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BBA - Biomembranes (v.1828, #5)
Structural diversity and mode of action on lipid membranes of three lactoferrin candidacidal peptides by Tânia Silva; Adao Regina Adão; Kamran Nazmi; Jan G.M. Bolscher; Sérgio S. Funari; Uhrikova Daniela Uhríková; Margarida Bastos (pp. 1329-1339).
The structure and membrane interactions of three antimicrobial peptides from the lactoferrin family were investigated through different techniques. Circular dichroism shows that the peptides adopt a secondary structure in the presence of DMPC/DMPG, and DSC reveals that they all interact with these membranes, albeit differently, whereas only LFchimera has an effect in pure zwitterionic membranes of DMPC. DSC further shows that membrane action is weakest for LFcin17-30, increases for LFampin265-284 and is largest for LFchimera. These differences are clearly reflected in a different structure upon interaction, as revealed by SAX. This technique shows that LFcin17-30 only induces membrane segregation (two lamellar phases are apparent upon cooling from fluid phase), whereas LFampin265-284 induces micellization of the membrane with structure compatible to a micellar cubic phase of space group Pm3n, and LFchimera leads to membrane destruction through the formation of two cubic phases, Pn3m and Im3m. These structural results show a remarkable parallel with the ones obtained previously by freeze fracture microscopy of the effect of these peptides against Candida albicans.Display Omitted► Three peptides from the lactoferrin family interact differently with model membranes. ► DSC, CD and SAXD results taken together help to differentiate their mode of action. ► SAXD shows a different structure for each peptide/membrane assembly. ► The peptide's effect on model membranes and C. albicans show remarkable agreement.
Keywords: Abbreviations; AMPs; antimicrobial peptides; CD; circular dichroism; DMPC; dimyristoylphosphatidylcholine; DMPG; dimyristoylphosphatidylglycerol; DSC; differential scanning calorimetry; IMPs; intra-membranous particles; LC; 50; lethal concentration that causes 50% of cell death; LFampin265-284; lactoferrampin 265–284; LFchimera; LFcin17-30-K-LFampin265-284; LFcin17-30; lactoferricin 17–30; LUVs; large unilamellar vesicles; MLVs; multilamellar vesicles; P:L; peptide-to-lipid molar ratio; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; SAXD; small angle X-ray diffraction; WAXD; wide angle X-ray diffractionAntimicrobial peptides; Lactoferrin peptides; DSC; CD; SAXD
Effect of phospholipid composition on discoidal HDL formation by Masakazu Miyazaki; Yoko Tajima; Yasushi Ishihama; Tetsurou Handa; Minoru Nakano (pp. 1340-1346).
Discoidal high-density lipoprotein (HDL) particles are known to fractionalize into several discrete populations. Factors regulating their size are, however, less understood. To reveal the effect of lipid composition on their formation and characteristics, we prepared several reconstituted HDLs (rHDLs) with 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoserine (POPS), 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE), and sphingomyelin at phospholipid to apolipoprotein A-I ratios of 100 and 25. When reconstitution was conducted at 37°C, the efficiency of rHDL formation from POPC was decreased as compared with that conducted at 4°C. Moreover, large rHDLs with a Stokes diameter of 9.6nm became dominant over small rHDL with a diameter of 7.9nm, which was distinctly observed at 4°C. The aminophospholipids POPS and POPE promoted the formation of small rHDLs at 37°C, but fluorescence experiments revealed that they did so in a different fashion: Fluorescence lifetime data suggested that the head group of POPS reduces hydrophobic hydration, especially in small rHDLs, suggesting that this lipid stabilizes the saddle-shaped bilayer structure in small rHDLs. Fluorescence lifetime and anisotropy data showed that incorporation of POPE increases acyl chain order and water penetration into the head group region in large rHDLs, suggesting that POPE destabilizes the planar bilayer structure. These results imply that these aminophospholipids contribute to the formation of small rHDLs under biological conditions.Display Omitted► HDLs were reconstituted with several lipids. ► Efficiency of small rHDL formation from POPC was decreased at 37°C than at 4°C. ► POPS stabilized the saddle-shaped bilayer structure in small rHDLs and promoted the small rHDL formation. ► POPE destabilizes the planar bilayer structure and promoted the small rHDL formation.
Keywords: Abbreviations; apoA-I; apolipoprotein A-I; C; 10; dipyPC; 1,2-bis(1-pyrenedecanoyl)-; sn; -glycero-3-phosphocholine; dansyl PE; dansyl phosphatidylethanolamine; DPH; 1,6-diphenyl-1,3,5-hexatriene; LPR; lipid-to-protein ratio; PL; phospholipid; rHDL; reconstituted HDL; Rho-DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl)Apolipoprotein A-I; Phosphatidylcholine; Phosphatidylethanolamine; Phosphatidylserine; Sphingomyelin
Biophysical changes induced by xenon on phospholipid bilayers by Ryan D. Booker; Amadeu K. Sum (pp. 1347-1356).
