DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study

Cationic lipids-DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA-cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel-fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge-charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets. (c) 2009 Elsevier B.V. All rights reserved.

Lipid bilayer pre-transition as the beginning of the melting process

We investigate the bilayer pre-transition exhibited by some lipids at temperatures below their main phase transition, and which is generally associated to the formation of periodic ripples in the membrane. Experimentally we focus on the anionic lipid dipalmytoylphosphatidylglycerol (DPPG) at different ionic strengths, and on the neutral lipid dipalmytoylphosphatidylcholine (DPPC). From the analysis of differential scanning calorimetry traces of the two lipids we find that both pre- and main transitions are part of the same melting process. Electron spin resonance of spin labels and excitation generalized polarization of Laurdan reveal the coexistence of gel and fluid domains at temperatures between the pre- and main transitions of both lipids, reinforcing the first finding. Also, the melting process of DPPG at low ionic strength is found to be less cooperative than that of DPPC. From the theoretical side, we introduce a statistical model in which a next-nearest-neighbor competing interaction is added to the usual two-state model. For the first time, modulated phases (ordered and disordered lipids periodically aligned) emerge between the gel and fluid phases as a natural consequence of the competition between lipid-lipid interactions. (C) 2009 Elsevier B.V. All rights reserved.

On the interaction of the anthraquinone barbaloin with negatively charged DMPG bilayers

Barbaloin is a bioactive glycosilated 1,8-dihydroxyanthraquinone present in several exudates from plants, Such as Aloe vera, which are used for cosmetic or food purposes. It has been shown that barbaloin interacts with DMPG (dimyristoylphosphatidylglycerol) model membranes, altering the bilayer structure (Alves, D. S.; Perez-Fons, L.; Estepa, A.; Micol, V. Biochem. Pharm. 2004, 68, 549). Considering that ESR (electron spin resonance) of spin labels is one of the best techniques to monitor structural properties at the molecular level, the alterations caused by the anthraquinone barbaloin on phospholipid bilayers will be discussed here via the ESR signal of phospholipid spin probes intercalated into the membranes. In DMPG at high ionic strength (10 mM Hepes pH 7.4 + 100 mM NaCl), a system that presents a gel-fluid transition around 23 degrees C, 20 mol % barbaloin turns the gel phase more rigid, does not alter much the fluid phase packing, but makes the lipid thermal transition less sharp. However, in a low-salt DMPG dispersion (10 mM Hepes pH 7.4 + 2 mM NaCl), which presents a rather complex gel-fluid thermal transition (Lamy-Freund, M. T.; Riske, K. A. Chem. Phys. Lipids 2003, 122, 19), barbaloin strongly affects bilayer structural properties, both in the gel and fluid phases, extending the transition region to much higher temperature values. The position of barbaloin in DMPG bilayers will be discussed on the basis of ESR results, in parallel with data from sample viscosity, DSC (differential scanning calorimetry), and SAXS (small-angle X-ray scattering).

Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Multiple stages of detergent-erythrocyte membrane interaction-A spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate- and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9 mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5 mM detergent. Between 3.2 and 10 mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10 mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs: the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5 mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111). (C) 2010 Elsevier B.V. All rights reserved.

Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle

Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

A short-term, high-fat diet up-regulates lipid metabolism and gene expression in human skeletal muscle

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle.
Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and ß-oxidation in skeletal muscle.
Design: Fourteen well-trained male cyclists and triathletes with a mean (± SE) age of 26.9 ± 1.7 y, weight of 73.7 ± 1.7 kg, and peak oxygen uptake of 67.0 ± 1.3 mL ˙ kg-1 ˙ min-1 consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70–75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), ß-hydroxyacyl-CoA dehydrogenase (ß-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm.
Results: The gene expression of FAT/CD36 and &szlig; -HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet.
Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle.

Sulfatide-tenascin interaction mediates binding to the extracellular matrix and endocytic uptake of liposomes in glioma cells

Tenascin-C is an extracellular matrix glycoprotein, whose expression is highly restricted in normal adult tissues, but markedly up-regulated in a range of tumors, and therefore serves as a potential receptor for targeted anticancer drug or gene delivery. We describe here a liposomal carrier system in which the targeting ligand is sulfatide. Experiments with tenascin-C-expressing glioma cells demonstrated that binding of liposomes to the extracellular matrix relied essentially on the sulfatide-tenascin-C interaction. Following binding to the extracellular matrix, the sulfatide-containing liposomes were internalized via both caveolae/lipid raft- and clathrin-dependent pathways, which would ensure direct cytoplasmic release of the cargoes carried in the liposomes. Such natural lipid-guided intracellular delivery targeting at the extracellular matrix glycoproteins of tumor cells thus opens a new direction for development of more effective anticancer chemotherapeutics in future.

Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.

Neurotoxic, redox-competent Alzheimer's --amyloid is released from lipid membrane by methionine oxidation

The amyloid β peptide is toxic to neurons, and it is believed that this toxicity plays a central role in the progression of Alzheimer's disease. The mechanism of this toxicity is contentious. Here we report that an Aβ peptide with the sulfur atom of Met-35 oxidized to a sulfoxide (Met(O)Aβ) is toxic to neuronal cells, and this toxicity is attenuated by the metal chelator clioquinol and completely rescued by catalase implicating the same toxicity mechanism as reduced Aβ. However, unlike the unoxidized peptide, Met(O)Aβ is unable to penetrate lipid membranes to form ion channel-like structures, and β-sheet formation is inhibited, phenomena that are central to some theories for Aβ toxicity. Our results show that, like the unoxidized peptide, Met(O)Aβ will coordinate Cu₂₊ and reduce the oxidation state of the metal and still produce H2O2. We hypothesize that Met(O)Aβ production contributes to the elevation of soluble Aβ seen in the brain in Alzheimer's disease.

