Synthesis and properties of cyclic gomesin and analogues

Gomesin (Gm) was the first antimicrobial peptide (AMP) isolated from the hemocytes of a spider, the Brazilian mygalomorph Acanthoscurria gomesiana. We have been studying the properties of this interesting AMP, which also displays anticancer, antimalarial, anticryptococcal and anti-Leishmania activities. In the present study, the total syntheses of backbone-cyclized analogues of Gm (two disulfide bonds), [Cys(Acm)2,15]-Gm (one disulfide bond) and [Thr2,6,11,15,d-Pro9]-Gm (no disulfide bonds) were accomplished, and the impact of cyclization on their properties was examined. The consequence of simultaneous deletion of pGlu1 and Arg16-Glu-Arg18-NH2 on Gm antimicrobial activity and structure was also analyzed. The results obtained showed that the synthetic route that includes peptide backbone cyclization on resin was advantageous and that a combination of 20% DMSO/NMP, EDC/HOBt, 60?degrees C and conventional heating appears to be particularly suitable for backbone cyclization of bioactive peptides. The biological properties of the Gm analogues clearly revealed that the N-terminal amino acid pGlu1 and the amidated C-terminal tripeptide Arg16-Glu-Arg18-NH2 play a major role in the interaction of Gm with the target membranes. Moreover, backbone cyclization practically did not affect the stability of the peptides in human serum; it also did not affect or enhanced hemolytic activity, but induced selectivity and, in some cases, discrete enhancements of antimicrobial activity and salt tolerance. Because of its high therapeutic index, easy synthesis and lower cost, the [Thr2,6,11,15,d-Pro9]-Gm analogue remains the best active Gm-derived AMP developed so far; nevertheless, its elevated instability in human serum may limit its therapeutic potential. Copyright (c) 2012 European Peptide Society and John Wiley & Sons, Ltd.

Functional single-nucleotide polymorphisms in the DEFB1 gene are associated with systemic lupus erythematosus in Southern Brazilians

Systemic lupus erythematosus (SLE) is an autoimmune disease that results in inflammation and tissue damage. The etiology of SLE remains unknown, but recent studies have shown that the innate immune system may have a role in SLE pathogenesis through the secretion of small cationic peptides named defensins. The aim of the study was to determine the possible involvement in SLE of three functional single nucleotide polymorphisms (SNPs) (c.-52G>A, c.-44C>G and c.-20G>A) in the 5'UTR region of DEFB1 gene, by analyzing them in a population of 139 SLE patients and 288 healthy controls. The c.-52G>A SNP showed significant differences in allele and genotype frequency distribution between SLE patients and controls (p = 0.01 and p = 0.02 respectively) indicating protection against SLE (A allele, OR = 0.68, AA genotype OR = 0.51). Significant differences were also observed for c.-44C>G SNP, the C/G genotype being associated with susceptibility to SLE (OR = 1.60, p = 0.04). Moreover, statistically significant differences between patients and controls were found for two DEFB1 haplotypes (GCA and GGG, p = 0.01 and p = 0.02 respectively). When considering DEFB1 SNPs and SLE clinical and laboratory manifestations, significant association was found with neuropsychiatric disorders, immunological alterations and anti-DNA antibodies. In conclusion, our results evidence a possible role for the c.-52G>A and c.-44C>G DEFB1 polymorphisms in SLE pathogenesis, that can be considered as possible risk factors for development of disease and disease-related clinical manifestations. Additional studies are needed, to corroborate these results as well as functional studies to understand the biological role of these SNPs in the pathogenesis of SLE. Lupus (2012) 21, 625-631.

