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BBA - Biomembranes (v.1778, #5)

Editorial Board (pp. ii).

Structural polymorphism of two CPP: An important parameter of activity by Sébastien Deshayes; Marc Decaffmeyer; Robert Brasseur; Annick Thomas (pp. 1197-1205).

Despite numerous investigations, the important structural features of Cell Penetrating Peptides (CPPs) remain unclear as demonstrated by the difficulties encountered in designing new molecules. In this study, we focused our interest on Penetratin and Transportan and several of their variants. Penetratin W48F and Penetratin W48F/W56F exhibit a reduced and a complete lack of cellular uptake, respectively; TP07 and TP10 present a similar cellular uptake as Transportan and TP08, TP13 and TP15 display no or weak internalization capacity. We applied the algorithmic method named PepLook to analyze the peptide polymorphism. The study reveals common conformational characteristics for the CPPs and their permeable variants: they all are polymorphic. Negative, non permeable, mutants share the opposite feature since they are monomorphic. Finally, we support the hypothesis that structural polymorphism may be crucial since it provides peptides with the possibility of adapting their conformation to medium hydrophobicity and or to partner diversity.

Keywords: Cell penetrating peptides; Carrier peptides; Transportan; Penetratin; PepLook; Structure; Polymorphism

Regulation of connexons composed of human connexin26 (hCx26) by temperature by Melanie Steffens; Friederike Göpel; Anaclet Ngezahayo; Carsten Zeilinger; Arne Ernst; Hans-Albert Kolb (pp. 1206-1212).

This report shows that temperature is a latent regulator of the voltage-dependent conductance of hemichannels composed of hCx26. The latter were expressed in Xenopus oocytes by injection of a mixture of hCx26 cRNA and antisense of endogenous Cx38 (anti-Cx38). At 24–25 °C, voltage clamp of oocytes at potentials above −40 mV evoked outward currents which were not observed in control oocytes. These currents were reversibly affected by change in temperature. Increasing temperature of the bath solution amplified gradually, whereas decreasing bath temperatures below 20 °C reduced the current. Furthermore analysis revealed that temperature-dependent increase of the conductance of the hemichannels did not correlate with a change of the apparent gating charge, whereas the half-activation voltage V_1/2 of the hemichannel was affected by a temperature change. It is proposed that this finding correlates with a temperature-dependent transition into an open state above 20 °C. In addition, a temperature-dependent release of Lucifer Yellow from loaded liposomes containing reconstituted purified and hCx26 hemichannels was observed, which indicate that a temperature-dependent regulation of the permeability of hCx26 hemichannels is not related to intracellular mediators. The involvement of temperature to modulate hemichannels as well as of the corresponding gap junction channel composed of hCx26 at physiological condition is discussed.

Keywords: Connexin26; Connexon; Gap junction; Electrophysiology; Voltage; Temperature; Recombinant connexon; Folding

Resonance energy transfer study of lysozyme–lipid interactions by Galyna P. Gorbenko; Valeriya M. Ioffe; Julian G. Molotkovsky; Paavo K.J. Kinnunen (pp. 1213-1221).

Resonance energy transfer (RET) between the tryptophan residues of lysozyme as donors and anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as acceptors has been examined to gain insight into molecular level details of the interactions of lysozyme with the lipid bilayers composed of PC with 10, 20, or 40 mol% PG. Energy transfer efficiency determined from the enhanced acceptor fluorescence was found to increase with content of the acidic lipid and surface coverage. The results of RET experiments performed with lipid vesicles containing 40 mol% PG were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for an interfacial location of the two predominant lysozyme fluorophores, Trp62 and Trp108, was obtained. The RET enhancement observed while employing AV-PG instead of AV-PC as an energy acceptor was interpreted as arising from the ability of lysozyme to bring about local demixing of the neutral and charged lipids in PC/PG model membranes.

Keywords: Resonance energy transfer; Lysozyme; Protein–lipid interaction; Tryptophan bilayer location; Lipid demixing

Effect of the headgroup variation on the gene transfer properties of cholesterol based cationic lipids possessing ether linkage by Avinash Bajaj; Santosh K. Mishra; Paturu Kondaiah; Santanu Bhattacharya (pp. 1222-1236).

