Mixture Of Phosphatidylserine Research Articles

Influence of the Immune Checkpoint Inhibitors on the Hemostatic Potential of Blood Plasma

Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized classical treatment approaches of various cancer entities, but are also associated with a number of side effects. One of these may be life-threatening clotting disorders with the risk of thrombotic or hemorrhagic complications, the mechanisms of which are still poorly understood. In the present study, we analyzed the direct effects of pembrolizumab, nivolumab, and ipilimumab on platelet aggregation as well as plasma coagulation followed by fibrinolysis in an ex vivo model. Methods: Microplate spectrometry was used to analyze aggregation, coagulation, and fibrinolysis in platelet-free (PFP) and platelet-rich (PRP) healthy donor plasma samples treated with pembrolizumab, nivolumab, ipilimumab, and appropriate isotype controls. Aggregation was induced by TRAP-6. Clotting of PFP and PRP followed by lysis was initiated with a tissue factor in a mixture of phosphatidylserine:phosphatidylcholine and the addition of t-PA. Among other parameters, the area under the curve (AUC) was used to compare the effect of ICIs on aggregation, coagulation, and fibrinolysis. Results: Upon direct contact with platelets, pembrolizumab stimulated platelet aggregation in PRP, while nivolumab and ipilimumab promoted disaggregation with corresponding changes in the AUC. Pembrolizumab and nivolumab, both PD-1 receptor inhibitors, had no effect on the plasma coagulation cascade. Ipilimumab, a CTLA-4 receptor inhibitor, significantly increased the rate of PRP clotting. When clotting was followed by lysis, all ICIs were found to prolong the growth of the PRP-derived fibrin clot and delay its elimination. This was manifested by an increase in AUC relative to control PRP. Conclusion: This study characterizes the potential impact of pembrolizumab, nivolumab, and ipilimumab on hemostasis. Nivolumab and ipilimumab are able to reduce aggregation and increase the procoagulant properties of platelets, which can cause side effects associated with hemostatic imbalance leading to thrombosis or bleeding. The observed ICI-specific effects may contribute to our understanding of the mechanisms by which ICI affects platelets and suggest how, in a clinical setting, to reduce coagulation disorders during ICI treatment in the future.

Anionic Lipids Take Charge: Juxtamembrane Domain Interactions with Cellular Membrane

Nearly half of the human integral membrane proteome consists of single-pass transmembrane domains. Of these, receptor tyrosine kinases (RTKs), a class with twenty families of cell-surface receptors, are key regulators of vital cellular processes and targets of drug development efforts. The fibroblast growth factor receptors (FGFRs) a family of RTKs that influences cell growth, proliferation, differentiation, is also activated in a number of cancers. FGFRs dynamic architecture, that contains extracellular, single-pass transmembrane (TM), juxtamembrane (JM), and kinase domains, is tightly intertwined with cellular membrane and is a challenging target for structural studies. Recent experimental studies revealed that highly charged FGFR3 JM domains are involved in stabilization of unliganded FGFR3 dimers, which is achieved in part through interactions with charged lipids of the inner leaflet of the cellular membrane. The mechanism of JM-lipid interaction is unknown. We use all-atom molecular dynamics with highly mobile membrane mimetic (HMMM) model to capture spontaneous JM-lipid encounter complexes. To investigate the role of the anionic lipids we performed extensive simulations of multiple replicas of pure phosphatidylcholine (PC), binary mixtures of phosphatidylserine (PS) and PC, and tertiary mixtures of PC, PS, and phosphatidylinositol 4,5-bisphosphate (PIP2) lipid bilayers. Simulations revealed formation of stable anionic lipid conformations coordinated by charged JM residues. Lipid type-specific geometries lead to characteristic modes of JM-lipid interactions revealing the role of the charged lipid headgroups in establishing specific interactions. Knowledge of these dynamic structures will facilitate our understanding of mechanisms of RTK activation.

