Pleiotropic effects of statins via interaction with the lipid bilayer: A combined approach.

Statins are effective inhibitors of cholesterol biosynthesis, largely used for prevention of cardiovascular diseases induced by hypercholesterolemia. However, their use in different clinical trials clearly indicate that the general benefits observed with statins are also related to effects beyond the cholesterol lowering. Increasing evidences suggest that some of these cholesterol-independent or "pleiotropic" effects of statins involve the interaction and modification of the membrane bilayers. In this manuscript, using a combined approach based on biophysical (electrochemical impedance spectroscopy on tethered bilayer lipid membranes) and biological methods (hemolysis on erythrocytes and immunocytochemistry on cancer cells), we demonstrate that lipophilic, but not hydrophilic statins are capable of reducing the damage caused by cholesterol-dependent cytolysins. This protection correlates with statins lipophilicity and capacity to interact with the lipid bilayer. Our data suggests lipophilic statins associate with membranes and interfere with the ability of cholesterol dependent cytolysins, to bind to membrane cholesterol. Evaluation of the capacity of statins to modulate cell membrane properties is essential for developing a correct therapeutic approach for cardiovascular diseases as well as for understanding the potential of this class of drugs as adjuvants for drug delivery.

The seafood Musculus senhousei shows anti-influenza A virus activity by targeting virion envelope lipids.

Marine environments are known to be a new source of structurally diverse bioactive molecules. In this paper, we identified a porphyrin derivative of Pyropheophorbide a (PPa) from the mussel Musculus senhousei (M. senhousei) that showed broad anti-influenza A virus activity in vitro against a panel of influenza A viral strains. The analysis of the mechanism of action indicated that PPa functions in the early stage of virus infection by interacting with the lipid bilayer of the virion, resulting in an alteration of membrane-associated functions, thereby blocking the entry of enveloped viruses into host cells. In addition, the anti-influenza A virus activity of PPa was further assessed in mice infected with the influenza A virus. The survival rate and mean survival time of mice were apparently prolonged compared with the control group which was not treated with the drug. Therefore, PPa and its derivatives may represent lead compounds for controlling influenza A virus infection.

Synergy between plant phenols and carotenoids in stabilizing lipid-bilayer membranes of giant unilamellar vesicles against oxidative destruction.

We have investigated the synergism between plant phenols and carotenoids in protecting the phosphatidylcholine (PC) membranes of giant unilamellar vesicles (GUVs) from oxidative destruction, for which chlorophyll-a (Chl-a) was used as a lipophilic photosensitizer. The effect was examined for seven different combinations of ß-carotene (ß-CAR) and plant phenols. The light-induced change in GUV morphology was monitored via conventional optical microscopy, and quantified by a dimensionless image-entropy parameter, ΔE. The ΔE-t time evolution profiles exhibiting successive lag phase, budding phase and ending phase could be accounted for by a Boltzmann model function. The length of the lag phase (LP in s) for the combination of syringic acid and ß-CAR was more than seven fold longer than for ß-CAR alone, and those for other different combinations followed the order: salicylic acid < vanillic acid < syringic acid > rutin > caffeic acid > quercetin > catechin, indicating that moderately reducing phenols appeared to be the most efficient membrane co-stabilizers. The same order held for the residual contents of ß-CAR in membranes after light-induced oxidative degradation as determined by resonance Raman spectroscopy. The dependence of LP on the reducing power of phenols coincided with the Marcus theory plot for the rate of electron transfer from phenols to the radical cation ß-CAR˙+ as a primary oxidative product, suggesting that the plant phenol regeneration of ß-CAR plays an important role in stabilizing the GUV membranes, as further supported by the involvement of CAR˙+ and the distinct shortening of its lifetime as shown by transient absorption spectroscopy.

Life During Wartime: A Personal Recollection of the Circa 1990 Prestegard Lab and Its Contributions to Membrane Biophysics.

A subjective account is presented of challenges and excitement of being a postdoctoral trainee in the lab of James H. Prestegard at Yale University in New Haven, Connecticut from 1989 to 1991. This includes accounts of the early development of bicelles and of oriented sample NMR results that contributed to our modern understanding of the properties of the water-lipid interface of disordered phase biological membranes.

Synthesis and Characterization of Peptide-Chitosan Conjugates (PepChis) with Lipid Bilayer Affinity and Antibacterial Activity.