Eight cholesterol based cationic lipids differing in the headgroup have been synthesized based on the ether linkage between the cationic headgroup and the cholesterol backbone. All the lipids formed stable suspensions in water. Transfection efficacies were examined in the absence and presence of serum using their optimized liposomal (lipid:DOPE) formulations. Our results showed that the transfection activities depend on the nature of the headgroup. Lipid bearing 4- N, N′-dimethylaminopyridine (DMAP) as headgroup showed the maximum transfection efficacy in the presence of serum. Importantly, the optimized formulation for this cationic lipid does not require DOPE, which is being used by most commercially available formulations. Cytotoxicity studies showed that the introduction of the positive charge decreases the cell viability of the cationic lipid formulations. Gel electrophoresis and Ethidium bromide exclusion assay revealed the different DNA binding abilities of formulations depending upon the headgroup of the cholesteryl lipid.

Keywords: Abbreviations; DC-Chol; 3β-[; N; ,(; N; ,; N; -dimethylaminoethane)-carbamoyl] cholesterol; DOPE; 1,2-dioleoyl-; l; -; α; -glycero-3-phosphatidylethanolamine; DOTMA; N; -[1-(2,3-dioleyloxy)propane]-; N; ,; N; ,; N; -trimethylammonium chloride; DOTAP; N; -[1-(2,3-dioleoyloxy)propane]-; N; ,; N; ,; N; -trimethylammonium chloride; MFI; mean fluorescence intensity; FACS; Fluorescence activated cell sortingCationic lipid; Liposome; Gene transfection; Serum

High yield cell-free production of integral membrane proteins without refolding or detergents by Jessica J. Wuu; James R. Swartz (pp. 1237-1250).

Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570±50 μg/mL and 130±30 μg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.

Keywords: Integral membrane protein; Transporter; Cell-free protein synthesis; In vitro protein synthesis

Cholesterol homeostasis in T cells. Methyl-β-cyclodextrin treatment results in equal loss of cholesterol from Triton X-100 soluble and insoluble fractions by Saleemulla Mahammad; Ingela Parmryd (pp. 1251-1258).

Methyl-β-cyclodextrin (MBCD) is frequently used to acutely deplete cells of cholesterol. A widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts and that sensitivity to MBCD is proof of lipid raft involvement in a cellular process. To analyse any MBCD preference systematically, progressive cholesterol depletion of Jurkat T cells was performed using MBCD and [³H]-cholesterol. It was found that at 37 °C, MBCD extracts similar proportions of cholesterol from the Triton X-100 resistant (lipid raft enriched) as it does from other cellular fractions and that the cells rapidly reestablish the relative differences in cholesterol concentration between different compartments. Moreover, cells restore the cholesterol level in the plasma membrane by mobilising cholesterol from intracellular cholesterol stores. Interestingly, mere incubation at 0 °C caused a loss of plasma membrane cholesterol with a concomitant increase in cholesteryl esters and adiposomes. Moreover, only 35% of total cholesterol could be extracted by MBCD at 0 °C and was accompanied by a complete loss of plasma membrane and endocytotic recycling centre filipin staining. This study clearly shows that MBCD does not specifically extract cholesterol from any cellular fraction, that cholesterol redistributes upon temperature changes and that intracellular cholesterol stores can be used to replenish plasma membrane cholesterol.

Keywords: Adiposomes; Cholesterol; Cholesteryl esters; Detergent resistant membranes; Filipin; Lipid rafts; Plasma membrane; Subcellular fractionation

Lysophosphatidylcholine–arbutin complexes form bilayer-like structures by M.A. Frías; B. Winik; M.B. Franzoni; P.R. Levstein; A. Nicastro; A.M. Gennaro; S.B. Diaz; E.A. Disalvo (pp. 1259-1266).