Temperature-Dependence of Cationic Lipid Bilayer Intermixing: Possible Role of Interdigitation

In this work, we investigated the properties of a fusogenic cationic lipid, diC14-amidine, and show that this lipid possesses per se the capacity to adopt either an interdigitated structure (below and around its transition temperature) or a lamellar structure (above the transition temperature). To provide experimental evidence of this lipid bilayer organization, phospholipids spin-labeled at different positions of the hydrocarbon chain were incorporated into the membrane and their electron spin resonance (ESR) spectra were recorded at different temperatures. For comparison, similar experiments were performed with dimyristoyl phosphatidylcholine, a zwitterionic lipid (DMPC) which adopts a bilayer organization over a broad temperature range. Lipid mixing between diC14-amidine and asolectin liposomes was more efficient below (10-15 degrees C) than above the transition temperature (above 25 degrees C). This temperature-dependent "fusogenic" activity of diC14-amidine liposomes is opposite to what has been observed so far for peptides or virus-induced fusion. Altogether, our data suggest that interdigitatiori is a highly fusogenic state and that interdigitation-mediated fusion occurs via an unusual temperature-dependent mechanism that remains to be deciphered.

Therapeutic use of a cationic antimicrobial peptide from the spider Acanthoscurria gomesiana in the control of experimental candidiasis

Background: Antimicrobial peptides are present in animals, plants and microorganisms and play a fundamental role in the innate immune response. Gomesin is a cationic antimicrobial peptide purified from haemocytes of the spider Acanthoscurria gomesiana. It has a broad-spectrum of activity against bacteria, fungi, protozoa and tumour cells. Candida albicans is a commensal yeast that is part of the human microbiota. However, in immunocompromised patients, this fungus may cause skin, mucosal or systemic infections. The typical treatment for this mycosis comprises three major categories of antifungal drugs: polyenes, azoles and echinocandins; however cases of resistance to these drugs are frequently reported. With the emergence of microorganisms that are resistant to conventional antibiotics, the development of alternative treatments for candidiasis is important. In this study, we evaluate the efficacy of gomesin treatment on disseminated and vaginal candidiasis as well as its toxicity and biodistribution. Results: Treatment with gomesin effectively reduced Candida albicans in the kidneys, spleen, liver and vagina of infected mice. The biodistribution of gomesin labelled with technetium-99 m showed that the peptide is captured in the kidneys, spleen and liver. Enhanced production of TNF-alpha, IFN-gamma and IL-6 was detected in infected mice treated with gomesin, suggesting an immunomodulatory activity. Moreover, immunosuppressed and C. albicans-infected mice showed an increase in survival after treatment with gomesin and fluconazole. Systemic administration of gomesin was also not toxic to the mice Conclusions: Gomesin proved to be effective against experimental Candida albicans infection. It can be used as an alternative therapy for candidiasis, either alone or in combination with fluconazole. Gomesin's mechanism is not fully understood, but we hypothesise that the peptide acts through the permeabilisation of the yeast membrane leading to death and/or releasing the yeast antigens that trigger the host immune response against infection. Therefore, data presented in this study reinforces the potential of gomesin as a therapeutic antifungal agent in both humans and animals.

Interaction of cationic water-soluble meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) with ionic and nonionic micelles: aggregation and binding

The equilibrium of meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (TMPyP) in aqueous solution in the presence of surfactants was studied by optical spectroscopic techniques and SAXS (small angle X-ray scattering). Anionic SDS (sodium dodecyl sulfate), zwitterionic HPS (N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate) and nonionic TRITON X-100 (t-octyl-phenoxypolyethoxyethanol), surfactants were used. TMPyP is characterized by a protonation equilibrium with a pK(a) around 1.0, associated with the diacid-free base transition, and a second pK(a) around 12.0 related with the transition between the free base and the monoanion form. Three independent species were observed for TMPyP at pH 6.0 as a function of SDS concentration: free TMPyP, TMPyP-SDS aggregates and porphyrin monomer bound to micelles. For HPS and TRITON X-100, the equilibrium of TMPyP as a function of pH is quite similar to that obtained in pure aqueous solution: no aggregation was observed, suggesting that electrostatic contribution is the major factor in the interaction between TMPyP and surfactants. SAXS data analysis demonstrated a prolate ellipsoidal shape for SDS micelles; no significant changes in shape and size were observed for SDS-TMPyP co-micelles. Moreover, the ionization coefficient, alpha, decreases with the increase of the porphyrin concentration, suggesting the ""screening"" of the anionic charge of SDS by the cationic porphyrin. These results are consistent with optical absorption, fluorescence and RLS (resonance light scattering) spectroscopies data, allowing to conclude that neutral surfactants present a smaller interaction with the cationic porphyrin as compared with an ionic surfactant. Therefore, the interaction of TMPyP with the ionic and nonionic surfactants is predominantly due to the electrostatic contribution. Copyright (c) 2008 Society of Porphyrins & Phthalocyanines.