Calcium-Induced Sculpturing of the Plasma Membrane: Lipid Microclusters Cast by Anionic Lipids

The plasma membrane of the cell is a complex, tightly-controlled heterogeneous environment. While anionic lipids constitute a small part of all phospholipids in the plasma membrane, they play key functional roles in regulation of vital cellular processes such as blood clotting, signaling, vesicle fusion, and apoptosis. Lipid composition and distribution in the plasma membrane are highly dynamic, with anionic phosphatidylserine (PS) lipids typically found on the interior leaflet. In turn, the concentration of divalent Ca2+ ions, an important cellular messenger, is three orders of magnitude greater outside of the cell, spatially separating the ions from anionic lipids. PS lipids can become externalized to the outer leaflet of the plasma membrane in the case of cell injury, disease, or apoptotic changes allowing them to interact with Ca2+, resulting in changes in the plasma membrane structure and initiation of signaling cascades. Nonetheless, the molecular details of lipid-Ca2+ interactions are not completely understood. We performed molecular dynamics simulations of binary mixtures of PS and phosphatidylcholine (PC) lipids using highly mobile membrane mimetic model (HMMM) that enabled extended sampling. Simulations revealed that lipid microclusters are formed by higher order (three or more) lipid oligomers coordinated by Ca2+, with two distinct conformations of the PS headgroup serving as building blocks. In parallel, 13C-dephased 31P-observed solid-state nuclear magnetic resonance (SSNMR) rotational-echo double-resonance (REDOR) experiments were performed with spin-diluted Nanodisc samples to report on 31P-13C distances. Experimental data are consistent with both intra- and inter-molecular lipid headgroup characteristic distances of approximately 4.8 +/- 0.5 A that match well with all-atom MD results. To our knowledge, this is one of the first joint MD and high-resolution SSNMR studies to characterize how lipid microclusters are formed at the atomic level and how they reshape the plasma membrane.

Tight binding of NAP-22 with acidic membrane lipids

Recovery of various signal transduction molecules in the detergent-resistant membrane microdomain (DRM) fraction suggests the importance of this region in cellular functions. Insolubility of the outer leaflet of DRM to the non-ionic detergent is ascribed to the tight association of cholesterol and sphingolipid. Since, poor localization of sphingolipid is observed in the inner leaflet, the physicochemical background of the insolubility of the inner leaflet is hence still an enigma. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the DRM of the neuronal cell membrane. A previous study showed the presence of several lipids in a NAP-22 fraction after the process of extraction and column chromatography. In this study, the effect of lipid extraction on NAP-22 was studied through native-gel electrophoresis, ultracentrifugation, and electron microscopic observation. The mobility of NAP-22 in native-PAGE was shifted from low to high after delipidation. Delipidated NAP-22 bound phosphatidylserine (PS), phosphatidylinosotol, and ganglioside. Some part of the mixture of PS and NAP-22 was recovered in the insoluble fraction after Triton X-100 treatment and the addition of cholesterol enhanced the amount of NAP-22 in the insoluble fraction.

Interaction of an Antitumor Peptide with Lipids of the Cancer Plasma Membrane - Formation of Membrane Domains and Influence of Cholesterol

R-DIM-P-LF11-322, derived from the cationic human host defense peptide lactoferricin, exhibits selective antitumor activity against several human cancer cell types. We have shown that cancer cells expose the negatively charged phosphatidylserine (PS) on the outer leaflet of the plasma membrane (1), which in healthy cells only comprises neutral lipids as phosphatidylcholine (PC) or cholesterol. Therefore the membrane interaction of R-DIM-P-LF11-322 with membrane mimics composed of mixtures of PS, PC and cholesterol was studied. Lipid analysis of normal melanocytes and melanoma revealed a slight increase of PC and cholesterol in the cancer lipid extracts.Tryptophan fluorescence showed that the peptide penetrated only in the cancer mimics containing ratios of PS. Addition of cholesterol slightly reduced the affinity of the peptide.Calorimetric studies with peptide and mixed liposomes of PS and PC indicated complete perturbation of the PS fraction leaving PC unaffected. Interestingly upon addition of cholesterol the peptide induced separation into PC and PS enriched domains, but seemed to be slightly reduced in its effect.Studies using giant unilamellar vesicles showed that the peptide interacts on cancer model membranes slightly stronger in the absence of cholesterol by formation of extremely deformed and destroyed vesicles. In the presence of cholesterol the peptide however still induced shrinking of the vesicles and formation of many small PS domains.In summary the antitumor peptide strongly interacts only with its target PS on cancer cells. However, cholesterol seems to play a role in the modulation of membrane domain formation and reducing the membrane affinity, which may represent a mechanism for cancer cells to protect themselves against antitumor peptides.Acknowledgment: Austrian Science Foundation FWF (grant no: P24608)(1) Riedl et al. BBA 1808: 2638-2645, 2011.

