Dioleoylphosphatidylserine Research Articles

A novel light-dependent activation of DAGK and PKC in bovine photoreceptor nuclei

In this work, we describe a selective light-dependent distribution of the lipid kinase 1,2-diacylglycerol kinase (EC 2.7.1.107, DAGK) and the phosphorylated protein kinase C alpha (pPKCα) in a nuclear fraction of photoreceptor cells from bovine retinas. A nuclear fraction enriched in small nuclei from photoreceptor cells (PNF), was obtained when a modified nuclear isolation protocol developed by our laboratory was used. We measured and compared DAGK activity as phosphatidic acid (PA) formation in PNF obtained from retinas exposed to light and in retinas kept in darkness using [γ-32P]ATP or [3H]DAG. In the absence of exogenous substrates and detergents, no changes in DAGK activity were observed. However, when DAGK activity assays were performed in the presence of exogenous substrates, such as stearoyl arachidonoyl glycerol (SAG) or dioleoyl glycerol (DOG), and different detergents (used to make different DAGK isoforms evident), we observed significant light effects on DAGK activity, suggesting the presence of several DAGK isoforms in PNF. Under conditions favoring DAGKζ activity (DOG, Triton X-100, dioleoyl phosphatidylserine and R59022) we observed an increase in PA formation in PNF from retinas exposed to light with respect to those exposed to darkness. In contrast, under conditions favoring DAGKɛ (SAG, octylglucoside and R59022) we observed a decrease in its activity. These results suggest different physiological roles of the above-mentioned DAGK isoforms. Western blot analysis showed that whereas light stimulation of bovine retinas increases DAGKζ nuclear content, it decreases DAGKɛ and DAGKβ content in PNF. The role of PIP2-phospholipase C in light-stimulated DAGK activity was demonstrated using U73122. Light was also observed to induce enhanced pPKCα content in PNF. The selective distribution of DAGKζ and ɛ in PNF could be a light-dependent mechanism that in vertebrate retina promotes selective DAG removal and PKC regulation.

In vivo optical imaging of brain tumors and arthritis using fluorescent SapC-DOPS nanovesicles.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

<em>In Vivo</em> Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10° steps over 380°. A rectangular region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

Bending rigidity of phosphatidylserine-containing lipid bilayers in acidic aqueous solutions

The elastic properties of anion lipid-containing bilayers were studied in aqueous solutions with fixed ionic strength at pH ≤5. Giant vesicles from stearoyl-oleoyl-phosphatidylcholine (SOPC) with various contents of dioleoyl-phosphatidylserine (DOPS) were obtained via electroformation and observed in phase contrast and fluorescence. Two structural phases were found to coexist in vesicle membranes. Thermal fluctuation spectroscopy of quasispherical vesicles with homogeneous liquid membranes was applied for the quantification of their bending rigidity. The white-noise contribution, significantly influencing the calculated value of the bending modulus, was taken into account in the analysis of the thermal shape fluctuations of vesicles. Electrokinetic measurements with SOPC/DOPS liposomes provided information about their surface potential dependence on pH and also as a function of the DOPS molar content in bilayers. The experimentally obtained values for the bending rigidity of SOPC bilayers containing various amounts of DOPS are reported and discussed in the light of the existing theory for the electrostatic contribution to the bending modulus of charged lipid membranes.

Dermcidin, an anionic antimicrobial peptide: influence of lipid charge, pH and Zn2+on its interaction with a biomimetic membrane

The mechanism of membrane permeabilization by dermcidin (DCD-1L), an antimicrobial peptide present in human sweat, was investigated at a mercury-supported monolayer of dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) and at a mercury-supported tethered bilayer lipid membrane (tBLM) consisting of a thiolipid (DPTL) with a DOPC or DOPS monolayer self-assembled on top of it. In an unbuffered solution of pH 5.4, DCD-1L is almost neutral and permeabilizes a DPTL/DOPS tBLM at transmembrane potentials, ϕtrans, which are physiological. In a pH 7 buffer solution DCD-1L bears two negative charges and has no effect on a DPTL/DOPC tBLM, whereas it permeabilizes a DPTL/DOPS tBLM only outside the physiological ϕtrans range; however, the presence of zinc ion induces DCD-1L to permeabilize the DPTL/DOPS tBLM at physiological ϕtrans values. The effect of zinc ions suggests a DCD-1L conformation with its positive N-terminus embedded in the lipid bilayer and the negative C terminus floating on the membrane surface. This conformation can be stabilized by a zinc ion bridge between the His(38) residue of the C terminus and the carboxyl group of DOPS. Chronocoulometric potential jumps from ϕtrans ≅ +160 mV to sufficiently negative values yield charge transients exhibiting a sigmoidal shape preceded by a relatively long "foot". This behavior is indicative of ion-channel formation characterized by disruption of DCD-1L clusters adsorbed on top of the lipid bilayer, incorporation of the resulting monomers and their aggregation into hydrophilic pores by a mechanism of nucleation and growth.

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.

New proteoliposome vaccine formulation from N. meningitidis serogroup B, without aluminum hydroxide, retains its antimeningococcal protectogenic potential as well as Th-1 adjuvant capacity

Proteoliposomes purified from the Outer Membrane of Neisseria meningitidis B, have been successfully used as core for adjuvants and vaccine formulations. We have tried to increase their structural definition and to conserve their efficacy and stability avoiding the addition of the aluminum hydroxide to the final formulation. Liposomal particle systems were prepared from components of defined molecular structure, such as a Neisseria meningitidis B protein complex, extracted and purified without forming vesicle structures. Liposomes were prepared from a mixture of dioleoyl phosphatidyl serine and cholesterol, using the classical dehydration-rehydration method. Transmission Electron Microscopy (TEM) was used to characterize the liposomes. BALB/c mice were used for animal testing procedures. Analysis of specific IgG response, serum bactericidal activity as well as DTH reaction was carried out. Isolation and purification of mRNA and real-time PCR, was performed to determine the dominating Th lymphokine pattern. The new antimeningococcal formulation without aluminum hydroxide prepared with components of defined molecular structure assembled itself into Neoproteoliposomes (NPL) ranging from 50 to 70 nm in diameter. The extraction and purification of selected membrane proteins to provide the antigen for this new formulation (PD-Tp), as well as the NPL-formulation favors a Th1 response pattern, suggested by the higher percentages of DTH, increased expression of proinflamatory lymphokine mRNAs when administered by intramuscular and intranasal routes. It stimulates a systemic bactericidal antibody response against Neisseria meningitidis B and immunologic memory similar to the Cuban VA-MENGOC-BC® vaccine, even at lower dosages and is less reactogenic at the injection site in comparison with the formulation with aluminum hydroxide. This new adjuvant formulation could be applicable to the development of new and improved vaccines against meningococcal disease, and eventually as modulators of the immune response against other diseases.

Interactions of antileishmanial drugs with monolayers of lipids used in the development of amphotericin B–miltefosine-loaded nanocochleates

The emergence of strains of Leishmania resistant to existing drugs complicates the treatment of life-threatening visceral leishmaniasis. The development of new lipid formulation (nanocochleates), containing two active drugs: amphotericin B (AmB) and miltefosine (hexadecylphosphocholine, HePC), could increase effectiveness, decrease toxicity and reduce the risk of appearance of resistance. Nanocochleates are cigar-shaped structures of rolled negatively charged lipid bilayers bridged by calcium, prepared from dioleoylphosphatidylserine (DOPS) and cholesterol (Cho) and able to accommodate drugs. To determine the interaction, the orientation and the stability of the amphiphilic drugs in the lipid mixture and the optimal drugs/lipids ratio, the Langmuir film balance and BAM (Brewster angle microscopy) were used. The drugs were mixed with the lipids (DOPS or 9DOPS/1Cho) and spread at the air-water interface. A stability study showed that DOPS maintained HePC at the interface at low molar fraction of HePC; this effect became more marked in the presence of Cho. The fact that HePC can be stably associated with the monolayer at low molar fraction (below 10%) suggests that in the nanocochleates HePC is inserted between the lipid molecules rather than between the bilayers. Phase diagrams and BAM images showed that, even at low pressure, DOPS maintains AmB at low molar fraction (below 10%) in the “erect” rather than the horizontal form at the interface and that the presence of Cho reinforces this effect. These results allowed us to predict the organization and the orientation of these drugs in the nanocochleates and to determine the optimal drugs/lipids ratio.

A dual-scale model for the caveolin-mediated vesiculation

Caveolae-mediated vesiculation refers to the ∼100 nm cave-like plasma membrane invaginations, which function as macromolecular transport vesicles that regulate cellular function. The initiation of vesiculation is closely associated with protein–protein and protein–membrane interactions, yet their roles in the caveolae budding process are not well-understood. We here propose a dual-scale model, which allows us to identify a unique role for the caveolin–dioleoylphosphatidylserine (DOPS) interaction and the high-order oligomer of caveolins. An energy analysis reveals that the protein–lipid affinity and oligomerization can provide enough energy to increase the local curvature within membrane vesicle formation. Membrane evolution simulations also propose that the radius of the mature vesicle is intrinsic and relies on the molecular interactions, regardless of the oligomer number and surface tension. By introducing molecular interactions at the nanoscale into cellular events occurring in the microscale, the present dual-scale model sheds light on how molecular interactions can change the membrane vesiculation.

Abstract 1840: SapC-DOPS nanovesicles: Acidic lipid domain-targeted agents for cancer therapy

Abstract Introduction: Acidic phosphatidylserine-enriched membrane microdomains (PS-EMMs) are exposed on the membrane surface of cancer cells and tumor blood vessels. We have explored a novel PS-EMMs targeting mechanism by saposin C (SapC)-coupled dioleoylphosphatidylserine (DOPS) nanovesicles that are found to specifically target and destroy cancer and tumor-associated endothelial cells with little or no systemic toxicity (Qi et al. Clin. Cancer Res. 2009; Kaimal et al. Mol. Imaging Biol. 2011). Methods: We have developed a novel biotheragnositic agent, SapC-DOPS, which we assembled into cancer-selective nanovesicles. Surface exposed PS levels were analyzed by a flow cytometric method using fluorescently labeled annexin-V. Deparafinized tissue slides were stained using far-red fluorophore (CellVue Maroon, CVM) labeled SapC-DOPS nanovesicles, SapC-DOPS-CVM was used to determine cancer-selective targeting via PS-mediated membrane fusion in human cancer cells, patient-derived cancer tissue specimens, and preclinical animal tumor models, including brain, pancreatic, lung, and pediatric cancers. The cytotoxicity of SapC-DOPS was measured using the MTT assay. Live imaging for visualization of SapC-DOPS PS-targeting and survival curves were demonstrated in transgenic and xenograft mouse tumor models. Results: We demonstrated the specific targeting of SapC-DOPS in paraffin embedded patient-derived tumor tissue sections. Based on MTT assay and annexin-V labeled flow cytometry, we revealed that SapC-DOPS cytotoxic effects correlated to the level of surface exposed PS in a variety of human cancer cell lines. Kinetic studies indicated that SapC-DOPS fused with tumor cell membrane within a few minutes, followed by internalization into the cells. In vivo cancer cell targeting of SapC-DOPS was blocked by pretreatment with PS-specific binding proteins (Lact-C2 and beta-2 glycoprotin-1). Increased PS expression and externalization on cancer cell membrane surfaces after chemotherapy or extracellular stresses enhanced the SapC-DOPS response. SapC-DOPS showed cancer-selective targeting in numerous spontaneously formed and xenografted tumors in living mice using either a double-tracking optical or a contrast-enhanced magnetic resonance imaging system. Furthermore, we observed that tumor-bearing animals treated with intravenous SapC-DOPS had significant tumor reduction and increased survival. Tumor-free survivals were confirmed by tumor-luminescence imaging and at necropsy. Conclusion: We have demonstrated a novel biomarker for numerous human cancers that can be effectively targeted by using cancer-selective SapC-DOPS nanovesicles. The direct confirmation of tumor-targeting by SapC-DOPS provides powerful new evidence in support of our goal to develop PS-EMMs targeted nanovesicles as novel therapeutic agents for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1840. doi:1538-7445.AM2012-1840