Evaluation of the in vitro Activity of Dermaseptin 01, a Cationic Antimicrobial Peptide, against Schistosoma mansoni

Schistosomiasis is a neglected tropical disease that remains a considerable public health problem worldwide. Since the mainstay of schistosomiasis control is chemotherapy with a single drug, praziquantel, drug resistance is a concern. Here, we examined the in vitro effects of dermaseptin 01 (DS 01), an antimicrobial peptide found in the skin secretion of frogs of the genus Phyllomedusa, on Schistosoma mansoni adult worms. DS 01 at a concentration of 100 mg/ml reduced the worm motor activity and caused the death of all worms within 48 h in RPMI 1640 medium. At the highest sublethal concentration of antimicrobial peptide (75 mg/ml), a 100% reduction in egg output of paired female worms was observed. Additionally, DS 01 induced morphological alterations on the tegument of S. mansoni, and a quantitative analysis carried out by confocal microscopy revealed extensive destruction of the tubercles in a dose-dependent manner over the concentration range of 50-200 mu g/ml. It was the first time that an anthelmintic activity towards schistosomes has been reported for a dermaseptin.

A cyclic bipolar wind in the interacting binary V 393 Scorpii

V393 Scorpii is a double periodic variable characterized by a relatively stable non-orbital photometric cycle of 253 d. Mennickent et al. argue for the presence of a massive optically thick disc around the more massive B-type component and describe the evolutionary stage of the system. In this paper, we analyse the behaviour of the main spectroscopic optical lines during the long non-orbital photometric cycle. We study the radial velocity of the donor determining its orbital elements and find a small but significant orbital eccentricity (e = 0.04). The donor spectral features are modelled and removed from the spectrum at every observing epoch using the light-curve model given by Mennickent et al. We find that the line emission is larger during eclipses and mostly comes from a bipolar wind. We also find that the long cycle is explained in terms of a modulation of the wind strength; the wind has a larger line and continuum emissivity at the high state. We report the discovery of highly variable chromospheric emission in the donor, as revealed by the Doppler maps of the emission lines Mg II 4481 and C I 6588. We discuss notable and some novel spectroscopic features like discrete absorption components, especially visible at blue depressed O I 7773 absorption wings during the second half-cycle, Balmer double emission with V/R curves showing 'Z-type' and 'S-type' excursions around secondary and main eclipses, respectively, and H beta emission wings extending up to +/- 2000 km s(-1). We also discuss possible causes for these phenomena and for their modulations with the long cycle.

Pressure Dependence of N-15 Chemical Shifts in Model Peptides Ac-Gly-Gly-X-Ala-NH2

High pressure NMR spectroscopy has developed into an important tool for studying conformational equilibria of proteins in solution. We have studied the amide proton and nitrogen chemical shifts of the 20 canonical amino acids X in the random-coil model peptide Ac-Gly-Gly-X-Ala-NH2, in a pressure range from 0.1 to 200 MPa, at a proton resonance frequency of 800 MHz. The obtained data allowed the determination of first and second order pressure coefficients with high accuracy at 283 K and pH 6.7. The mean first and second order pressure coefficients <B-1(15N)> and <B-2(15N)> for nitrogen are 2.91 ppm/GPa and -2.32 ppm/GPa(2), respectively. The corresponding values <B-1(1H)> and <B-2(1H)> for the amide protons are 0.52 ppm/GPa and -0.41 ppm/GPa(2). Residual dependent (1)J(1H15N)-coupling constants are shown.