Improved membrane fluidity of ionic polysaccharide bead-supported phospholipid bilayer membrane systems

Supported phospholipid bilayer membranes on polysaccharide-based cationic polymer beads (cationic group: –[OCH2CH(OH)CH2]2N+(CH3)3·X−, 45–165μm in diameter) were prepared using small unilamellar vesicles from mixtures of phosphatidylserine (PS) and phosphatidylcholine (PC). Confocal fluorescence microscopic observations with a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the phospholipid-bead complexes were along the outer surface of the beads. The fluidity of the phospholipid bilayer membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were lower than that for the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supports. Such less fluid membranes in the complexes appeared to be due to the immobilization of the phospholipid bilayer membranes by electrostatic attractive forces between PS and the bead. The D values for the PS/PC-bead complexes were dependent on the phospholipid composition; the PS(100mol%)/PC(0mol%)-bead complex had the least fluid membranes among the PS/PC-bead complexes tested in this study. The phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer beads were much more fluid than those on a polystyrene-based one. Furthermore, such fluid phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer bead were maintained for 10 days, even though the complex sample was stood in plain buffer (pH 8.5) at ambient temperature.

Molecular Basis of Phospholipid Synergy in Promoting Blood Coagulation Reactions

Abstract 1109Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Many coagulation proteins bind to PS-containing membrane bilayers in a calcium-dependent manner via gamma-carboxyglutamate-rich (GLA) domains. Furthermore, certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. We have recently been investigating the molecular basis of lipid-Ca2+ and lipid-GLA interactions in blood clotting using solid-state NMR (SSNMR) studies that employ isotopically labeled PS. Our studies have revealed that Ca2+ induces two predominant conformations of the PS headgroup (Boettcher et al., Biochem. 50:2264–73, 2011). We now report the quantification of the intermolecular and intramolecular atomic distances within Ca2+-induced PS nanoclusters using SSNMR. We have also expanded SSNMR studies to phospholipid bilayers composed of trinary mixtures of PS, PC, and PE as a means to further investigate the impact of PE on PS headgroup conformations in an intact lipid bilayer (PE-PS synergy). Initial results suggest that incorporation of PE into the bilayer alters the Ca2+: PS ratio required to fully saturate the L-serine headgroups of PS. Additionally, PE appears to affect the predominant PS headgroup conformations in the presence of Ca2+, and may induce a third L-serine conformation. SSNMR studies utilizing isotopically labeled PE headgroups are currently underway which should provide novel information concerning PE's interactions with PS, Ca2+, and/or GLA domains.Previous SSNMR studies from our lab have also indicated that a fraction of L-serine headgroups in PS adopt a new conformation when bovine prothrombin fragment 1 is bound to PS/PC bilayers (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). These data support the idea that GLA domains contain an L-serine-specific binding site. We have now bound human prothrombin fragment 1 to membranes containing labeled PS headgroups for use in SSNMR studies. In addition, we have also employed surface plasmon resonance (SPR) experiments to investigate the interactions of both human and bovine prothrombin with bilayers containing PS and PE. These data demonstrate that binding affinities and binding footprints are similar for both prothrombin proteins, and that PE enhances binding affinities and increases the number of GLA binding sites for both proteins. Together, results from SSNMR and SPR studies suggest that different GLA domains use similar molecular mechanisms to interact with the membrane surface. Disclosures:No relevant conflicts of interest to declare.

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H–13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.