Direct measurement of the force between two lipid bilayers and observation of their fusion

The force between twophosphatidylcholine bilayers is measured as a function of their separation, showing a strong hydration repulsion at short range, as previously reported by LeNeveu et al. (LeNeveu, D.M., Rand, R.P., Parsegian, V.A. and Gingell, D. (1977) (Biophys. J. 18, 209-230). The experimental technique also allows direct observation of the molecular process by which two bilayers fuse into one.

First-principles based force-field for the interaction of proteins with Au(100)(5 x 1): an extension of GolP-CHARMM

Noncovalent recognition between peptides and inorganic materials is an established phenomenon. Key to exploiting these interactions in a wide range of materials self-assembly applications would be to harness the facet-selective control of peptide binding onto these materials. Fundamental understanding of what drives facet-selectivity in peptide binding is developing, but as yet is not sufficient to enable design of predictable facet-specific sequences. Computational simulation of the aqueous peptide-gold interface, commonly used to understand the mechanisms driving adsorption at an atomic level, has thus far neglected the role that surface reconstruction might play in facet specificity. Here the polarizable GolP-CHARMM suite of force fields is extended to include the reconstructed Au(100) surface. The force field, compatible with the bio-organic force field CHARMM, is parametrized using first-principles data. Our extended force field is tailored to reproduce the heterogeneity of weak chemisorbing N and S species to specific locations in the Au(100)(5 × 1) surface identified from the first-principles calculations. We apply our new model to predict and compare the three-dimensional structure of liquid water at Au(111), Au(100)(1 × 1), and Au(100)(5 × 1) interfaces. Using molecular dynamics simulations, we predict an increased likelihood for water-mediated peptide adsorption at the aqueous-Au(100)(1 × 1) interface compared with the Au(100)(5 × 1) interface. Therefore, our findings suggest that peptide binding can discriminate between the native and reconstructed Au(100) interfaces and that the role of reconstruction on binding at the Au(100) interface should not be neglected. © 2013 American Chemical Society.

Comparative study of materials-binding peptide interactions with gold and silver surfaces and nanostructures : A thermodynamic basis for biological selectivity of inorganic materials

Controllable 3D assembly of multicomponent inorganic nanomaterials by precisely positioning two or more types of nanoparticles to modulate their interactions and achieve multifunctionality remains a major challenge. The diverse chemical and structural features of biomolecules can generate the compositionally specific organic/inorganic interactions needed to create such assemblies. Toward this aim, we studied the materials-specific binding of peptides selected based upon affinity for Ag (AgBP1 and AgBP2) and Au (AuBP1 and AuBP2) surfaces, combining experimental binding measurements, advanced molecular simulation, and nanomaterial synthesis. This reveals, for the first time, different modes of binding on the chemically similar Au and Ag surfaces. Molecular simulations showed flatter configurations on Au and a greater variety of 3D adsorbed conformations on Ag, reflecting primarily enthalpically driven binding on Au and entropically driven binding on Ag. This may arise from differences in the interfacial solvent structure. On Au, direct interaction of peptide residues with the metal surface is dominant, while on Ag, solvent-mediated interactions are more important. Experimentally, AgBP1 is found to be selective for Ag over Au, while the other sequences have strong and comparable affinities for both surfaces, despite differences in binding modes. Finally, we show for the first time the impact of these differences on peptide mediated synthesis of nanoparticles, leading to significant variation in particle morphology, size, and aggregation state. Because the degree of contact with the metal surface affects the peptide's ability to cap the nanoparticles and thereby control growth and aggregation, the peptides with the least direct contact (AgBP1 and AgBP2 on Ag) produced relatively polydispersed and aggregated nanoparticles. Overall, we show that thermodynamically different binding modes at metallic interfaces can enable selective binding on very similar inorganic surfaces and can provide control over nanoparticle nucleation and growth. This supports the promise of bionanocombinatoric approaches that rely upon materials recognition.

Peptide-based biosensors

Peptides have been used as components in biological analysis and fabrication of novel biosensors for a number of reasons, including mature synthesis protocols, diverse structures and as highly selective substrates for enzymes. Bio-conjugation strategies can provide an efficient way to convert interaction information between peptides and analytes into a measurable signal, which can be used for fabrication of novel peptide-based biosensors. Many sensitive fluorophores can respond rapidly to environmental changes and stimuli manifest as a change in spectral characteristics, hence environmentally-sensitive fluorophores have been widely used as signal markers to conjugate to peptides to construct peptide-based molecular sensors. Additionally, nanoparticles, fluorescent polymers, graphene and near infrared dyes are also used as peptide-conjugated signal markers. On the other hand, peptides may play a generalist role in peptide-based biosensors. Peptides have been utilized as bio-recognition elements to bind various analytes including proteins, nucleic acid, bacteria, metal ions, enzymes and antibodies in biosensors. The selectivity of peptides as an enzymatic substrate has thus been utilized to construct enzyme sensors or enzyme-activity sensors. In addition, progress on immobilization and microarray techniques of peptides has facilitated the progress and commercial application of chip-based peptide biosensors in clinical diagnosis.