Inhibition of thimet oligopeptidase by siRNA alters specific intracellular peptides and potentiates isoproterenol signal transduction

Mammalian cells have a large number of intracellular peptides that are generated by extralysosomal proteases. In this study, the enzymatic activity of thimet oligopeptidase (EP24.15) was inhibited in human embryonic kidney (HEK) 293 cells using a specific siRNA sequence. The semi-quantitative intracellular peptidome analyses of siRNA-transfected HEK293 cells shows that the levels of specific intracellular peptides are either increased or decreased upon EP24.15 inhibition. Decreased expression of EP24.15 was sufficient to potentiate luciferase gene reporter activation by isoproterenol (1-10 mu M). The protein kinase A inhibitor KT5720 (1 mu M) reduced the positive effect of the EP24.15 siRNA on isoproterenol signaling. Thus, EP24.15 inhibition by siRNA modulates the levels of specific intracellular peptides and isoproterenol signal transduction. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Angiotensin-(3-4) counteracts the Angiotensin II inhibitory action on renal Ca2+-ATPase through a cAMP/PKA pathway

We recently demonstrated that Angiotensin-(3-4) [Ang-(3-4)], an Ang II-derived dipeptide, overcomes inhibition of plasma membrane Ca2+-ATPase promoted by nanomolar concentrations of Ang II in basolateral membranes of renal proximal tubule cells, with involvement of a so far unknown AT(2)R-dependent and NO-independent mechanism. The present study investigates the signaling pathway triggered by Ang-(3-4) that is responsible for counteracting the inhibitory effect of Ang II, and attempts to elucidate the functional interaction of the dipeptide with Ang II at the level of AT(2)R. Stimulation by cholera toxin of G(s)alpha protein structurally linked to AT(2)R as revealed by their co-immunoprecipitation mimicked the effect of Ang-(3-4) on Ca2+-ATPase activity. Furthermore, addition of dibutyril-cAMP (db-cAMP) mimicked Ang-(3-4), whereas the specific PKA inhibitor, PKAi((5-24)) peptide, suppressed the counter-regulatory effect of Ang-(3-4) and the AT(2)R agonist, CGP42112A. Membrane-associated PKA activity was stimulated by Ang-(3-4) or CGP42112A to comparable levels as db-cAMP, and the Ang-(3-4) effect was abrogated by the AT(2)R antagonist PD123319, whereas the AT(1)R antagonist Losartan had no effect. Ang-(3-4) stimulated PKA-mediated phosphorylation of Ca2+-ATPase and activated PKA to comparable levels. Binding assays demonstrated that Ang-(3-4) could not displace H-3-Ang II from HEK 293T cells expressing AT(2)R, but 10(-10) mol/L Ang-(3-4) resulted in the appearance of a probable higher-affinity site (picomolar range) for Ang II. The results presented herein demonstrate that Ang-(3-4), acting as an allosteric enhancer, suppresses Ang II-mediated inhibition of Ca2+-ATPase through an AT(2)R/cAMP/PKA pathway, after inducing conformational changes in AT(2)R that results in generation of higher-affinity sites for Ang II. (C) 2012 Elsevier B.V. All rights reserved.

Headgroup specificity for the interaction of the antimicrobial peptide tritrpticin with phospholipid Langmuir monolayers

We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE similar to DPPC << DPPA < DPPG). The stronger interaction with negatively charged phospholipids agrees with data for vesicles and planar lipid bilayers, and with AMPs greater activity against bacterial membranes versus mammalian cell membranes. Considerable expansion of negatively charged monolayers occurred at 10 and 30 mol% TRP3, especially at low surface pressure. Moreover, a difference was observed between PA and PG, demonstrating that the interaction, besides being modulated by electrostatic interactions, displays specificity with regard to headgroup, being more pronounced in the case of PG, present in large quantities in bacterial membranes. In previous studies, it was proposed that the peptide acts by a toroidal pore-like mechanism [1,2]. Considering the evidence from the literature that PG shows a propensity to form a positive curvature as do toroidal pores, the observation of TRP3's preference for the PG headgroup and the dramatic increase in area promoted by this interaction represent further support for the toroidal pore mechanism of action proposed for TRP3. (C) 2012 Elsevier B.V. All rights reserved.

Co-occurrence of microcystin and microginin congeners in Brazilian strains of Microcystis sp.

Species of Microcystis are the most common bloom-forming cyanobacteria in several countries. Despite extensive studies regarding the production of bioactive cyanopeptides in this genus, there are limited data on isolated strains from Brazil. Three Microcystis sp. strains were isolated from the Salto Grande Reservoir (LTPNA01, 08 and 09) and investigated for the presence of mcy genes, microcystins and other cyanopeptides. Microcystin and microginin production was confirmed in two isolates using high-resolution tandem mass spectrometry after electrospray ionization (ESI-Q-TOF), and the structures of two new microginin congeners were proposed (MG756 Ahda-Val-Leu-Hty-Tyr and MG770 MeAhda-Val-Leu-Hty-Tyr). The biosynthesis profile of the identified cyanopeptides was evaluated at different growth phases via a newly developed HPLC-UV method. Results demonstrated no substantial differences in the production of microcystins and microginins after data normalization to cell quota, suggesting a constitutive biosynthesis. This study represents the first confirmed co-production of microginins and microcystins in Brazilian strains of Microcystis sp. and highlights the potential of Brazilian cyanobacteria as a source of natural compounds with pharmaceutical interest.

Natural intracellular peptides can modulate the interactions of mouse brain proteins and thimet oligopeptidase with 14-3-3e and calmodulin

Protein interactions are crucial for most cellular process. Thus, rationally designed peptides that act as competitive assembly inhibitors of protein interactions by mimicking specific, determined structural elements have been extensively used in clinical and basic research. Recently, mammalian cells have been shown to contain a large number of intracellular peptides of unknown function. Here, we investigate the role of several of these natural intracellular peptides as putative modulators of protein interactions that are related to Ca2+-calmodulin (CaM) and 14-3-3 epsilon, which are proteins that are related to the spatial organization of signal transduction within cells. At concentrations of 1-50 mu M, most of the peptides that are investigated in this study modulate the interactions of CaM and 14-3-3 epsilon with proteins from the mouse brain cytoplasm or recombinant thimet oligopeptidase (EP24.15) in vitro, as measured by surface plasmon resonance. One of these peptides (VFDVELL; VFD-7) increases the cytosolic Ca2+ concentration in a dose-dependent manner but only if introduced into HEK293 cells, which suggests a wide biological function of this peptide. Therefore, it is exciting to suggest that natural intracellular peptides are novel modulators of protein interactions and have biological functions within cells.

Broad and Cross-Clade CD4(+) T-Cell Responses Elicited by a DNA Vaccine Encoding Highly Conserved and Promiscuous HIV-1 M-Group Consensus Peptides

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-gamma secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-gamma and TNF-alpha, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications

We developed cationic liposomes containing DNA through a conventional process involving steps of (i) preformation of liposomes, (ii) extrusion, (iii) drying and rehydration and (iv) DNA complexation. Owing to its high prophylactic potentiality against tuberculosis, which had already been demonstrated in preclinical assays, we introduced modifications into the conventional process towards getting a simpler and more economical process for further scale-up. Elimination of the extrusion step, increasing the lipid concentration (from 16 to 64 mM) of the preformed liposomes and using good manufacturing practice bulk lipids (96-98% purity) instead of analytical grade purity lipids (99.9-100%) were the modifications studied. The differences in the physico-chemical properties, such as average diameter, zeta potential, melting point and morphology of the liposomes prepared through the modified process, were not as significant for the biological properties, such as DNA loading on the cationic liposomes, and effective immune response in mice after immunisation as the control liposomes prepared through the conventional process. Beneficially, the modified process increased productivity by 22% and reduced the cost of raw material by 75%.