Phospholipid Synergy in Prothrombinase Activity

Abstract 1175Most steps in the blood coagulation cascade obligatorily take place on membrane surfaces and are dependent on the exposure of phosphatidylserine (PS). Previous studies from our lab and others have shown that phosphatidylethanolamine (PE) poorly supports clotting reactions by itself, but strongly synergizes with PS to promote several membrane-dependent steps in the blood clotting cascade, although the mechanism for PE-PS synergy has been unclear. We recently put forward a new mechanistic explanation – which we termed the ABC or Anything But Choline hypothesis – for how PS and PE synergize to enhance factor X (fX) activation by the factor VIIa-tissue factor complex (Tavoosi et al., J. Biol. Chem. 286:23247–53, 2011). The membrane contribution to this reaction is dominated by the affinity of fX for the membrane surface; since fX binds to membranes via its gamma-carboxyglutamate-rich (GLA) domain, the ABC hypothesis therefore focuses on the mechanisms by which GLA domains engage the phospholipid bilayer. We identified two main types of GLA domain-phospholipid interactions: a single phospho-L-serine-specific binding site in each GLA domain; and multiple ”phosphate-specific” interactions in which the phosphate groups of non-phosphatidylcholine phospholipids form coordination complexes with the tightly bound calcium ions in GLA domains.In the current study, we test the ABC hypothesis in the context of the prothrombinase complex – i.e., activation of prothrombin by the membrane-bound complex of fXa and factor Va (fVa). Using a variety of approaches including surface plasmon resonance analyses, we measured the contributions of varying phospholipid compositions to the membrane binding affinities of fXa, fVa and prothrombin, as well as to the enzymatic activity of prothrombinase. Our results suggest that phospholipid synergy in prothrombinase activity differs in certain respects from that observed for the factor VIIa-tissue factor complex. Not only did PS synergize with PE for enhancing the activity of prothrombinase, but phosphatidylglycerol (PG) and phosphatidylacid (PA) also synergized with PE, albeit more weakly than with PS (i.e., significantly higher levels of PG or PA in the presence of PE were required to achieve prothrombinase activities comparable to mixtures of PS and PE). In contrast, PE failed to synergize with either PG or PA to support fX activation by the factor VIIa-tissue factor complex. These differences primarily arise from differential membrane binding of the substrates for these two complexes (fX for factor VIIa-tissue factor and prothrombin for prothrombinase). The data suggest that the phospho-L-serine-specific binding site in the GLA domain of prothrombin may not be as stringent as that of fX, as high levels of PG or PA can substitute for PS in membrane binding of prothrombin but not for fX. This study provides further insights into the membrane’s role in regulating blood clotting reactions, specifically the binding interactions between GLA domains and membrane surfaces. Disclosures:No relevant conflicts of interest to declare.

Phosphatidylinositol 4,5-Bisphosphate Alters Synaptotagmin 1 Membrane Docking and Drives Opposing Bilayers Closer Together

Synaptotagmin 1 (syt1) is a synaptic vesicle-anchored membrane protein that acts as the calcium sensor for the synchronous component of neuronal exocytosis. Using site-directed spin labeling, the position and membrane interactions of a fragment of syt1 containing its two C2 domains (syt1C2AB) were assessed in bilayers containing phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol 4,5-bisphosphate (PIP(2)). Addition of 1 mol % PIP(2) to a lipid mixture of PC and PS results in a deeper membrane penetration of the C2A domain and alters the orientation of the C2B domain so that the polybasic face of C2B comes into the proximity of the bilayer interface. The C2B domain is found to contact the membrane interface in two regions, the Ca(2+)-binding loops and a region opposite the Ca(2+)-binding loops. This suggests that syt1C2AB is configured to bridge two bilayers and is consistent with a model generated previously for syt1C2AB bound to membranes of PC and PS. Point-to-plane depth restraints, obtained by progressive power saturation, and interdomain distance restraints, obtained by double electron-electron resonance, were obtained in the presence of PIP(2) and used in a simulated annealing routine to dock syt1C2AB to two membrane interfaces. The results yield an average structure different from what is found in the absence of PIP(2) and indicate that bilayer-bilayer spacing is decreased in the presence of PIP(2). The results indicate that PIP(2), which is necessary for bilayer fusion, alters C2 domain orientation, enhances syt1-membrane electrostatic interactions, and acts to drive vesicle and cytoplasmic membrane surfaces closer together.