Arbutin is known to suppress melanin production in murine B16 melanoma cells and inhibit phospholipase action. This encourages the possibility to stabilize it in lipid aggregates for its administration in medical applications. Thus, it was of interest to demonstrate that monomyristoylphosphatidylcholine (14:0 lysoPC) and arbutin may form association complexes. This was studied by Electron Microscopy (EM),³¹P Nuclear Magnetic Resonance (³¹P NMR), Electronic Paramagnetic Resonance (EPR) and Fourier Transform Infrared Spectroscopy (FTIR). EM images show the formation of particles of c.a. 6 nm in diameter. For a 1:1 lysoPC–arbutin molar ratio³¹P NMR shows a spectrum with a shoulder that resembles the axially symmetric spectrum characteristic of vesicles. The addition of La³⁺ ions to the arbutin–lysoPC complex allows one to distinguish two phosphorous populations. These results suggest that arbutin–lysoPC forms vesicles with bilayers stabilized in an interdigitated array. FTIR spectroscopy shows that arbutin interacts with the hydrated population of the carbonyl groups and with the phosphates through the formation of hydrogen bonds. It is interpreted that hydrophobic interactions among the phenol group of arbutin and the acyl chain of lysoPC are responsible for the decrease in acyl chain mobility observed at the 5th C level by EPR. A model proposing the formation of interdigitated bilayers of arbutin-lysoPC could explain the experimental results.

Keywords: LysoPC; Arbutin; Interdigitation; FTIR interactions; ³¹; P NMR; Electron Microscopy; Electron Paramagnetic Resonance

Calcium influx into phospholipid vesicles caused by dynorphin neuropeptides by Loïc Hugonin; Vukojevic Vladana Vukojević; Georgy Bakalkin; Graslund Astrid Gräslund (pp. 1267-1273).

Dynorphins, endogeneous opioid peptides, function as ligands to the opioid kappa receptors but also induce non-opioid excitotoxic effects. Dynorphin A can increase the intra-neuronal calcium concentration through a non-opioid and non-NMDA mechanism. In this investigation, we show that big dynorphin, dynorphin A and to some extent dynorphin A (1–13), but not dynorphin B, allow calcium to enter into large unilamellar phospholipid vesicles with partly negative headgroups. The effects parallel the previously studied potency of dynorphins to translocate through biological membranes and to cause calcein leakage from large unilamellar phospholipid vesicles. There is no calcium ion influx into vesicles with zwitterionic headgroups. We have also investigated if the dynorphins can translocate through the vesicle membranes and estimated the relative strength of interaction of the peptides with the vesicles by fluorescence resonance energy transfer. The results show that dynorphins do not translocate in this membrane model system. There is a strong electrostatic contribution to the interaction of the peptides with the membrane model system.

Keywords: Abbreviations; CPP; cell penetrating peptide; Big Dyn; big dynorphin; Dyn A; Dynorphin A; Dyn A (1–13); dynorphin A (1–13); Dyn B; Dynorphin B; LUVs; large unilamellar phospholipid vesicles; POPC. Palmitoyl-2-oleoyl-phosphatidylcholine; POPG; 1-palmitoyl-2-oleoyl-phosphatidylglycerol; dansyl-DHPE; N; -(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine triethylammonium salt; EDTA; ethylenediaminetetraacetic acid; EGTA; ethylene glycol bis(8-aminoethyl ether)-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; FRET; fluorescence resonance energy transferDynorphin; Cell penetrating peptides; Phospholipid membranes; Unilamellar vesicles; Fluorescence; Calcium influx; Energy transfer

Effect of acylation on the interaction of the N-Terminal segment of pulmonary surfactant protein SP-C with phospholipid membranes by I. Plasencia; F. Baumgart; D. Andreu; D. Marsh; Perez-Gil J. Pérez-Gil (pp. 1274-1282).

SP-C, the smallest pulmonary surfactant protein, is required for the formation and stability of surface-active films at the air–liquid interface in the lung. The protein consists of a hydrophobic transmembrane α-helix and a cationic N-terminal segment containing palmitoylated cysteines. Recent evidence suggests that the N-terminal segment is of critical importance for SP-C function. In the present work, the role of palmitoylation in modulating the lipid–protein interactions of the N-terminal segment of SP-C has been studied by analyzing the effect of palmitoylated and non-palmitoylated synthetic peptides designed to mimic the N-terminal segment on the dynamic properties of phospholipid bilayers, recorded by spin-label electron spin resonance (ESR) spectroscopy. Both palmitoylated and non-palmitoylated peptides decrease the mobility of phosphatidylcholine (5-PCSL) and phosphatidylglycerol (5-PGSL) spin probes in dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglycerol (DPPG) bilayers. In zwitterionic DPPC membranes, both peptides have a greater effect at temperatures below than above the main gel-to-liquid-crystalline phase transition, the palmitoylated peptide inducing greater immobilisation of the lipid than does the non-palmitoylated form. In anionic DPPG membranes, both palmitoylated and non-palmitoylated peptides have similar immobilizing effects, probably dominated by electrostatic interactions. Both palmitoylated and non-palmitoylated peptides have effects comparable to whole native SP-C, as regards improving the gel phase solubility of phospholipid spin probes and increasing the polarity of the bilayer surface monitored by pK shifts of fatty acid spin probes. This indicates that a significant part of the perturbing properties of SP-C in phospholipid bilayers is mediated by interactions of the N-terminal segment. The effect of SP-C N-terminal peptides on the chain flexibility gradient of DPPC and DPPG bilayers is consistent with the existence of a peptide-promoted interdigitated phase at temperatures below the main gel-to-liquid-crystalline phase transition. The palmitoylated peptide, but not the non-palmitoylated version, is able to stably segregate interdigitated and non-interdigitated populations of phospholipids in DPPC bilayers. This feature suggests that the palmitoylated N-terminal segment stabilizes ordered domains such as those containing interdigitated lipids. We propose that palmitoylation may be important to promote and facilitate association of SP-C and SP-C-containing membranes with ordered lipid structures such as those potentially existing in highly compressed states of the interfacial surfactant film.

Keywords: Protein palmitoylation; Lipid–protein interactions; Interdigitated phase; Lipid domains; Monolayer; Surface tension

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro by Héctor Molina; Lorena Azocar; Meenakshisundaram Ananthanarayanan; Marco Arrese; Juan Francisco Miquel (pp. 1283-1291).

The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known.To determine whether the Na⁺/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains.Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na⁺-dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated.Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells.Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.

Keywords: Abbreviations; BS; bile salts; BSEP; bile salt export pump; Cav-1; caveolin-1; MR; membrane rafts; HEK 293; Human embryonic kidney 293; MCD; Methyl-β-Cyclodextrin; Ntcp; S; odium-; T; aurocholate; C; otransporting; P; olypeptideBile acid; Caveolin; Lipid microdomain; Lipid raft; Membrane transport

Flip-flop of hydroxy fatty acids across the membrane as monitored by proton-sensitive microelectrodes by Elena E. Pohl; Anna M. Voltchenko; Anne Rupprecht (pp. 1292-1297).

Hydroxyl group-containing fatty acids play an important role in anti-inflammatory action, neuroprotection, bactericide and anti-cancer defense. However, the mechanism of long-chain hydroxy fatty acids (HFA) transport across plasma membranes is still disputed. Two main hypotheses have been suggested: firstly, that protonated HFAs traverse across the membranes spontaneously and, secondly, that the transport is facilitated by proteinaceous carriers. Here, we demonstrate that the protonated HFA are able to move across planar lipid bilayers without protein assistance. This transport step is accompanied by the acidification of the buffer in receiving compartment and the pH augmentation in the donating compartment. The latter contained liposomes doped with HFA. As revealed by scanning pH-sensitive microelectrodes, the pH shift occurred only in the immediate vicinity of the membrane, while bulk pH remained unchanged. In concurrence with the theoretical model of weak acid transport, the pH value at maximum proton flux was almost equal to the pK of the studied HFA. Intrinsic p K_i values were calculated from the electrophoretic mobilities of HFA-containing liposomes and were 5.4, 6.5, 6.9 and 6.3 for 2-hydroxyhexadecanoic, 16-hydroxyhexadecanoic, 12-hydroxydodecanoic and 9,10,16-trihydroxyhexadecanoic acids, respectively.

Keywords: Abbreviations; BLM; bilayer lipid membrane; FA; long-chain fatty acids; HFA; long-chain hydroxy fatty acids; FH; protonated form of fatty acid; FA; ⁻; deprotonated form of fatty acid; USL; unstirred layer; PTFE; polytetrafluoroethylene; DPhPC; Diphytanoyl-phosphatidylcholine; TES; N; -[Tris(hydroxymethyl)methyl]-2-aminoethane-sulfonic acid; THPA; 9,10,16-trihydroxyhexadecanoic acid (9,10,16-trihydroxypalmitic, aleuritic acid); 12-HLA; 12-hydroxydodecanoic acid (12-hydroxylauric, sabinic acid); 16-HPA; 16-hydroxyhexadecanoic acid (16-hydroxypalmitic, juniperic acid); 2-HPA; 2-hydroxyhexadecanoic acid (2-hydroxypalmitic acid); PEG-PE; 1,2-Dipalmitoyl-; sn; -Glycero-3-Phosphoethanolamine-; N; -[Nethoxy(Polyethyleneglycol) - 3000]; LUV; large unilamellar vesicles; SUV; small unilamellar vesicles; UCP; uncoupling proteins; K; _ow; octanol/water distribution coefficient; P; permeability coefficient2-hydroxyhexadecanoic acid; 16-hydroxyhexadecanoic acid; 12-hydroxydodecanoic acid; 9,10,16-trihydroxyhexadecanoic acid; Long-chain fatty acid; Proton flux; Bilayer membrane; Membrane permeability coefficient

Biophysical characterization and membrane interaction of the most membranotropic region of the HIV-1 gp41 endodomain by Miguel R. Moreno; Ana J. Pérez-Berná; Jaime Guillén; José Villalaín (pp. 1298-1307).

The membrane fusion protein of HIV-1 is the envelope transmembrane gp41 glycoprotein, which is the responsible of the membrane fusion between the virus and the target cell. Gp41 has an unusual cytoplasmic tail, the endodomain, containing highly helicoidal segments with large hydrophobic moments, the so called lentivirus lytic peptides or LLPs. According to our previous work, one of the most membranotropic regions along the whole gp41 glycoprotein was located in the LLP3 region of the gp41. In order to get new insights into the viral membrane fusion mechanism, a peptide pertaining to the LLP3 domain has been studied by infrared, fluorescence and calorimetry regarding its structure, its ability to induce membrane rupture and aggregation, as well as its affinity towards specific phospholipids. Our results demonstrate that this peptide interacts with phospholipid-containing model membranes, affects the phase-behavior of membrane phospholipids and induces leakage and aggregation of liposomes. The membrane-perturbing properties of LLP3, together with the possibility that the Kennedy sequence could be part of an external loop, open the possibility that these domains might function in modulating viral membrane fusion or budding, synergistically with other membranotropic regions of the gp41 glycoprotein.

Keywords: Abbreviations; 16NS; 16-Doxyl-stearic acid; 5NS; 5-Doxyl-stearic acid; BPI; Bovine brain; l; -α-phosphadidylinositol; BPS; Bovine brain; l; -α-phosphatidylserine; CF; 5-Carboxyfluorescein; Chol; Cholesterol; DEPE; 1,2-Dielaidoyl-; sn; -glycero-3-phosphatidylethanolamine; DMPA; 1,2-Dimyristoyl-; sn; -glycero-phosphatidic acid; DMPC; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylcholine; DMPG; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylglycerol; DMPS; 1,2-Dimyristoyl-; sn; -glycero-phosphatidylserine; DSC; Differential Scanning Calorimetry; EPC; Egg; l; -α-phosphatidylcholine; HIV; Human immunodeficiency virus; LLPs; Lentivirus Lytic Peptides; LUV; Large Unilamellar Vesicles; MLV; Multilamellar Vesicles; NBD-PE; N; -(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-; sn; -glycero-phosphoethanolamine; N; -RhB-PE; Lissamine™; rhodamine B 1,2-dihexadecanoyl-; sn; -glycero-3-phosphoethanolamine; POPC; 1-Palmitoyl-2-oleoyl-; sn; -glycero-3-phosphatidylcholine; SM; Egg sphingomyelin; SUV; Small Unilamellar Vesicles; ThT; Thioflavine T; TPE; Egg trans-sterified; l; -α-phosphatidylethanolaminePeptide–lipid interaction; Gp41 endodomain; HIV; LLP3

Binding of phospholipids to β-Lactoglobulin and their transfer to lipid bilayers by P.A.T. Martins; F. Gomes; W.L.C. Vaz; M.J. Moreno (pp. 1308-1315).

The bovine milk lipocalin, β-Lactoglobulin (β-LG), has been associated with the binding and transport of small hydrophobic and amphiphilic compounds, whereby it is proposed to increase their bioavailability. We have studied the binding of the fluorescent phospholipid-derivative, NBD-didecanoylphosphatidylethanolamine (NBD-diC₁₀PE) to β-LG by following the increase in amphiphile fluorescence upon binding to the protein using established methods. The equilibrium association constant, K_B, was (1.2±0.2)×10⁶ M−¹ at 25 °C, pH 7.4 and I=0.15 M. Dependence of K_B on pH and on the monomer–dimer equilibrium of β-LG gave insight on the nature of the binding site which is proposed to be the hydrophobic calyx formed by the β-barrel in the protein. The monomer–dimer equilibrium of β-LG was re-assessed using fluorescence anisotropy of Tryptophan. The equilibrium constant for dimerization, K_D, was (7.0±1.5)×10⁵ M−¹ at 25 °C, pH 7.4, and 0.15 M ionic strength. The exchange of NBD-diC₁₀PE between β-LG and POPC lipid bilayers was followed by the change in NBD fluorescence. β-LG was shown to be a catalyst of phospholipid exchange between lipid bilayers, the mechanism possibly involving adsorption of the protein at the bilayer surface.

Keywords: Phospholipid exchange; Phospholipid binding; β-Lactoglobulin; Protein aggregation; POPC bilayers; Protein adsorption

B₂ receptor-mediated dual effect of bradykinin on proximal tubule Na⁺-ATPase: Sequential activation of the phosphoinositide-specific phospholipase Cβ/protein kinase C and Ca²⁺-independent phospholipase A₂ pathways by J.D. Líbano-Soares; E. Gomes-Quintana; H.K. Melo; E.P. Queiroz-Madeira; R.G. Roubach; A.G. Lopes; C. Caruso-Neves (pp. 1316-1323).

In a previous paper we showed that bradykinin (BK), interacting with its B₂ receptor, inhibits proximal tubule Na⁺-ATPase activity but does not change (Na⁺+K⁺)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B₂-mediated modulation of proximal tubule Na⁺-ATPase by BK. To abolish B₁ receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg⁹-[Leu⁸]-BK (DALBK), a specific antagonist of B₁ receptor. A dual effect on the Na⁺-ATPase activity through the B₂ receptor was found: short incubation times (1–10 min) stimulate the enzyme activity; long incubation times (10–60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C β (PI-PLCβ)/protein kinase C (PKC); its inhibitory action is mediated by Ca²⁺-independent phospholipase A₂ (iPLA₂). Prior activation of the PI-PLCβ/PKC pathway is required to activate the iPLA₂-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B₂ receptor and activation of membrane-associated iPLA₂.

Keywords: Abbreviations; AA; arachidonic acid; ACA; N; -(; p; -amylcinnamoyl) anthranilic acid; BEL; bromoenol lactone; BK; bradykinin; BLM; basolateral membranes; Cph C; calphostin C; cPLA; ₂; cytosolic PLA; ₂; DALBK; (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg; ⁹; -[Leu; ⁸; ]-BK; DPPC; 1,2-dipalmitoyl-phosphatidylcholine; EGTA; ethylene glycol-bis (β-aminoethyl ether)-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid; GDPβS; guanosine 5′-; O; -(2-thiodiphosphate); HEPES; N; -2-hydroxyethylpiperazine; N; ′-2-ethanesulfonic acid; iPLA; ₂; Ca; +; ²; -independent PLA; ₂; PACOCF; ₃; palmitoyl trifluoromethyl ketone; PI-PLCβ; phosphoinositide-specific phospholipase C β; PKC; protein kinase C; PLA; ₂; phospholipase A; ₂; PMA; phorbol 12-myristate 13-acetate; sPLA; ₂; secretory PLA; ₂; TLC; thin layer chromatography; Tris; tris(hydroxymethyl)aminomethaneSecond sodium pump; Extracellular volume regulation; Sodium transport; Kinase; Receptors

Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers by Sylvain Bussières; Thierry Buffeteau; Bernard Desbat; Rock Breton; Christian Salesse (pp. 1324-1334).

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all- trans-retinol into all- trans-retinyl ester. The enzymatic activity of a truncated form of LRAT (tLRAT) which contains the residues required for catalysis but which is lacking N- and C-terminal hydrophobic segments has been shown to depend on the detergent used for its solubilization. Moreover, it is unknown whether tLRAT can bind membranes in the absence of these hydrophobic segments. The present study has allowed to measure the membrane binding and hydrolytic action of tLRAT in lipid monolayers by use of polarization modulation infrared reflection absorption spectroscopy and Brewster angle microscopy. Moreover, the proportion of the secondary structure components of tLRAT was determined in three different detergents by infrared absorption spectroscopy, vibrational circular dichroism and electronic circular dichroism which allowed to explain its detergent dependent activity. In addition, the secondary structure of tLRAT in the absence of detergent was very similar to that in Triton X-100 thus suggesting that, compared to the other detergents assayed, the secondary structure of this protein is very little perturbed by this detergent.

Keywords: Abbreviations; LRAT; Lecithin retinol acyltransferase; VCD; vibrational circular dichroism; ECD; electronic circular dichroism; tLRAT; recombinant truncated form of LRAT; RPE; retinal pigment epithelium; OG; n; -octyl-β-; d; -glucopyranoside; SDS; sodium dodecyl sulfate; BSA; bovine serum albumin; DMPC; 1,2-Dimyristoyl-; sn; -Glycero-3-Phosphocholine; cmc; critical micellar concentration; PM-IRRAS; polarization modulation infrared reflection absorption spectroscopy; PC/FA; Principal Component method of Factor Analysis; PLS; Partial Least-Square Analysis; S.D.; standard deviationLecithin retinol acyltransferase; Vibrational circular dichroism; Electronic circular dichroism; Infrared spectroscopy; PM-IRRAS; Protein secondary structure

Membrane processes and biophysical characterization of living cells decorated with chromatic polydiacetylene vesicles by Natalie Groysman; Zulfiya Orynbayeva; Marina Katz; Sofiya Kolusheva; Marina Khanin; Michael Danilenko; Raz Jelinek (pp. 1335-1343).

The structural complexity of the cell membrane makes analysis of membrane processes in living cells, as compared to model membrane systems, highly challenging. Living cells decorated with surface-attached colorimetric/fluorescent polydiacetylene patches might constitute an effective platform for analysis and visualization of membrane processes in situ. This work examines the biological and chemical consequences of plasma membrane labeling of promyelocytic leukemia cells with polydiacetylene. We show that the extent of fusion between incubated lipid/diacetylene vesicles and the plasma membrane is closely dependent upon the lipid composition of both vesicles and cell membrane. In particular, we find that cholesterol presence increased bilayer fusion between the chromatic vesicles and the plasma membrane, suggesting that membrane organization plays a significant role in the fusion process. Spectroscopic data and physiological assays show that decorating the cell membrane with the lipid/diacetylene patches reduces the overall lateral diffusion within the membrane bilayer, however polydiacetylene labeling does not adversely affect important cellular metabolic pathways. Overall, the experimental data indicate that the viability and physiological integrity of the surface-engineered cells are retained, making possible utilization of the platform for studying membrane processes in living cells. We demonstrate the use of the polydiacetylene-labeled cells for visualizing and discriminating among different membrane interaction mechanisms of pharmaceutical compounds.

Keywords: Abbreviations; PDA; polydiacetylene; DNP; 2,4-dinitrophenol; THF; tetrahydrofuran; MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Fura-2AM; acetoxymethyl ester; JC-1; 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide; MBCD; methyl-β-cyclodextrin; DMPE; 1,2-dimyristoyl-; sn; -glycero-3-phosphoethanolamine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-[phospho-; rac; -(1-glycerol)] (sodium salt); DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; Sph; sphingomyelin (egg, chicken); chol; cholesterol; NBD-PE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-; N; -(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt); Rdh-PE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-; N; -(lissamine rhodamine B sulfonyl) (ammonium salt), fMLP,; N; -formyl-methionyl-leucyl-phenylalanine.Plasma membrane; Membrane sensor; Lipid fusion; Polydiacetylene; Membrane permeation; FRET; Biomimetic chemistry

Molecular organization of the lipid matrix in intact Stratum corneum using ATR-FTIR spectroscopy by Mila Boncheva; Fabienne Damien; Valéry Normand (pp. 1344-1355).

