Our results indicate that three important fractions: (1) albumin, immunoglobulin etc.; (2) PCC; and (3) FVIII can be separated in one chromatographic AEX column and the delicate and troublesome cryoprecipitation can be eliminated, making the purification of blood products faster and cheaper.

Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity.

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

Antimicrobial compounds produced by Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product.

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

Biochemical and functional properties of a new l-amino acid oxidase (LAAO) from Micrurus lemniscatus snake venom.

This study reports the purification of ML-LAAO, a new LAAO from the venom of Micrurus lemniscatus snake (ML-V), using size exclusion chromatography. ML-LAAO is a 69-kDa glycoprotein that represents ~2.0% of total venom proteins. This enzyme exhibited optimal activity at pH 8.5, displaying high specificity toward hydrophobic l-amino acids. MALDI TOF/TOF and Blast analysis identified internal segments in ML-LAAO that share high sequence identity with homologous snake venom LAAOs. Western blot analysis on two-dimensional SDS-PAGE of ML-V, using anti-LAAO revealed the presence of ML-LAAO isoforms (pI 6.3-8.9). ML-LAAO blocked aggregation induced by collagen on washed platelets in a rather weak manner, it did not, however, inhibit platelet aggregation induced by ADP on platelet-rich plasma. In addition, this enzyme displayed in vitro antibacterial activity against Staphylococcus aureus (MIC/MBC of 0.39 µg/mL) and in vitro leishmanicidal action against Leishmania amazonensis and L. chagasi (IC50 values of 0.14 and 0.039 µg/mL, respectively). These activities were significantly reduced by catalase, suggesting that hydrogen peroxide production is involved in some way. The data presented here revealed that ML-LAAO has bactericidal and leishmanicidal effects, suggesting that it may have therapeutic potential.

Mo-HLPs: New flocculating agents identified from Moringa oleifera seeds belong to the hevein-like peptide family.

Cationic peptides found in Moringa oleifera seeds belong to different protein families and are described as the main flocculating agents of the species. In this study we report the identification and isolation of four new flocculant peptides, called Mo-HLPs 1-4, belonging to the family of hevein-like peptides, previously only known for their members' antimicrobial activity. Purification of the peptides followed two sequential membrane ultrafiltration steps and separation by reverse-phase liquid chromatography. Proteomic analyses showed that Mo-HLPs are extremely basic (pI >10) cysteine-rich molecules with molecular masses between 4.5 and 4.8 kDa and with a highly conserved chitin-binding domain. Searches in BLAST revealed high similarity of Mo-HLPs with hevein and other hevein-like peptides and 90% identity with morintides, which are members of the 8C-hevein-like subfamily found in M. oleifera leaves. Mo-HLPs microflocculation assays showed distinct coagulation/flocculation efficiencies, promoting turbidity reduction levels between 67 and 89% in synthetic turbid water. Activity variations were attributed to the substitution of some amino acids among the isoforms, which may have altered the final net charge of the molecules. The identification of Mo-HLPs represents the discovery of a new group of cationic peptides involved in the flocculation properties of M. oleifera seeds. SIGNIFICANCE: The study reveals the presence of hevein-like peptides in Moringa oleifera seeds. It is reported for the first time that members of this family have properties to act as flocculating agents of importance for water treatment processes. The identification of these peptides as well as new functional assignment broadens the horizon for speculation on new species which could act as sources of green coagulants for sustainable water treatment, and contributes to the knowledge about occurrence, distribution, molecular and active diversity of peptides belonging to the hevein-like family.

Novel Ocellatin Peptides Mitigate LPS-induced ROS Formation and NF-kB Activation in Microglia and Hippocampal Neurons.

Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.

Structure and membrane interactions of the antibiotic peptide dermadistinctin K by multidimensional solution and oriented 15N and 31P solid-state NMR spectroscopy.

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an alpha-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with (15)N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting (15)N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled (31)P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Effect of cholesterol on the interaction of the amphibian antimicrobial peptide DD K with liposomes.

DD K is an antimicrobial peptide previously isolated from the skin of the amphibian Phyllomedusa distincta. The effect of cholesterol on synthetic DD K binding to egg lecithin liposomes was investigated by intrinsic fluorescence of tryptophan residue, measurements of kinetics of 5(6)-carboxyfluorescein (CF) leakage, dynamic light scattering and isothermal titration microcalorimetry. An 8 nm blue shift of tryptophan maximum emission fluorescence was observed when DD K was in the presence of lecithin liposomes compared to the value observed for liposomes containing 43 mol% cholesterol. The rate and the extent of CF release were also significantly reduced by the presence of cholesterol. Dynamic light scattering showed that lecithin liposome size increase from 115 to 140 nm when titrated with DD K but addition of cholesterol reduces the liposome size increments. Isothermal titration microcalorimetry studies showed that DD K binding both to liposomes containing cholesterol as to liposomes devoid of it is more entropically than enthalpically favored. Nevertheless, the peptide concentration necessary to furnish an adjustable titration curve is much higher for liposomes containing cholesterol at 43 mol% (2 mmol L(-1)) than in its absence (93 micromol L(-1)). Apparent binding constant values were 2160 and 10,000 L mol(-1), respectively. The whole data indicate that DD K binding to phosphatidylcholine liposomes is significantly affected by cholesterol, which contributes to explain the low hemolytic activity of the peptide.

Solution NMR structures of the antimicrobial peptides phylloseptin-1, -2, and -3 and biological activity: the role of charges and hydrogen bonding interactions in stabilizing helix conformations.

Phylloseptins are antimicrobial peptides of 19-20 residues which are found in the skin secretions of the Phyllomedusa frogs that inhabit the tropical forests of South and Central Americas. The peptide sequences of PS-1, -2, and -3 carry an amidated C-terminus and they exhibit 74% sequence homology with major variations of only four residues close to the C-terminus. Here we investigated and compared the structures of the three phylloseptins in detail by CD- and two-dimensional NMR spectroscopies in the presence of phospholipid vesicles or in membrane-mimetic environments. Both CD and NMR spectroscopies reveal a high degree of helicity in the order PS-2> or =PS-1>PS-3, where the differences accumulate at the C-terminus. The conformational variations can be explained by taking into consideration electrostatic interactions of the negative ends of the helix dipoles with potentially cationic residues at positions 17 and 18. Whereas two are present in the sequence of PS-1 and -2 only one is present in PS-3. In conclusion, the antimicrobial phylloseptin peptides adopt alpha-helical conformations in membrane environments which are stabilized by electrostatic interactions of the helix dipole as well as other contributions such hydrophobic and capping interactions.

Anti-band 3 and anti-spectrin antibodies are increased in Plasmodium vivax infection and are associated with anemia.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

Venomic analyses of Scolopendra viridicornis nigra and Scolopendra angulata (Centipede, Scolopendromorpha): shedding light on venoms from a neglected group.

Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a 'structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds.