Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane

Under many apoptotic conditions, Bax undergoes conformational rearrangements, leading to its insertion in the mitochondrial outer membrane as a transmembrane oligomer. At the same time, mitochondria undergo fragmentation and activated Bax was reported to localize to fission sites. We studied how lipid composition and membrane curvature regulate Bax activation. When isolated mitochondria were incubated with phospholipase A2, which led to phosphatidylethanolamine and cardiolipin hydrolysis, tBid and Bax insertion were hindered. We thus studied in liposomes how phosphatidylethanolamine, cardiolipin, and its hydrolysis products affect Bax activation. Whereas phosphatidylethanolamine, a lipid with negative curvature, did not affect Bax insertion, it inhibited Bax oligomerization. Conversely, Bax insertion required cardiolipin, and was not blocked by cardiolipin hydrolysis products. These experiments support a direct role for cardiolipin in the recruitment and activation of Bax. To examine if the increase in membrane curvature that accompanies mitochondrial fission participates in Bax activation, we studied how liposome size affects the process, and observed that it was inhibited in small liposomes (相似文献   

tBid forms a pore in the liposome membrane

We investigated the ability of tBid (truncated form of Bid) to bind and permeabilize the liposomes (large unilamellar vesicles, LUVs) and release fluorescent marker molecules (fluorescein-isothiocyanate-conjugated dextrans, FITC-dextrans) of various molecular diameters (FD-20, FD-70, FD-250S) from LUVs. Obtained data showed that tBid was more efficient in promoting leakage of FITC-dextrans from LUVs composed of cardiolipin and dioleoylphosphatidylcholine (DOPC) than LUVs made of dioleoylphosphatidic acid or dioleoylphosphatidylglycerol and DOPC. The leakage efficiency was reduced with increasing amount of dioleoylphosphatidylethanolamine or dielaidoylphosphatidylethanolamine. Phospholipid monolayer assay and fluorescence quenching measurements revealed that tBid inserted deeply into the hydrophobic acyl chain of acidic phospholipids. Taking into account the tBid three-dimensional structure, we propose that tBid could penetrate into the hydrophobic core of membrane, resulting in the leakage of entrapped content from LUVs via a pore-forming mechanism.     

Design and function of a conformationally restricted analog of the influenza virus fusion peptide.

A conformationally restricted analog of the N-terminal 12-residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of Calpha-methyl-valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer-to-hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.     

Liposome composition effects on lipid mixing between cells expressing influenza virus hemagglutinin and bound liposomes

The involvement of contacting and distal lipid monolayers in different stages of protein-mediated fusion was studied for fusion mediated by influenza virus hemagglutinin. Inclusion of non-bilayer lipids in the composition of the liposomes bound to hemagglutinin-expressing cells affects fusion triggered by low pH. Lysophosphatidylcholine added to the outer membrane monolayers inhibits fusion. The same lipid added to the inner monolayer of the liposomes promotes both lipid and content mixing. In contrast to the inverted cone-shaped lysophosphatidylcholine, lipids of the opposite effective shape, oleic acid or cardiolipin with calcium, present in the inner monolayers inhibit fusion. These results along with fusion inhibition by a bipolar lipid that does not support peeling of one monolayer of the liposomal membrane from the other substantiate the hypothesis that fusion proceeds through a local hemifusion intermediate. The transition from hemifusion to the opening of an expanding fusion pore allows content mixing and greatly facilitates lipid mixing between liposomes and cells.     

Dolichol induces membrane leakage of liposomes composed of phosphatidylethanolamine and phosphatidylcholine

Dolichol promotes the leakage of membranes in liposomes composed of phosphatidylethanolamine and phosphatidylcholine but not liposomes composed only of phosphatidylcholine. The membrane leakage was assayed by measuring the entrapment of TEMPOcholine, a cationic spin probe, in liposomes using ESR methods. The percent of membrane leakage induced by dolichol was found to be linearly proportional to the concentrations of dolichol. It is proposed that dolichol enhances the formation of non-bilayer configurations in liposomes containing phosphatidylethanolamine, thereby membrane leakage.     

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.     

Novel lipid transfer property of two mitochondrial proteins that bridge the inner and outer membranes

This study provides evidence of a novel function for mitochondrial creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D). Both are basic peripheral membrane proteins with symmetrical homo-oligomeric structure, which in the case of MtCK was already shown to allow crossbridging of lipid bilayers. Here, different lipid dilution assays clearly demonstrate that both kinases also facilitate lipid transfer from one bilayer to another. Lipid transfer occurs between liposomes mimicking the lipid composition of mitochondrial contact sites, containing 30 mol % cardiolipin, but transfer does not occur when cardiolipin is replaced by phosphatidylglycerol. Ubiquitous MtCK, but not NDPK-D, shows some specificity in the nature of the lipids transferred and it is not active with phosphatidylcholine alone. MtCK can undergo reversible oligomerization between dimeric and octameric forms, but only the octamer can bridge membranes and promote lipid transfer. Cytochrome c, another basic mitochondrial protein known to bind to anionic membranes but not crosslinking them, is also incapable of promoting lipid transfer. The lipid transfer process does not involve vesicle fusion or loss of the internal contents of the liposomes.     

Transbilayer lipid diffusion promoted by Bax: implications for apoptosis

It is known that the proapoptotic protein Bax facilitates the formation of pores in bilayers, resulting in the release of proteins from the intermitochondrial space. We demonstrate that another consequence of the interaction of Bax with membranes is an increase in the rate of lipid transbilayer diffusion. We use two independent assays for transbilayer diffusion, one involving the formation of asymmetric liposomes by placing a pyrene-labeled lipid into the outer monolayer of preformed vesicles and another assay based on the initial preparation of liposomes having an asymmetric transbilayer distribution of lipids. With both methods we find that oligomeric BaxDeltaC or full-length Bax in the presence of tBid, but not monomeric full-length Bax, strongly promotes the rate of transbilayer diffusion. Although biological membranes exhibit rates of lipid transbilayer diffusion of minutes or less, they are able to maintain an asymmetric distribution of lipids across the bilayer. In the case of mitochondria, cardiolipin is sequestered on the inner leaflet of the inner mitochondrial membrane. However, during apoptosis this lipid translocates to the outer surface of the outer mitochondrial membrane. This phenomenon must involve an increase in the rate of transbilayer diffusion. The results of the present paper demonstrate that an activated form of Bax can cause this increased rate.     

The interaction of adriamycin with small unilamellar vesicle liposomes. A fluorescence study

The interaction of the antineoplastic agent adriamycin with sonicated liposomes composed of phosphatidylcholine alone and with small amounts (1-6%) of cardiolipin has been studied by fluorescence techniques. Equilibrium binding data show that the presence of cardiolipin increases the amount of drug bound to liposomes when the bilayer is below its phase transition temperature and when the ionic strength is relatively low (0.01 M). At higher ionic strength (0.15 M) and above the Tm (i.e. conditions which are closer to the physiological state) the binding of the drug to the two liposome types is nearly the same. Thus the differences in the interactions of adriamycin with cardiolipin-containing membranes, as opposed to those composed of phosphatidylcholine alone, are not due simply to increased binding but rather to an altered membrane structure when this lipid is present. Quenching of adriamycin fluorescence by iodide shows that bound drug is partially, but not completely, buried in the liposomal membrane. Both in the presence and absence of cardiolipin the bulk of the adriamycin is more accessible to the quencher below the Tm than above it; that is, a solid membrane tends to exclude the drug from deep penetration. Above the Tm, the presence of cardiolipin alters the nature of liposome-adriamycin interaction. Here the fluorescence quenching data suggest that the presence of small amounts of cardiolipin (3%) in a phosphatidylcholine matrix creates two types of binding environments for drug, one relatively exposed and the other more deeply buried in the membrane. The temperature dependence of the adriamycin fluorescence and the liposome light scattering reveal that cardiolipin alters the thermal properties of the bilayer as well as its interaction with adriamycin. At low ionic strength lateral phase separations may occur with both pure phosphatidylcholine and when 3% cardiolipin is present; under these conditions the bound adriamycin exists in two kinds of environment. It is notable that only adriamycin fluorescence reveals this phenomenon; thebulk property of liposome light scattering reports only on the overall membrane phase change. These data suggest that under certain conditions the drug binding sites in the membranes are decoupled from the bulk of the lipid bilayer.     

Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid

Cationic cell-penetrating peptides (CPPs) are a promising vehicle for the delivery of macromolecular drugs. Although many studies have indicated that CPPs enter cells by endocytosis, the mechanisms by which they cross endosomal membranes remain elusive. On the basis of experiments with liposomes, we propose that CPP escape into the cytosol is based on leaky fusion (i.e., fusion associated with the permeabilization of membranes) of the bis(monoacylglycero)phosphate (BMP)-enriched membranes of late endosomes. In our experiments, prototypic CPP HIV-1 TAT peptide did not interact with liposomes mimicking the outer leaflet of the plasma membrane, but it did induce lipid mixing and membrane leakage as it translocated into liposomes mimicking the lipid composition of late endosome. Both membrane leakage and lipid mixing depended on the BMP content and were promoted at acidic pH, which is characteristic of late endosomes. Substitution of BMP with its structural isomer, phosphatidylglycerol (PG), significantly reduced both leakage of the aqueous probe from liposomes and lipid mixing between liposomes. Although affinity of binding to TAT was similar for BMP and PG, BMP exhibited a higher tendency to support the inverted hexagonal phase than PG. Finally, membrane leakage and peptide translocation were both inhibited by inhibitors of lipid mixing, further substantiating the hypothesis that cationic peptides cross BMP-enriched membranes by inducing leaky fusion between them.     

Phosphorylation of mitochondrial phospholipid scramblase 3 by protein kinase C-delta induces its activation and facilitates mitochondrial targeting of tBid

Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Phosphatidylserine Decarboxylase 1 (Psd1) Promotes Mitochondrial Fusion by Regulating the Biophysical Properties of the Mitochondrial Membrane and Alternative Topogenesis of Mitochondrial Genome Maintenance Protein 1 (Mgm1)

Non–bilayer-forming lipids such as cardiolipin, phosphatidic acid, and phosphatidylethanolamine (PE) are proposed to generate negative membrane curvature, promoting membrane fusion. However, the mechanism by which lipids regulate mitochondrial fusion remains poorly understood. Here, we show that mitochondrial-localized Psd1, the key yeast enzyme that synthesizes PE, is required for proper mitochondrial morphology and fusion. Yeast cells lacking Psd1 exhibit fragmented and aggregated mitochondria with impaired mitochondrial fusion during mating. More importantly, we demonstrate that a reduction in PE reduces the rate of lipid mixing during fusion of liposomes with lipid compositions reflecting the mitochondrial membrane. This suggests that the mitochondrial fusion defect in the Δpsd1 strain could be due to the altered biophysical properties of the mitochondrial membrane, resulting in reduced fusion kinetics. The Δpsd1 strain also has impaired mitochondrial activity such as oxidative phosphorylation and reduced mitochondrial ATP levels which are due to a reduction in mitochondrial PE. The loss of Psd1 also impairs the biogenesis of s-Mgm1, a protein essential for mitochondrial fusion, further exacerbating the mitochondrial fusion defect of the Δpsd1 strain. Increasing s-Mgm1 levels in Δpsd1 cells markedly reduced mitochondrial aggregation. Our results demonstrate that mitochondrial PE regulates mitochondrial fusion by regulating the biophysical properties of the mitochondrial membrane and by enhancing the biogenesis of s-Mgm1. While several proteins are required to orchestrate the intricate process of membrane fusion, we propose that specific phospholipids of the mitochondrial membrane promote fusion by enhancing lipid mixing kinetics and by regulating the action of profusion proteins.     

tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation

Bid is a Bcl-2 family protein that promotes apoptosis by activating Bax and eliciting mitochondrial outer membrane permeabilization (MOMP). Full-length Bid is cleaved in response to apoptotic stimuli into two fragments, p7 and tBid (p15), that are held together by strong hydrophobic interactions until the complex binds to membranes. The detailed mechanism(s) of fragment separation including tBid binding to membranes and release of the p7 fragment to the cytoplasm remain unclear. Using liposomes or isolated mitochondria with fluorescently labeled proteins at physiological concentrations as in vitro models, we report that the two components of the complex quickly separate upon interaction with a membrane. Once tBid binds to the membrane, it undergoes slow structural rearrangements that result in an equilibrium between two major tBid conformations on the membrane. The conformational change of tBid is a prerequisite for interaction with Bax and is, therefore, a novel step that can be modulated to promote or inhibit MOMP. Using automated high-throughput image analysis in cells, we show that down-regulation of Mtch2 causes a significant delay between tBid and Bax relocalization in cells. We propose that by promoting insertion of tBid via a conformational change at the mitochondrial outer membrane, Mtch2 accelerates tBid-mediated Bax activation and MOMP. Thus the interaction of Mtch2 and tBid is a potential target for therapeutic control of Bid initiated cell death.     

Analysis of the perturbation of phospholipid model membranes by rhodanese and its presequence.

The ability of the cytoplasmically synthesized mitochondrial enzyme rhodanese and its putative import signal sequence to interact with model phospholipid membranes was characterized. Membrane perturbation assays were used to test a current hypothesis that the initial step in protein translocation may involve binding of signal sequences with membrane lipids. Here we show comparative studies on the effect of native and various forms of denatured rhodanese, as well as two peptides, rho(1-23) and rho(11-23), derived from its NH2-terminal sequence, on the perturbation of 6-carboxyfluorescein-containing large unilamellar vesicles composed of either cardiolipin, phosphatidylcholine, or phosphatidylserine. We monitored the degree of perturbation by measuring dye leakage and found differential perturbation by either peptide or protein. Unfolded rhodanese perturbed vesicles in the order phosphatidylserine > cardiolipin > phosphatidylcholine. Denatured rhodanese was approximately 25 times more effective (on a molar basis) than rho(1-23) in the disruption of anionic liposomes. Rho(11-23) was unable to perturb liposomes. We found an inverse correlation between degree of activity of rhodanese folding intermediates and their ability to perturb liposomes. On urea denaturation, enzymatic activity was completely lost before membrane perturbation ability reached significant levels. Analysis of the peptides by circular dichroism showed that anionic liposomes can induce alpha-helical structure only in rho(1-23) and denatured rhodanese. Intrinsic peptide fluorescence studies showed that only rho(1-23) and denatured rhodanese partitioned into these model membranes. Results obtained here imply that peptides from naturally occurring alpha-helical structures may need adjacent motifs for helical structure induction in lipid environments, and the subsequent secondary structure may, in turn, promote partitioning of these segments into the lipid phase and ultimately lead to membrane perturbation.     

Fusion of phosphatidylethanolamine-containing liposomes and mechanism of the L alpha-HII phase transition

The initial kinetics of fusion and leakage of liposomes composed of N-methylated dioleoylphosphatidylethanolamine (DOPE-Me) have been correlated with the phase behavior of this lipid. Gagné et al. [Gagné, J., Stamatatos, L., Diacovo, T., Hui, S. W., Yeagle, P., & Silvius, J. (1985) Biochemistry 24, 4400-4408] have shown that this lipid is lamellar (L alpha) below 20 degrees C, is hexagonal (HII) above 70 degrees C, and shows isotropic 31P NMR resonances at intermediate temperatures. This isotropic state is also characterized by complex morphological structures. We have prepared DOPE-Me liposomes at pH 9.5 and monitored the temperature dependence of the mixing of aqueous contents, leakage, and changes in light scattering upon reduction of the pH to 4.5. At and below 20 degrees C, where the lipid is in the L alpha phase, there is very little aggregation or destabilization of the liposomes. Between 30 and 60 degrees C, i.e., where the lipid is in the isotropic state, the initial rates of liposome fusion (mixing of aqueous contents) and leakage increase. At temperatures approaching that where the hexagonal HII phase transition occurs, the initial rates and extents of fusion decrease, whereas leakage is enhanced. Similar results were found for dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine (2:1) liposomes. These results clearly establish a common mechanism between the appearance of the isotropic state (between the L alpha and HII phases) and the promotion of liposome fusion. We propose a simple model to explain both the observed behavior of phosphatidylethanolamine-containing membranes with respect to liposome fusion and/or lysis and the beginning of the L alpha-HII phase transition.     

Membrane features and activity of GPI-anchored enzymes: alkaline phosphatase reconstituted in model membranes

The influence of membrane lipid environment on the activity of GPI-anchored enzymes was investigated with human placental alkaline phosphatase reconstituted by a detergent-dialysis technique in liposomes composed of palmitoyloleoylphosphatidylcholine, alone or in mixture with lipids enriched along with the protein within lipid rafts: cholesterol, sphingomyelin, and GM1 ganglioside. The highest V max was recorded for a phosphatidylcholine/10% GM1 mixture (143 +/- 5 nmol of substrate hydrolyzed per minute per microgram of protein), while the lowest for a phosphatidylcholine/30% cholesterol mixture and for raft-mimicking 1:1:1 phosphatidylcholine/sphingolipid/cholesterol liposomes (M:M:M) (57 +/- 3 and 52 +/- 3, respectively). No significant differences in K m were detected. The protein segregation, assessed using the chemical cross-linker bis(sulfosuccinimidyl)suberate, increased with the protein:lipid ratio, within the 1:1200-1:4800 protein:lipid molar ratio range, but did not affect enzyme activity. The activity decreased when the order of the lipid bilayers was increased, higher for those containing cholesterol, as judged by steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. Finally, the GPI-enzyme activity was affected by membrane curvature. This result was suggested by a strong inverse correlation (Pearson's correlation coefficient = 0.91; p < 0.0001) between activity and liposome diameter, measured by laser light scattering and ranging between 59 +/- 6 nm for a phosphatidylcholine/10% GM1 mixture (displaying the highest activity) and 188 +/- 25 nm for a phosphatidylcholine/30% cholesterol mixture and 185 +/- 23 nm for raft-mimicking liposomes (displaying the lowest activities). The activity-membrane curvature relationship was further confirmed by comparing the activity of proteoliposomes having different sizes but identical lipid compositions. These data open the possibility that the activity of GPI-anchored enzymes may be modulated by membrane microenvironment features, in particular by membrane curvature and cholesterol-enriched ordered microenvironments, such as those of lipid rafts.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: Implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Conformation of membrane-associated proapoptotic tBid

The proapoptotic Bcl-2 family protein Bid is cleaved by caspase-8 to release the C-terminal fragment tBid, which translocates to the outer mitochondrial membrane and induces massive cytochrome c release and cell death. In this study, we have characterized the conformation of tBid in lipid membrane environments, using NMR and CD spectroscopy with lipid micelle and lipid bilayer samples. In micelles, tBid adopts a unique helical conformation, and the solution NMR (1)H/(15)N HSQC spectra have a single well resolved resonance for each of the protein amide sites. In lipid bilayers, tBid associates with the membrane with its helices parallel to the membrane surface and without trans-membrane helix insertion, and the solid-state NMR (1)H/(15)N polarization inversion with spin exchange at the magic angle spectrum has all of the amide resonances centered at (15)N chemical shift (70-90 ppm) and (1)H-(15)N dipolar coupling (0-5 kHz) frequencies associated with NH bonds parallel to the bilayer surface, with no intensity at frequencies associated with NH bonds in trans-membrane helices. Thus, the cytotoxic activity of tBid at mitochondria may be similar to that observed for antibiotic polypeptides, which bind to the surface of bacterial membranes as amphipathic helices and destabilize the bilayer structure, promoting the leakage of cell contents.     

Interaction of Na2B12H11SH with dimyristoyl phosphatidylcholine liposomes

Previous investigations have revealed that the boron cluster compound Na₂B₁₂H₁₁SH (BSH) is very potent in causing major structural rearrangements of and leakage from phosphatidylcholine liposomes. This somewhat unexpected finding is interesting from a fundamental point of view and may also constitute the basis of future important pharmaceutical/medical applications of BSH. In order to further explore the BSH-lipid interaction, we have studied the effects caused by BSH on dimyristoyl phosphatidylcholine (DMPC) liposomes.Cryo-transmission electron microscopy showed that BSH induces aggregation, membrane rupture and increasing wall thickness of the liposomes. Differential scanning calorimetry revealed a BSH dependent shift of the gel to liquid crystalline phase transition temperature of DMPC. The zeta potential of the liposomes decreases with increasing BSH concentrations, and an apparent dissociation constant of 0.23 mM was found.BSH caused leakage of liposome-encapsulated carboxyfluorescein; leakage was higher at 23 °C (near the phase transition temperature) than at 15 °C and 37 °C. It induced lipid mixing only at very high concentrations.