Structural and dynamic changes in cell membrane properties induced by xenon, a volatile anesthetic molecule, may affect the function of membrane-mediated proteins, providing a hypothesis for the mechanism of general anesthetic action. Here, we use molecular dynamics simulation and differential scanning calorimetry to examine the biophysical and thermodynamic effects of xenon on model lipid membranes. Our results indicate that xenon atoms preferentially localize in the hydrophobic core of the lipid bilayer, inducing substantial increases in the area per lipid and bilayer thickness. Xenon depresses the membrane gel–liquid crystalline phase transition temperature, increasing membrane fluidity and lipid head group spacing, while inducing net local ordering effects in a small region of the lipid carbon tails and modulating the bilayer lateral pressure profile. Our results are consistent with a role for nonspecific, lipid bilayer-mediated mechanisms in producing xenon's general anesthetic action.Display Omitted► We use MD and DSC to examine the effects of xenon on lipid membranes. ► Xenon atoms preferentially localize in the hydrophobic core of the lipid bilayer. ► Xenon induces substantial structural changes in the lipid bilayers. ► Xenon depresses the membrane gel–liquid crystalline phase transition temperature. ► Our results are consistent with a lipid bilayer-mediated mechanism of anesthesia.
Keywords: General anesthesia; Lipid membrane; Phase transition; Simulation; Molecular dynamics
The electrical response of bilayers to the bee venom toxin melittin: Evidence for transient bilayer permeabilization by Gregory Wiedman; Katherine Herman; Peter Searson; William C. Wimley; Kalina Hristova (pp. 1357-1364).
Melittin is a 26-residue bee venom peptide that folds into amphipathic α-helix and causes membrane permeabilization via a mechanism that is still disputed. While an equilibrium transmembrane pore model has been a central part of the mechanistic dialogue for decades, there is growing evidence that a transmembrane pore is not required for melittin's activity. In part, the controversy is due to limited experimental tools to probe the bilayer's response to melittin. Electrochemical impedance spectroscopy (EIS) is a technique that can reveal details of molecular mechanism of peptide activity, as it yields direct, real-time measurements of membrane resistance and capacitance of supported bilayers. In this work, EIS was used in conjunction with vesicle leakage studies to characterize the response of bilayers of different lipid compositions to melittin. Experiments were carried out at low peptide to lipid ratios between 1:5000 and 1:100. The results directly demonstrate that the response of the bilayer to melittin at these concentrations cannot be explained by an equilibrium transmembrane pore model.Display Omitted► Melittin was studied by electrochemical impedance spectroscopy. ► Mellitin causes a drop in membrane resistance, followed by recovery. ► Melittin does not alter the bilayer capacitance. ► The results are not consistent with a simple transmembrane pore model.
Keywords: Melittin; Peptide; Pore; Impedance spectroscopy; Protein–lipid interactions
New generation of efficient peptide-based vectors, NickFects, for the delivery of nucleic acids by Piret Arukuusk; Parnaste Ly Pärnaste; Nikita Oskolkov; Dana-Maria Copolovici; Helerin Margus; Kärt Padari; Moll Kaidi Möll; Julia Maslovskaja; Radi Tegova; Gaily Kivi; Andres Tover; Margus Pooga; Mart Ustav; Ülo Langel (pp. 1365-1373).
Harnessing of a branched structure is a novel approach in the design of cell-penetrating peptides and it has provided highly efficient transfection reagents for intracellular delivery of nucleic acids. The new stearylated TP10 analogs, NickFects, condense plasmid DNA, splice correcting oligonucleotides and short interfering RNAs into stable nanoparticles with a size of 62–160nm. Such nanoparticles have a negative surface charge (−11 to −18mV) in serum containing medium and enable highly efficient gene expression, splice correction and gene silencing. One of the novel peptides, NickFect51 is capable of transfecting plasmid DNA into a large variety of cell lines, including refractory suspension and primary cells and in several cases exceeds the transfection level of commercially available reagent Lipofectamine™ 2000 without any cytotoxic side effects. Additionally we demonstrate the advantages of NickFect51 in a protein production system, QMCF technology, for expression and production of recombinant proteins in hardly transfectable suspension cells.Display Omitted► A novel approach in the design of cell-penetrating peptides. ► Efficient peptide-based non-toxic and versatile delivery vector for nucleic acids. ► Transfects pDNA into suspension and primary cells. ► Applicable in protein production.
Keywords: Abbreviations; CPP; cell-penetrating peptide; CR; charge ratio; DLS; dynamic light scattering; DIEA; diisopropylethylamine; EGFP; enhanced green fluorescent protein; HBTU; O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate; HOBT; hydroxybenzotriazole; LF2000; Lipofectamine™ 2000; FACS; fluorescence activated cell sorter; NF; NickFects; pGL3; luciferase expressing plasmid; pDNA; plasmid DNA; SCO; splice-correcting oligonucleotide; siRNA; small interfering RNA; TP10; transportan 10; TEM; transmission electron microscopyPlasmid transfection; Cell-penetrating peptide; Splice-correction; Gene silencing; Protein production; QMCF technology
Alternative cycling modes of the Na+/K+-ATPase in the presence of either Na+ or Rb+ by José L.E. Monti; Mónica R. Montes; Rolando C. Rossi (pp. 1374-1383).
A comprehensive study of the interaction between Na+ and K+ with the Na+/K+-ATPase requires dissecting the incidence of alternative cycling modes on activity measurements in which one or both of these cations are absent. With this aim, we used membrane fragments containing pig-kidney Na+/K+-ATPase to perform measurements, at 25°C and pH=7.4, of ATPase activity and steady-state levels of (i) intermediates containing occluded Rb+ at different [Rb+] in media lacking Na+, and (ii) phosphorylated intermediates at different [Na+] in media lacking Rb+. Most relevant results are: (1) Rb+ can be occluded through an ATPasic cycling mode that takes place in the absence of Na+ ions, (2) the kinetic behavior of the phosphoenzyme formed by ATP in the absence of Na+ is different from the one that is formed with Na+, and (3) binding of Na+ to transport sites during catalysis is not at random unless rapid equilibrium holds.Display Omitted► We present steady-state determinations of reaction intermediates and ATPase activity. ► We show occlusion of Rb+ (a K+ congener) during a cycling mode taking place at null [Na+]. ► Phosphoenzyme formed by ATP at null [Na+] differs from the one obtained with Na+. ► Binding of Na+ to transport sites during catalysis is not at random unless rapid equilibrium holds. ► We present a minimal kinetic model that reproduces the results.
Keywords: Na; +; /K; +; -ATPase; Steady-state kinetics; Phosphoenzyme; Rb; +; -occlusion; ATPase activity; Minimal model
Disrupting membrane raft domains by alkylphospholipids by A.B. Gomide; Thome C.H. Thomé; G.A. dos Santos; G.A. Ferreira; Faca V.M. Faça; E.M. Rego; L.J. Greene; R.G. Stabeli; P. Ciancaglini; R. Itri (pp. 1384-1389).
Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100μM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100μM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)–liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14–18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane.Display Omitted► ODPC disrupts raft domains. ► Alkylphospholipids act on lipid raft domains. ► Raft disruption by ALPS may be correlated with their toxicity upon neoplastic cells.
Keywords: Alkylphospholipid; Giant vesicles; Model membranes; Ld–Lo lipid domains; Lipid rafts
2NH and 3OH are crucial structural requirements in sphingomyelin for sticholysin II binding and pore formation in bilayer membranes by Terhi Maula; Y. Jenny E. Isaksson; Garcia-Linares Sara García-Linares; Sanna Niinivehmas; Pentikainen Olli T. Pentikäinen; Mayuko Kurita; Shou Yamaguchi; Tetsuya Yamamoto; Shigeo Katsumura; José G. Gavilanes; Martinez-del-Pozo Álvaro Martínez-del-Pozo; J. Peter Slotte (pp. 1390-1395).
Sticholysin II (StnII) is a pore-forming toxin from the sea anemone Stichodactyla heliantus which belongs to the large actinoporin family. The toxin binds to sphingomyelin (SM) containing membranes, and shows high binding specificity for this lipid. In this study, we have examined the role of the hydrogen bonding groups of the SM long-chain base (i.e., the 2NH and the 3OH) for StnII recognition. We prepared methylated SM-analogs which had reduced hydrogen bonding capability from 2NH and 3OH. Both surface plasmon resonance experiments, and isothermal titration calorimetry measurements indicated that StnII failed to bind to bilayers containing methylated SM-analogs, whereas clear binding was seen to SM-containing bilayers. StnII also failed to induce calcein release (i.e., pore formation) from vesicles made to contain methylated SM-analogs, but readily induced calcein release from SM-containing vesicles. Molecular modeling of SM docked to the phosphocholine binding site of StnII indicated that the 2NH and 3OH groups were likely to form a hydrogen bond with Tyr135. In addition, it appeared that Tyr111 and Tyr136 could donate hydrogen bonds to phosphate oxygen, thus stabilizing SM binding to the toxin. We conclude that the interfacial hydrogen bonding properties of SM, in addition to the phosphocholine head group, are crucial for high-affinity SM/StnII-interaction.Display Omitted► The structural requirements for SM binding to StnII were explored. ► StnII did not bind to bilayers in which SM hydrogen bonding was compromised. ► Molecular docking of SM with StnII revealed the likely residues for interactions.