ATR-FTIR spectroscopy is useful in investigating the lateral organization of Stratum corneum (SC) lipids in full-thickness skin. Based on studies of the thermotropic phase transitions in n-tricosane and in excised human skin, the temperature dependence of the CH₂ scissoring bandwidth emerged as a measure of the extent of orthorhombic and hexagonal phases. This dependence provides a simpler measure of the lateral order in lipid assemblies than the common spectroscopic approaches based on difference spectra, curve fitting of the CH₂ scissoring region, and the position of the CH₂ stretching vibrations. It has the advantages of ease of determination, relatively low variability, and high discriminative power for the type of lateral intermolecular chain packing. A comparison of the lateral organization of the lipids at the SC surface of mammalian skin using the scissoring bandwidth revealed considerable differences between human abdominal skin (containing mostly orthorhombic phases), porcine ear skin (containing mostly hexagonal phases), and reconstructed human epidermis (containing mostly disordered phases). This parameter also correctly described the different effects of propylene glycol (minimally disturbing) and oleic acid (formation of a highly disordered phase) on the SC lipids in excised human skin. The procedure described here is applicable to in vivo studies in the areas of dermatology, transdermal drug delivery, and skin biophysics.

Keywords: Human skin; Infrared spectroscopy; Lipid organization; Skin biophysics

Role of ceramide in membrane protein organization investigated by combined AFM and FCS by Salvatore Chiantia; Jonas Ries; Grzegorz Chwastek; Dolores Carrer; Zaiguo Li; Robert Bittman; Petra Schwille (pp. 1356-1364).

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside G_M1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

Keywords: Ceramide; Raft; Liquid-ordered; Membrane protein; GPI anchor; Fluorescence correlations spectroscopy; Atomic force microscopy

α,α′-trehalose 6,6′-dibehenate in non-phospholipid-based liposomes enables direct interaction with trehalose, offering stability during freeze-drying by Dennis Christensen; Daniel Kirby; Camilla Foged; Else Marie Agger; Peter Andersen; Yvonne Perrie; Hanne Mørck Nielsen (pp. 1365-1373).

Trehalose is a well known protector of biostructures like liposomes and proteins during freeze-drying, but still today there is a big debate regarding its mechanism of action. In previous experiments we have shown that trehalose is able to protect a non-phospholipid-based liposomal adjuvant (designated CAF01) composed of the cationic dimethyldioctadecylammonium (DDA) and trehalose 6,6′-dibehenate (TDB) during freeze-drying [D. Christensen, C. Foged, I. Rosenkrands, H.M. Nielsen, P. Andersen, E.M. Agger, Trehalose preserves DDA/TDB liposomes and their adjuvant effect during freeze-drying, Biochim. Biophys. Acta, Biomembr. 1768 (2007) 2120–2129]. Furthermore it was seen that TDB is required for the stabilizing effect of trehalose. Herein, we show using the Langmuir–Blodgett technique that a high concentration of TDB present at the water-lipid interface results in a surface pressure around 67 mN/m as compared to that of pure DDA which is approximately 47 mN/m in the compressed state. This indicates that the attractive forces between the trehalose head group of TDB and water are greater than those between the quaternary ammonium head group of DDA and water. Furthermore, addition of trehalose to a DDA monolayer containing small amounts of TDB also increases the surface pressure, which is not observed in the absence of TDB. This suggests that even small amounts of trehalose groups on TDB present at the water-lipid interface associate free trehalose to the liposome surface, presumably by hydrogen bonding between the trehalose head groups of TDB and the free trehalose molecules. Hence, for CAF01 the TDB component not only stabilizes the cationic liposomes and enhances the immune response but also facilitates the cryo-/lyoprotection by trehalose through direct interaction with the head group of TDB. Furthermore the results indicate that direct interaction with liposome surfaces is necessary for trehalose to enable protection during freeze-drying.

Keywords: Liposome; Freeze-drying; Adjuvant; Vaccine; Dimethyldioctadecylammonium; DDA; Trehalose 6,6-dibehenate; TDB; Sugar; Trehalose; Drug delivery; CAF01

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BBA - Biomembranes (v.1778, #5)