Antimicrobial peptides appear among innovative biopolymers with potential therapeutic interest. Nevertheless, issues concerning efficiency, production costs, and toxicity persist. Herein, we show that conjugation of peptides with chitosans can represent an alternative in the search for these needs. To increase solubility, deacetylated and degraded chitosans were prepared. Then, they were functionalized via N-succinimidyl- S-acetylthiopropionate or via glutathione (GSH), an endogenous peptide linker. To the best of our knowledge, it is the first time that GSH is used as a thiolating agent for the conjugation of peptides. Next, thiolated chitosans were conjugated through a disulfide bond with designed short-chain peptides, one of them derived from the antimicrobial peptide Jelleine-I. Conjugates and respective reaction intermediates were characterized by absorciometry, attenuated total reflectance-Fourier transform infrared, and 1H NMR. Zeta potential measurements showed the cationic nature of these biomacromolecules and their preferential partitioning to Gram-positive bacterial-like model membranes. In vitro investigation using representative Gram-positive and -negative bacteria ( Staphylococcus aureus and Escherichia coli, respectively) showed that the conjugation strategies lead to enhanced activity in relation to the unconjugated peptide and to the unconjugated chitosan. The obtained products showed selectivity toward S. aureus at low cytotoxicity as determined in NIH/3T3 cells. Overall, our study demonstrates that an appropriate choice of antimicrobial peptide and chitosan characteristics leads to increased antimicrobial activity of the conjugated product and represents a strategy to modulate the activity and selectivity of antimicrobials resorting to low-cost chemicals. The present proposal starts from less expensive raw materials (chitosan and short-chain peptide), is based on aqueous solvents, and minimizes the use of reactants with a higher environmental impact. The final biopolymer contains the backbone of chitosan, just 3-6% peptide derived from royal jelly and GSH, all of them considered safe for human use or as a physiological molecule.

Peridinin Is an Exceptionally Potent and Membrane-Embedded Inhibitor of Bilayer Lipid Peroxidation.

Antilipoperoxidant protein dysfunction is associated with many human diseases, suggesting that bilayer lipid peroxidation may contribute broadly to pathogenesis. Small molecule inhibitors of this membrane-localized chemistry could in theory enable better understanding and/or treatment of such diseases, but currently available compounds have important limitations. Many biological questions thus remain unanswered, and clinical trials have largely been disappointing. Enabled by efficient, building block-based syntheses of three atypical carotenoid natural products produced by microorganisms that thrive in environments of extreme oxidative stress, we found that peridinin is a potent inhibitor of nonenzymatic bilayer lipid peroxidation in liposomes and in primary human endothelial cells. We also found that peridinin blocks monocyte-endothelial cell adhesion, a key step in atherogenesis. A series of frontier solid-state NMR experiments with a site-specifically 13C-labeled isotopolog synthesized using the same MIDA boronate building block-based total synthesis approach revealed that peridinin is completely embedded within and physically spans the hydrophobic core of POPC membranes, maximizing its effective molarity at the site of the targeted lipid peroxidation reactions. Alternatively, the widely used carotenoid astaxanthin is significantly less potent and was found to primarily localize extramembranously. Peridinin thus represents a promising and biophysically well-characterized starting point for the development of small molecule antilipoperoxidants that serve as more effective biological probes and/or therapeutics.

Toxicity of lupane derivatives on anionic membrane models, isolated rat mitochondria and selected human cell lines: Role of terminal alkyl chains.

Triterpenoids have multiple biological properties, although little information is available regarding their toxicity. The present study evaluates the toxicity of two new synthetic lupane derivatives using distinct biological models including synthetic lipids membranes, isolated liver and heart mitochondria fractions, and cell lines in culture. The two novel triterpenoids caused perturbations in the organization of synthetic lipid bilayers, leading to changes in membrane fluidity. Inhibition of cell proliferation and mitochondrial and nuclear morphological alterations were also identified. Inhibition of mitochondrial oxygen consumption, transmembrane electric potential depolarization and induction of the mitochondrial permeability transition pore was observed, although effects on isolated mitochondrial fractions were tissue-dependent (e.g. liver vs. heart). The size and length of hydrocarbon chains in the two molecules appear to be determinant for the degree of interaction with mitochondria, especially in the whole cell environment, where more barriers for diffusion exist. The results suggest that the positively charged triterpenoids target mitochondria and disrupt bioenergetics.

The permeability enhancing mechanism of menthol on skin lipids: a molecular dynamics simulation study.