Temperature dependence of syringomycin E channel functioning in planar lipid bilayers

327 We have studied conductivity and dwell time of ion channels produced by antifungal lipodepsipeptide syringomycin E (SRE) in planar lipid bilayers in the temperature range of 10 − 50 ° C. Membranes were formed from either phosphatidylethanolamine or from an equimomolar mixture of phosphatidylserine and phosphatidylethanolamine; membrane bathing solution contained 0.4 M LiCl or 0.4 M NaCl, pH 6. We have established that a 10 ° C increase in temperature produces ~1.5-fold increase in the single-channel conductance and ~3-fold decrease in the mean dwell time of a single channel. These changes correspond to ~7 kcal/mol activation energy for ion transport through the SRE channel. This value exceeds the activation energy barrier of the free ion diffusion in water by a factor of two (~3.3 kcal/mol). Higher value of the activation energy of ion transport in the SRE channel suggests specific interactions between the permeating ions and the channel walls; this suggestion is also supported by our earlier observation of the difference between SRE channel conductance and the conductance of the equivalent water volume. The decrease of the channel dwell time with the increase of temperature may be related to dissociation of the SRE aggregates (6 − 7 lipodepsipeptide molecules) forming the conducting channel.

Miscibility Phase Behavior Of GUV Membranes Containing Ternary Mixtures Of PS Lipids, PC Lipids, And Cholesterol

Vesicles containing ternary mixtures of phospholipids and cholesterol produce coexisting liquid phases over a range of compositions and temperatures. To date, most studies of membrane phase behavior have used uncharged lipids, which readily produce giant unilamellar vesicles through electroformation. My goal is to study the phase behavior of membranes containing charged lipids such as phosphatidylserine, which is found in the inner leaflet of cell plasma membranes. I present experimental protocols to prepare giant unilamellar vesicles containing ternary mixtures of phosphatidylserine (PS) lipids, phosphatidylcholine (PC) lipids, and cholesterol, based on earlier protocols by Akashi et al [1], Rodriguez et al [2], and Claessens et al [3]. I also detail the phase behavior of membranes of several compositions that incorporate PS lipids. [1] Akashi et al., “Formation of Giant Liposomes Promoted by Divalent Cations: Critical Role of Electrostatic Repulsion.” 74 (1998) 2973-2982. [2] Rodriguez et al., “Giant vesicles formed by gentle hydration and electroformation: A comparison by fluorescence microscopy.” Colloids and Surfaces B: Biointerfaces 42 (2005) 125-130. [3] Claessens et al., “Charged Lipid Vesicles: Effects of Salts on Bending Rigidity, Stability, and Size.” Biophysical Journal 87(6) (2004) 3882-3893.

Phospholipid Components of the Activated Platelet Surface Determine Multimerin 1 and Factor V/Va Binding, and Modulate Thrombin Generation in Plasma.

Multimerin 1 (MMRN1) is a factor V (FV) binding protein sequestered in endothelium and platelets, where it is costored with FV in α-granules for regulated release. Both MMRN1 and FV/Va bind to the outer surface of activated platelets and to phosphatidylserine (PS), with overlap in FV/Va binding sites for PS and MMRN1. Inhibitory effects of MMRN1 on FVa-dependent thrombin generation, and the activation dependent nature of MMRN1 binding to platelets led us to evaluate and compare MMRN1 and FV/Va binding to membranes of varying compositions. Binding was evaluated by established phospholipid binding ELISA, and by surface plasmon resonance (SPR) studies of liposome binding to immobilized MMRN1. Mixtures of PS: phosphatidylcholine (PC): phosphatidylethanolamine (PE): sphingomyelin (SM) and cholesterol representative of resting and activated platelet outer surfaces were investigated, including those typical of stimulation by thrombin, or thrombin plus collagen. Membrane compositions optimal for MMRN1 binding were compared to those that enhanced FV/Va binding and thrombin generation. Calibrated automated thrombograms were modified to compare functional effects of lipid mixtures on coagulation at low but saturating FV concentrations. ELISA results indicated that MMRN1 lipid binding was linear with PS content of PS:PC membranes from 0 to 100 mole% PS (R2=0.9). At 5% PS (where MMRN1 binding was ~18% of pure PS), addition of PE increased MMRN1 binding in a linear, dose-dependent manner (R2=0.92) up to 35% PE (the highest PE content tested), and this increased the MMRN1 binding relative to pure PS. A parallel trend was observed for FVa binding to these mixtures. While FV binding to PS:PC 5:95 was enhanced by the addition of increasing PE content (R2=0.98) up to 20 mole% PE (where the effect of PE appeared to saturate), significant binding (greater than 50% of binding to pure PS) occurred without PE, and to pure PC. Significant increases (p values <0.005) in MMRN1 and FV/Va binding were observed when a 25% PE was added to mixtures containing 5% PS. Incorporation of 33% cholesterol in the membrane had no significant effect on the binding of MMRN1 and FV; however, 68% cholesterol enhanced their binding. SPR measurements confirmed the relative binding affinities of MMRN1 for different membranes. There was no significant MMRN1 binding to membranes typical of the resting platelet surface (PS:PC:PE:SM 2:39:9:50), but MMRN1 binding was increasingly enhanced by membranes typical of thrombin stimulated platelets (5:40:25:30), randomized total platelet lipid contents (10:44:27:19) and thrombin plus collagen stimulated platelets (11:35:36:20), in that order. Lipid mixtures that were optimal for MMRN1, FV and FVa binding also enhanced coagulation, by increasing both the rates and total amounts of thrombin generated by plasma. Increases in thrombin generation were evident with increased membrane content of PS, PE or cholesterol. The results indicate that membrane lipid compositions characteristic of activated platelets enhance coagulation, and have parallel enhancing effects on MMRN1, FV and FVa binding. The data suggests that activation-induced differences in surface exposure of PS and PE, relative to PC and sphingomyelin, and local concentrations of cholesterol in the platelet membrane, alter the probability of MMRN1 and FVa binding, possibly by changing the outer membrane physical properties. These changes may influence the ability of MMRN1 to modulate FV/Va binding and function and possibly MMRN1 adhesive functions.

Improvement of a solid phase extraction method for separation of animal muscle phospholipid classes

The solid phase extraction (SPE) method used by Banni et al. (2001) [Banni, S., Carta, G., Angioni, E., Muru, E., Scanu, P., Melis, M. P., et al. (2001). Distribution of conjugated linoleic acid and metabolites in different lipid fraction in the rat liver. Journal of Lipid Research, 42, 1056–1061] for fractionation of liver phospholipids (PLs) into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) was modified to improve its separation efficiency. A mixture of PL standards, containing PC, PE, PS and PI, was separated into individual classes by using aminopropyl minicolumns, following the method of Banni et al. (2001). The obtained eluted fractions were further analysed by thin layer chromatography (TLC) and PL classes were identified. TLC revealed the co-elution of PC and PE, that of PE with PS and no elution of PI. The SPE method was subsequently modified in order to obtain a correct PL fractionation into PC, PE, PS and PI. The principal modifications consisted of increased solvent volumes for PC, PE and PS elution, and a different solvent mixture to allow PI elution. The effectiveness of the modified SPE method was checked by TLC, using both standards and muscle samples, showing a correct elution of PC, PE, PS and PI.

Phosphorylation of GRK1 and GRK7 by cAMP-dependent Protein Kinase Attenuates Their Enzymatic Activities

Phosphorylation of G protein-coupled receptors is a critical step in the rapid termination of G protein signaling. In rod cells of the vertebrate retina, phosphorylation of rhodopsin is mediated by GRK1. In cone cells, either GRK1, GRK7, or both, depending on the species, are speculated to initiate signal termination by phosphorylating the cone opsins. To compare the biochemical properties of GRK1 and GRK7, we measured the K(m) and V(max) of these kinases for ATP and rhodopsin, a model substrate. The results demonstrated that these kinases share similar kinetic properties. We also determined that cAMP-dependent protein kinase (PKA) phosphorylates GRK1 at Ser(21) and GRK7 at Ser(23) and Ser(36) in vitro. These sites are also phosphorylated when FLAG-tagged GRK1 and GRK7 are expressed in HEK-293 cells treated with forskolin to stimulate the endogenous production of cAMP and activation of PKA. Rod outer segments isolated from bovine retina phosphorylated the FLAG-tagged GRKs in the presence of dibutyryl-cAMP, suggesting that GRK1 and GRK7 are physiologically relevant substrates. Although both GRKs also contain putative phosphorylation sites for PKC and Ca(2+)/calmodulin-dependent protein kinase II, neither kinase phosphorylated GRK1 or GRK7. Phosphorylation of GRK1 and GRK7 by PKA reduces the ability of GRK1 and GRK7 to phosphorylate rhodopsin in vitro. Since exposure to light causes a decrease in cAMP levels in rod cells, we propose that phosphorylation of GRK1 and GRK7 by PKA occurs in the dark, when cAMP levels in photoreceptor cells are elevated, and represents a novel mechanism for regulating the activities of these kinases.