Keywords: Abbreviations; EqtII; equinatoxin II; ITC; isothermal titration calorimetry; LUV; large unilamellar vesicle; pbSM; procine brain sphingomyelin; POC; phosphocholine (binding site); POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; PSM; palmitoyl SM; SM; sphingomyelin; pbSM; porcine brain SM; sSM; truncated SM analog used for docking optimization; SPR; surface plasmon resonance; StnII; sticholysin IIMolecular docking; Membrane permeabilization; Isothermal titration calorimetry; Surface plasmon resonance
Targeting gemcitabine containing liposomes to CD44 expressing pancreatic adenocarcinoma cells causes an increase in the antitumoral activity by Elisa Dalla Pozza; Carlotta Lerda; Chiara Costanzo; Massimo Donadelli; Ilaria Dando; Elisa Zoratti; Maria Teresa Scupoli; Stefania Beghelli; Aldo Scarpa; Elias Fattal; Silvia Arpicco; Marta Palmieri (pp. 1396-1404).
Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.Display Omitted► CD44-mediated uptake of hyaluronated liposomes into cells ► Lipid-raft mediated uptake of hyaluronated liposomes into CD44 expressing cells ► In vitro and in vivo inhibition of cell proliferation by hyaluronated liposomes ► Differential effects of 4.8kDa MW or 12kDa MW hyaluronic acid charged liposomes.
Keywords: CD44; Hyaluronic acid-conjugated liposomes; Pancreatic cancer; Gemcitabine; Endocytosis; Lipid-raft
The role of membrane fatty acid remodeling in the antitumor mechanism of action of 2-hydroxyoleic acid by Maria Laura Martin; Barcelo-Coblijn Gwendolyn Barceló-Coblijn; Rodrigo F.M. de Almeida; Noguera-Salva Maria Antònia Noguera-Salvà; Teres Silvia Terés; Mónica Higuera; Gerhard Liebisch; Gerd Schmitz; Xavier Busquets; Escriba Pablo V. Escribá (pp. 1405-1413).
The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is a potent antitumor drug that we rationally designed to regulate the membrane lipid composition and structure. The lipid modifications caused by 2OHOA treatments induce important signaling changes that end up with cell death (Terés et al., 2012 [1]). One of these regulatory effects is restoration of sphingomyelin levels, which are markedly lower in cancer cells compared to normal cells (Barceló‐Coblijn et al., 2011 [2]). In this study, we report another important regulatory effect of 2OHOA on cancer cell membrane composition: a large increase in 2OHOA levels, accounting for ~15% of the fatty acids present in membrane phospholipids, in human glioma (SF767 and U118) and lung cancer (A549) cells. Concomitantly, we observed marked reductions in oleic acid levels and inhibition of stearoyl-CoA desaturase. The impact of these changes on the biophysical properties of the lipid bilayer was evaluated in liposomes reconstituted from cancer cell membrane lipid extracts. Thus, 2OHOA increased the packing of ordered domains and decreased the global order of the membrane. The present results further support and extend the knowledge about the mechanism of action for 2OHOA, based on the regulation of the membrane lipid composition and structure and subsequent modulation of membrane protein-associated signaling.Display Omitted► 2-Hydroxyoleic acid (2OHOA) caused a dramatic fatty acid remodeling in tumor cells. ► After 72h treatment, 2OHOA accounted for 15% of total fatty acids in phospholipids. ► SCD1 inhibition and 2OHOA incorporation accounted for the decrease in oleic acid. ► 2OHOA decreased membrane global order but increased the packing of ordered domains. ► These findings provide a new insight into the 2OHOA antitumor mechanism of action.
Keywords: Abbreviations; 2OHOA; 2-hydroxyoleic acid; DAG; diacylglycerol; DPH; 1,6-diphenyl-1,3,5-hexatriene; FAME; fatty acid methyl ester; HPTLC; high performance TLC; LUV; large unilamellar vesicle; MLV; multilamellar vesicle; MUFA; monounsaturated fatty acid; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; PS; phosphatidylserine; SCD1; stearoyl-CoA desaturase-1; SM; sphingomyelin; SMS; sphingomyelin synthase; TAG; triacylglycerol; t; -PnA; trans; -parinaric acidSphingolipid; Membrane lipid therapy; Cancer; Stearoyl-CoA desaturase
Corrigendum to “Membrane interactions of ionic liquids: Possible determinants for biological activity and toxicity” [Biochim. Biophys. Acta 1818 (2012) 2967–2974]
by Noga Gal; Danilo Malferrari; Sofiya Kolusheva; Paola Galletti; Emilio Tagliavini; Raz Jelinek (pp. 1414-1414).