The stratum corneum (SC), the outermost layer of skin, represents the primary barrier to molecules penetrating the skin. Menthol is widely used in clinical medicine as a penetration enhancer due to its high efficiency and relative safety. In this study, molecular dynamics simulations have been performed to investigate the effect of menthol molecules on the structural and permeability of both single component and ternary mixed bilayers. The lipid matrix is modeled as pure ceramide (CER2) or as a 2:2:1 mixture of CER2, cholesterol (CHOL), and free fatty acid (FFA). The effect of menthol on the SC bilayer was investigated at various concentrations of menthol. For both models, the area per lipid decreases and the membrane thickness increases with increased menthol concentration, which may be due to the fact that menthol molecules penetrate into the bilayer and aggregate at the bilayer center. As for ternary mixed bilayer at high concentration, the lipids rearranged, and one more layer formed inside the former two leaflets. Our simulation results are consistent with the experimental evidence that high concentrations of menthol fluidize the SC lipids and enhance permeability. The penetration enhancement of menthol may take place through direct interactions with lipids rather than by forming water pores. Graphical abstract The effect of menthol on the structural and permeability of skin lipids was investigated using a molecular dynamics simulation method. Increased menthol concentration makes the area per lipid decrease and the membrane thickness increase. Our results show that the penetration enhancement of menthol may take place through direct interactions with lipids.

Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes.

Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.

²H solid-state nuclear magnetic resonance investigation of whole Escherichia coli interacting with antimicrobial peptide MSI-78.

A key aspect of the activity of antimicrobial peptides (AMPs) is their interaction with membranes. Efforts to elucidate their detailed mechanisms have focused on applying biophysical methods, including nuclear magnetic resonance (NMR), to AMPs in model lipid systems. However, these highly simplified systems fail to capture many of the features of the much more complex cell envelopes with which AMPs interact in vivo. To address this issue, we have designed a procedure to incorporate high levels of (2)H NMR labels specifically into the cell membrane of Escherichia coli and used this approach to study the interactions between the AMP MSI-78 and the membranes of intact bacteria. The (2)H NMR spectra of these membrane-deuterated bacteria can be reproduced in the absence and presence of MSI-78. Because the (2)H NMR data provide a quantitative measure of lipid disorder, they directly report on the lipid bilayer disruption central to the function of AMPs, in the context of intact bacteria. Addition of MSI-78 to the bacteria leads to decreases in the order of the lipid acyl chains. The molar peptide:lipid ratios required to observe the effects of MSI-78 on acyl chain order are approximately 30 times greater than the ratios needed to observe effects in model lipid systems and approximately 100 times less than the ratios required to observe inhibition of cell growth in biological assays. The observations thus suggest that MSI-78 disrupts the bilayer even at sublethal AMP levels and that a large fraction of the peptide does not actually reach the inner membrane.

Quinacrine blocks PrP (106-126)-formed channels.

We investigated the action of the acridine derivative, quinacrine (QC), which has been shown to act as a noncompetitive channel inhibitor. The main effects of QC are voltage- and concentration-dependent changes in the kinetics of the prion protein fragment (PrP[106-126])-formed cation channels. The current-voltage relationships show that the maximal current (I) was not affected whereas the physiologically important mean current (I') was reduced as a result of changes in channel kinetics. These findings suggest that QC acts on the open state of the channels. The half-inhibitory concentration (IC50) for the dose-dependent effects of [QC]cis on the kinetic parameters of the PrP(106-126)-formed cation channel shows a reduction in the ratios Po(QC)/Po, Fo(QC)/Fo, and To(QC)/To, whereas Tc(QC)/Tc increases. Of these ratios, Po(QC)/Po was more sensitive than the others. The corresponding IC50 for these ratios were 51, 94, 86, and 250 microM QC, respectively. The QC-induced changes in the kinetic parameters were more apparent at positive voltages. IC50 values for Po were 95, 75, and 51 microM at +20, +80, and +140 mV, respectively. The fact that QC induced changes in the kinetics of this channel, although the conductance of the channel remained unchanged, indicates that QC may bind at the mouth of the channel via a mechanism known as fast channel block. The QC-induced changes in the kinetic parameters of this channel suggest that they are pathophysiologically significant because these channels could be the mechanisms by which amyloids induce membrane damage in vivo.

Factors determining vesicular lipid mixing induced by shortened constructs of influenza hemagglutinin.

