M. Antònia Busquets

A Langmuir Monolayer Study of the Interaction of E1(145−162) Hepatitis G Virus Peptide with Phospholipid Membranes

E1(145-162), a peptide corresponding to the structural protein E1 of the GB virus C, has been shown earlier to bind at pH 7.4 to vesicles containing 1,2-dimyiristoyl-sn-glycero-3-phospho-rac-(1-glycerol)] (DMPG) and 1,2-dimyiristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipids. To deepen the understanding of the interaction of E1(145-162) with the lipid membrane, in this paper, we report a detailed study of the surface properties of peptide, miscibility properties, and behavior of mixed monomolecular films of it and three phospholipids DMPG, DMPC, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPG). These studies were performed using the Langmuir balance by means of surface adsorption studies, surface pressure-mean molecular area compression isotherms, and penetration kinetics. The Brewster angle microscopy (BAM) was used to study the morphological properties of pure peptide and the mixed monolayers. The results show us that the peptide showed surface activity concentration dependent when injected beneath a buffered solution (HEPES/NaCl, pH 7.4). This tendency to accumulate into the air/water interface confirms its potential capacity to interact with membranes; the higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state and the lipids are charged negatively maybe due to its negative electric charge that interacts with the positive global charge of the peptide sequence. The area per molecule values obtained suggested that the main arrangement structure for E1(145-162) peptide is the alpha-helical at the air-water interface that agreed with computational prediction calculations. Miscibility studies indicated that mixtures become thermodynamically favored at low peptide molar fraction.

Analysis of HIV-1 fusion peptide inhibition by synthetic peptides from E1 protein of GB virus C

The aim of this study was to identify proteins that could inhibit the activity of the peptide sequence representing the N-terminal of the surface protein gp41 of HIV, corresponding to the fusion peptide of the virus (HIV-1 FP). To do this we synthesized and studied 58 peptides corresponding to the envelope protein E1 of the hepatitis G virus (GBV-C). Five of the E1 synthetic peptides: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18) and AQLVGELGSLYGPLSVSA (P22) were capable of inhibiting the leakage of vesicular contents caused by HIV-1 FP. A series of experiments were carried out to determine how these E1 peptides interact with HIV-1 FP. Our studies analyzed the interactions with and without the presence of lipid membranes. Isothermal titration calorimetry revealed that the binding of P7, P18 and P22 peptides to HIV-1 FP is strongly endothermic, and that binding is entropy-driven. Gibbs energy for the process indicates a spontaneous binding between E1 peptides and HIV-1 FP. Moreover, confocal microscopy of Giant Unilamellar Vesicles revealed that the disruption of the lipid bilayer by HIV-1 FP alone was inhibited by the presence of any of the five selected peptides. Our results highlight that these E1 synthetic peptides could be involved in preventing the entry of HIV-1 by binding to the HIV-1 FP. Therefore, the continued study into the interaction between GBV-C peptides and HIV-1 FP could lead to the development of new therapeutic agents for the treatment of AIDS.

Effect of E1(64–81) hepatitis G peptide on the in vitro interaction of HIV-1 fusion peptide with membrane models

One way to gain information about the fusogenic potential of virus-derived synthetic peptides is to examine their interfacial properties and subsequently to study them in monolayers and bilayers. Here, we characterize the physicochemical surface properties of the peptide E1(64-81), whose sequence is AQLVGELGSLYGPLSVSA. This peptide is derived from the E1 structural protein of GBV-C/HGV which was previously shown to inhibit leakage of vesicular contents caused by the HIV-1 fusion peptide (HIV-1 FP). Mixed isotherms of E1(64-81) and HIV-1 FP were obtained and their Brewster angle microscopy (BAM) and atomic force microscopy (AFM) images showed that the peptide mixture forms a different structure that is not present in the pure peptide images. Studies with lipid monolayers (1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DPPG)) show that both peptides interact with all the lipids assayed but the effect that HIV-1 FP has on the monolayers is reduced in the presence of E1(64-81). Moreover, differential scanning calorimetry (DSC) experiments show the capacity of HIV-1 FP to modify the properties of the bilayer structure and the capacity of E1(64-81) to inhibit these modifications. Our results indicate that E1(64-81) interacts with HIV-1 FP to form a new structure, and that this may be the cause of the previously observed inhibition of the activity of HIV-1 FP by E1(64-81).

Biophysical Investigations of GBV‐C E1 Peptides as Potential Inhibitors of HIV‐1 Fusion Peptide

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research.

Fluorescence study of the dynamic interaction between E1(145–162) sequence of hepatitis GB virus C and liposomes

The physicochemical characterization of the peptide sequence E1(145–162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium–DPH. The addition of the peptide E1(145–162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145–162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.