Study of interaction of long-chain n-alcohols with fluid DOPC bilayers by a lateral pressure sensitive fluorescence probe

The excimer 1,2-dipyrenedecanoyl-sn-glycero-3-phosphatidylcholine (dipy10PC) fluorescence probe was used to determine effects of aliphatic alcohols (CnH2n+1OH, n = 12-18 is the even number of carbons in alkyl chain) on fluid dioleoylphosphatidylcholine (DOPC) +dioleoylphosphatidylserine (DOPS) bilayers in multilamellar vesicles at molar ratio DOPC/DOPS = 24.7. The excimer to monomer fluorescence intensity ratio increases with the increase of CnH2n+1OH/DOPC molar ratio and decreases with the CnH2n+1OH alkyl chain length n at a constant CnH2n+1OH/DOPC = 0.4 molar ratio. These effects indicate changes in the bilayer lateral pressure on the level of pyrenyl moieties location.

Membrane Disruption by Antimicrobial Peptide Protegrin-1 is Tuned by Incorporation of Cholesterol and Phosphoethanolamine Lipids

Antimicrobial peptides (AMPs) are a class of small (less than 100 amino acid residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. To understand the mechanism of membrane disruption by AMPs, we investigated, via atomic force microscopy, topological changes induced by protegrin-1 (PG-1), an 18-residue, cationic, β-sheet AMP isolated from pig leukocytes, in supported phospholipid bilayers (SPBs). Lipid mixtures of dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylcholine (DOPC), and cholesterol were used to mimic eukaryotic cell membranes while bacterial cell membranes were emulated by substitution of cholesterol for dioleoylphosphatidylethanolamine (DOPE). We have previously shown that AMP disruption of zwitterionic dimyristoylphosphatidylcholine (DMPC) SPBs induce concentration dependent structural transformations that progress from fingerlike instabilities at bilayer edges, to the formation of sievelike nanoporous structures, and finally to a network of wormlike micellar structures. The observed transformations suggest that the peptides act to lower the interfacial energy of the bilayer in a manner similar to detergents. Detergent solubilization of membranes encompass processes such as pore formation, blebbing, budding, and vesiculation that share common saddle-splay (“negative Gaussian”) curved topologies. We pose that membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups enhance the efficacy of AMP disruption while those membranes containing cholesterol retard disruption. Results have shown that cholesterol incorporation shifts the disruption susceptibility of a bilayer to a higher peptide dosage regime while an opposite effect is observed in the presence of PE. The observed trend sheds light on a lingering debate as to how nature has evolved AMPs to discriminate between host and pathogen.

Kinetics of low pH-induced lamellar to bicontinuous cubic phase transition in dioleoylphosphatidylserine/monoolein

Recently, it has been well recognized that the modulation of electrostatic interactions due to surface charges can induce transitions between lamellar liquid-crystalline (L(α)) and inverse bicontinuous double-diamond cubic (Q(II)(D)) phases in biological lipids. To reveal their kinetic pathway and mechanism, we investigated the low pH-induced L(α) to Q(II)(D) phase transitions in 20%-dioleoylphosphatidylserine (DOPS)/80%-monoolein (MO) using time-resolved small-angle x-ray scattering and a rapid mixing method. At a final pH of 2.6-2.9, the L(α) phase was transformed completely into the hexagonal II (H(II)) phase within 2-10 s after mixing a low pH buffer with a suspension of multilamellar vesicles of 20%-DOPS∕80%-MO (the initial step). Subsequently, the H(II) phase slowly converted into the Q(II)(D) phase and completely disappeared within 15-30 min (the second step). The rate constants of the second step were obtained using the singular value decomposition analysis. On the basis of these data, we discuss the underlying mechanism of the kinetic pathway of the low pH-induced L(α) to Q(II)(D) phase transitions.