The HA2 subunit of influenza hemagglutinin is responsible for fusion of the viral and host-cell membranes during infection. An N-terminal 127 amino acid construct of HA2, FHA2-127, is shown to induce lipid mixing of large unilamellar vesicles under endosomal low pH conditions. Thus, FHA2 could serve as a good model system for biophysical studies of membrane fusion. With FHA2, we began to develop a mechanistic model which could explain how this short construct facilitates membrane fusion. In this endeavor, we studied the possible role of the kinked loop region (amino acids 105-113). A construct missing this loop, FHA2-90, although able to induce lipid mixing, has lost the sharp pH-dependent transition seen with FHA2-127 and native HA. In addition, FHA2-127 promotes extensive vesicle aggregation more effectively than FHA2-90 upon acidification. These data suggest that the kinked loop may play a pH-dependent regulatory role. To test this, we compared bis-ANS binding to the two constructs and observed that binding to FHA2-127 increases at a faster rate than FHA2-90 as the pH is decreased, indicating that the kinked loop not only is an ANS-binding site, but that it binds better at low pH. The pH dependence of this transition directly correlates with that observed in lipid mixing. Further, cysteine mutations of acidic residues in the kinked region are both fusion inactive and bind much less ANS, whereas a similar mutation of a threonine residue had little effect on fusion activity or ANS binding. This evidence lends further support to our idea that the kinked loop serves a regulatory role. To test the physiological relevance of the FHA2-127 fusion mechanism, we studied the effects of a G1E mutation, known to abolish fusion in native HA. We found that G1E-127 is fusion inactive as expected. This evidence indirectly suggests that the mechanism of FHA2-127 is perhaps physiologically relevant and from its study, we can learn much about the mechanism of native HA.

ATP-dependent potassium channel from rat liver mitochondria: inhibitory analysis, channel clusterization.

An inhibitor of potassium mitochondrial channels quinidine (0.1 mM) closed the ATP-sensitive potassium channels isolated from mitochondria and reconstituted into a bilayer lipid membrane. Inhibitors of cytoplasm membrane K-channels (ouabain, tetraethyl ammonium, and cesium ions) produced no effect on the ATP-sensitive mitochondria K-channels; 0.5-2 microM glybenclamide did not affect the reconstituted channels, either. The multiplicity of jumps of conductivity at small concentration of the protein and the maintenance of ion selectivity at switching various levels of conductivity lead us to assume that the large channels are clusters of elementary channels. The average frequency of channel-cluster switchovers between various levels of conductivity is much higher than at the elementary channel.

Stability of the sperm plasma membrane of hibernating bats (Myotis velifer) compared with other mammals.

Previous experiments have established that the long-lived spermatozoa of hibernating bats are resistant to the acrosome reaction and fertilization in vitro conventional techniques. We tested the hypothesis that the membranes of these spermatozoa are more resistant to perturbation than those of other mammals. We exposed them to non-specific bilayer destabilizing agents and abrupt changes in incubation temperature and tested their response by observing their status (motility and viability) after a time interval compared with other mammals (golden hamster, rabbit, human). The results did not support the hypothesis. The inherent longevity of bat spermatozoa may thus be a function of some component other than unique resilience of their plasma membrane.

The study of lipid-protein interactions: effect of melittin on phase transition of phosphatidylethanolamine and sensitivity of phospholipases to phase state.

The effects of melittin on the bilayer-to-inverted hexagonal (HII) phase transition of egg phosphatidylethanolamine (EPE) and the influence of the phase state of membrane matrix on hydrolysis of EPE by phospholipases have been studied. The phase transitions were measured using the fluorescent probe N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) and differential scanning calorimetry. In the presence of melittin at a lipid-to-melittin molar ratio (R1) of 200, 100, and 20, the phase transition of EPE disappeared, indicating that melittin stabilizes the bilayer structure. In the presence of 10 mol% of cholesterol, the phase transition temperature (TH) decreased and TH was observed even in the presence of melittin at R1 of 200 and 100. The fluorescence intensity of the tryptophan residue of melittin is sensitive to the phase transition and the wavelength of emission maxima shift from 352 to 337 nm upon addition of EPE and EPE-cholesterol (10 mol%) at R1 of 200. Kinetic parameters for phospholipase-catalyzed hydrolysis of EPE in bilayer and HII phases showed that HII phase of EPE is a poorer substrate for phospholipases and that cholesterol decreases the susceptibility of EPE to phospholipases.