Hyperici herba extract interaction with artificial lipid bilayers.

Hyperici herba (Hyp) is the aerial part collected during the flowering period from the well-known herb, Hypericum perforatum. Black lipid membrane experiments were performed to investigate the effect of the ethanolic Hyp extract on the electrical properties (capacitance and conductance) of artificial lipid bilayers. Hyp extract (1-10 microg mL(-1)) induced a concentration-dependent increase of both specific transmembrane capacitance and conductance in phosphatidylcholine (PC) membranes. The effect on conductance was enhanced when the Hyp extract (3 microg mL(-1)) was present on both sides of the membrane (Gm=77.89 +/- 8.81 nS cm(-2), n=5) compared with single-sided application (Gm=36.48 +/- 2.41 nS cm(-2), n=5). In bilayers containing PC and phosphatidylserine (PS), PC:PS, the Hyp extract effect was greater than on pure PC bilayers, although the surface charge was not the determining factor of this enhanced activity. Adding cholesterol to the PC:PS mixture reverted the conductance increase induced by the Hyp extract in a dose-dependent manner. The specific pattern of the Hyp extract interaction with lipid bilayers has possible consequences concerning its absorption and bioavailability, as well as its pharmacodynamic effects on neuronal excitability.

Lipid Antioxidant, Etoposide, Inhibits Phosphatidylserine Externalization and Macrophage Clearance of Apoptotic Cells by Preventing Phosphatidylserine Oxidation

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.

Fusion, leakage and surface hydrophobicity of vesicles containing phosphoinositides: influence of steric and electrostatic effects.

Calcium and lanthanum ion-induced fusion of lipid vesicles containing phosphatidylinositol (PI), phosphatidylinositol-4-monophosphate (PIP), phosphatidylinositol-4,5-bisphosphate (PIP2) or phosphatidylinositol-3,4,5-trisphosphate (PIP3) and its associated membrane properties, e.g., surface dielectric constant and vesicle leakage, were studied by fluorescence methods. The presence of poly-phosphorylated phosphoinositides (PPI) in lipid vesicles enhanced fusion, depending on the PPI phosphorylation level and the PPI concentration, as determined by the lipid mixing assay. This correlation held even at physiologically relevant small concentrations of PPI in vesicle membranes. However, the presence of nonphosphorylated PI inhibited fusion due to the steric effect of the inositol ring. The cation threshold concentration for the lipid mixing of vesicles made of mixtures of phosphatidylserine (PS) with PI increased with increasing PI contents. For all vesicle systems studied, a decrease in vesicle surface dielectric constant and an increase in vesicle leakage accompanied fusion. The presence of the nonphosphorylated inositol ring in PI did not interfere with the changes in the surface dielectric constant caused by fusogenic cations. Therefore, we deduce that the reduction of the surface dielectric constant is a necessary condition for membrane fusion to occur but it does not correlate with membrane fusion when interacting membranes are blocked for close approach as by the nonphosphorylated inositol ring.

A New High-Temperature Transition of Crystalline Cholesterol in Mixtures with Phosphatidylserine