Physicochemical characterization of GBV-C E1 peptides as potential inhibitors of HIV-1 fusion peptide: Interaction with model membranes

Four peptide sequences corresponding to the E1 protein of GBV-C: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10) and QAGLAVRPGKSAAQLVGE (P18) were studied as they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP). In this work, the surface properties of the E1 peptide sequences are investigated and their physicochemical characterization is done by studying their interaction with model membranes; moreover, their mixtures with HIV-1 FP were also studied in order to observe whether they are capable to modify the HIV-1 FP interaction with model membranes as liposomes or monolayers. Physicochemical properties of peptides (pI and net charge) were predicted showing similarities between P7 and P8, and P10 and HIV-1 FP, whereas P18 appears to be very different from the rest. Circular dichroism experiments were carried out showing an increase of the percentage of α-helix of P7 and P8 when mixed with HIV-1 FP corroborating a conformational change that could be the cause of their inhibition ability. Penetration experiments show that all the peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that the peptides interact with phospholipids and the E1 peptides modify the compression isotherms of HIV-1 FP, but there is one of the peptides that excelled as the best candidate for inhibiting the activity of HIV-1 FP, P7, and therefore, that could be potentially used in future anti-HIV-1 research.

Physicochemical study of hepatitis A and B peptide fragments with monolayers as membrane models

Abstract The surface behavior of two peptide fragments corresponding to immunologically active sequences of Hepatitis A and B proteins was determined. Their interactions with monolayers of different lipids were studied by measuring surface pressure changes in lipid monolayers previously spread at 5 mN/m. Results show that both peptides HBV-pre S (120–145) and HAV-VP1 (11–25) have by themselves surface activity in the absence of lipid monolayers. The HBV-peptide interacts strongly with lipids and forms monolayers, giving collapse pressures of the same order as the natural proteins.

Dual responsive gelatin-based nanoparticles for enhanced 5-fluorouracil efficiency

The very slow progress in the therapeutic efficacy of the treatment of severe diseases has suggested the use of a growing need for a multidisciplinary approach to the delivery of therapeutics to targets tissues. There has been increasing effort in the design of stimuli-responsive nanomaterials that they will be developed into effective drug delivery vehicles. Most commonly, effective drug delivery is associated with nanomaterial-facilitated accumulation and/or cellular internalization. Recent studies in our lab have demonstrated that gelatin-based NPs can be considered suitable pH responsive devices for the effective intracellular delivery of drugs. Concerning cancer treatment, ligands recognizing tumour-associated antigens expressed on the surface of the tumour cells have been employed. Some of the target structures suitable for tumour targeting belong to integrins which mediate cell adhesion to extracellular matrix and other cells. Interestingly, gelatin chains contain motifs such as RGD sequences that can be recognised by integrins. In this work the inclusion of the anticancer drug 5-fluorouracil (5-FU) on these gelatin-based NPs has been projected. These NPs may provide an opportunity to increase the therapeutic effect using a dual approach by: i) targeting the therapeutic drug to the tumour cells by the action of the naturally occurring RGD-motif on gelatin and ii) minimizing the non-productive trafficking from endosomes to lysosomes by releasing the cargo using the charge reversal approach after cellular internalization. In vitro cytotoxicity experiments of NPs on tumoral and non-tumoral cell lines have reported selectivity indexes higher than 30 demonstrating a great selectivity on the mode of action as a function of the cell line and the imposed compositions.

Interaction of lipidated GBV-C/HGV NS3 (513–522) and (505–514) peptides with phospholipids monolayer. An AFM study

Lipidation of a short hydrophilic peptide has the aim to make the molecule amphiphilic, which improves its insertion into lipid monolayer and at the same time, the tendency to self-assembly. These both properties of two positively charged, hepatitis G (GBV-C/HGV) related lipidated peptides--palmitic acid derivatives of the fragments: 505-514 and the 513-522 of the NS3 protein (respectively Palmitoyl-SAELSMQRRG and Palmitoyl-RGRTGRGRSG) were studied. First, using transmission electron microscope (TEM) and atomic force microscope (AFM) the tendency to self-assembly in water solution was examined. Both techniques confirmed the formation of fibrous aggregates of Palmitoyl-SAELSMQRRG in water solution. At the same conditions, any fibrous aggregates of Palmitoyl-RGRTGRGRSG were detected neither by TEM nor by AFM. Insertion of the lipidated peptides into phospholipids monolayer formed by zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or negatively charged 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) was investigated. Monolayers prepared by Langmuir-Blodgett method were visualized by AFM. The presence of lipidated peptides in phospholipid monolayers produced changes in the monolayers and different morphologies of the monolayers were obtained for each of the lipidated peptides.