Phosphatidylserine and cholesterol are two major components of the cytoplasmic leaflet of the plasma membrane. The arrangement of cholesterol is markedly affected by the presence of phosphatidylserine in model membranes. At relatively low mol fractions of cholesterol in phosphatidylserine, compared with other phospholipids, cholesterol crystallites are formed that exhibit both thermotropic phase transitions as well as diffraction of x-rays. In the present study we have observed and characterized a novel thermotropic transition occurring in mixtures of phosphatidylserine and cholesterol. This new transition is observed at 96°C by differential scanning calorimetry (DSC), using a heating scan rate of 2°C/min. Observation of the transition requires that the hydrated lipid mixture be incubated for several days, depending on the temperature of incubation. The rate of formation of the material exhibiting a transition at 96°C is more rapid at higher incubation temperatures. At 37°C the half-time of conversion is ∼7 days. Concomitant with the appearance of the 96°C peak the previously known transitions of cholesterol, occurring at ∼38°C and 75°C on heating scans of freshly prepared suspensions, disappear. These two transitions correspond to the polymorphic transition of anhydrous cholesterol and to the dehydration of cholesterol monohydrate, respectively. The loss of the 75°C peak takes a longer time than that of the 38°C peak, indicating that anhydrous cholesterol first gets hydrated to the monohydrate form exhibiting a transition at 75°C and subsequently is converted by additional time of incubation to an altered form of the monohydrate, showing a phase transition at 96°C. After several weeks of incubation at 37°C, only the form with a phase transition at 96°C remains. If such a sample undergoes several successive heating and cooling cycles, the 96°C peak disappears and the 38°C transition reappears on heating. For samples of 1-palmitoyl-2-oleoyl phosphatidylserine or of 1-stearoyl-2-oleoyl phosphatidylserine having mol fractions of cholesterol between 0.4 and 0.7, the 38°C transition that reappears after the melting of the 96°C component generally has the same enthalpy as do freshly prepared samples. This demonstrates that, at least for these samples, the amount of anhydrous cholesterol crystallites formed is indeed a property of the lipid mixture. We have also examined variations in the method of preparation of the sample and find similar behavior in all cases, although there are quantitative differences. The 96°C transition is partially reversible on cooling and reheating. This transition is also scan rate dependent, indicating that it is, at least in part, kinetically determined. The enthalpy of the 96°C transition, after incubation of the sample for 3 weeks at 37°C is dependent on the ratio of cholesterol to 1-palmitoyl-2-oleoyl phosphatidylserine or to 1-stearoyl-2-oleoyl phosphatidylserine, with the enthalpy per mole cholesterol increasing between cholesterol mol fractions of 0.2 and 0.5. Dimyristoyl phosphatidylserine at a 1:1 molar ratio with cholesterol, after incubation at 37°C, exhibits a transition at 95°C that reverses on cooling at 44°C, instead of 60°C, as observed with either 1-palmitoyl-2-oleoyl phosphatidylserine or 1-stearoyl-2-oleoyl phosphatidylserine. These findings along with the essential absence of the 96°C transition in pure cholesterol or in cholesterol/phosphatidylcholine mixtures, indicates that the phospholipid affects the characteristics of the transition, and therefore the cholesterol crystallites must be in direct contact with the phospholipid and are not simply in the form of pure crystals of cholesterol. These observations are particularly important in view of recent observations of the presence of cholesterol crystals in biological systems.

Discovery of a novel channel-forming protein in the cell wall of the non-pathogenic Nocardia corynebacteroides

Detergent extracts of whole cells of the Gram-positive, non-pathogenic, strictly aerobic bacterium Nocardia corynebacteroides contain channel-forming activity. The protein responsible for channel formation was identified using lipid bilayer experiments. It was purified to homogeneity and had an apparent molecular mass of about 134 kDa on SDS–PAGE when it was solubilized at 40°C. When the 134 kDa protein was heated to 100°C for 10 min in sample buffer, it dissociated into subunits with a molecular mass of about 23 kDa and focused at pI of 4.5 during isoelectric focusing. The pure 134 kDa protein was able to increase the specific conductance of artificial lipid bilayer membranes from phosphatidylcholine–phosphatidylserine mixtures by the formation of ion-permeable channels. The channels had an average single-channel conductance of 5.5 nS in 1 M KCl and were found to be cation-selective. Asymmetric addition of the 134 kDa protein to lipid bilayer membranes resulted in an asymmetric voltage-dependence. The analysis of the single-channel conductance as a function of cation radii using the Renkin correction factor and the effect of negative charges on channel conductance suggested that the diameter of the cell wall porin is about 1.0 nm. The channel characteristics of the cell wall channel of N. corynebacteroides were compared with those of other members of the mycolata. They share common features because they are composed of small molecular mass subunits and form large and water-filled channels.