Toll-like receptor 4 can recognize SapC-DOPS to stimulate macrophages to express several cytokines

SapC-DOPS is a newly combined compound consisting of saposin C and dioleoylphosphatidylserine (DOPS). Our recent study showed that SapC-DOPS exhibits anti-tumor activity. However, SapC-DOPS has recognition elements of Toll-like receptor (TLR) 2 and TLR4; therefore, we want to know whether SapC-DOPS can induce abnormal immunoreaction via identification TLRs. We investigated the capacity of SapC-DOPS to induce cytokines in vivo and in vitro and analyzed the involvement of TLR and NF-kB in these cytokines production. SapC-DOPS could activate the cytokine production by peripheral macrophages, enhance the expressions of TLR4 and stimulate the NF-κB nuclear translocation. PDTC, an NF-κB inhibitor, could decrease the SapC-DOPS inducible TNF-α and IL-1β production. SapC-DOPS was similar to LPS in the immune response and may induce the production of cytokines in macrophages via the TLR4 signaling pathway and, at least in part, the alteration of the NF-κB pathway.

A molecular dynamics study of the early stages of amyloid-β(1-42) oligomerization: The role of lipid membranes

As research progresses toward understanding the role of the amyloid-beta (Abeta) peptide in Alzheimer's disease, certain aspects of the aggregation process for Abeta are still not clear. In particular, the accepted constitution of toxic aggregates in neurons has shifted toward small oligomers. However, the process of forming these oligomers in cells is also not full clear. Even more interestingly, it has been implied that cell membranes, and, in particular, anionic lipids within those membranes, play a key role in the progression of Abeta aggregation, but the exact nature of the Abeta-membrane interaction in this process is unknown. In this work, we use a thermodynamic cycle and umbrella sampling molecular dynamics to investigate dimerization of the 42-residue Abeta peptide on model zwitterionic dipalmitoylphosphatidylcholine (DPPC) or model anionic dioleoylphosphatidylserine (DOPS) bilayer surfaces. We determined that Abeta dimerization was strongly favored through interactions with the DOPS bilayer. Further, our calculations showed that the DOPS bilayer promoted strong protein-protein interactions within the Abeta dimer, whereas DPPC favored strong protein-lipid interactions. By promoting dimer formation and subsequent dimer release into the solvent, the DOPS bilayer acts as a catalyst in Abeta aggregation.

Kinetics of Myosin-I-Membrane Detachment Under Load

Myo1c is a single-headed motor that links cell membranes to the underlying actin cytoskeleton. Actin binding occurs via the motor domain, while the tail domain interacts with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) through a putative pleckstrin homology (PH) domain. In this study, we measured the strength and lifetime of the myo1c attachment to spherical supported lipid bilayers (SSL) composed of 1 μm diameter silica or polystyrene beads coated with various mole fractions of PI(4,5)P2, dioleoyl-phosphatidylcholine (DOPC), and dioleoyl-phosphatidylserine (DOPS). The SSLs trapped by a laser beam were brought into contact with immobilized spherical (2 μm) silica pedestals sparsely labeled with the myo1c tail domain. Upon repeated contact and retraction cycles, binding events and subsequent rupture forces were measured. The most probable rupture forces (< 10 pN) from membranes containing 2% PI(4,5)P2 were found to be largely independent of the loading rate (pN/s). Nevertheless, the frequency of single myo1c molecules interacting with membranes containing 2% PI(4,5)P2 - 98% DOPC was considerably higher than with membranes that contained 20 - 80% DOPS. Lifetime measurements of myo1c attachment to 2% PI(4,5)P2 - 98% DOPC membranes under constant pulling forces (1 - 3 pN) were fitted to Bell's equation and the extrapolated duration of the bond at zero force was found to be ∼ 25 ms. Although phosphoinositide binding is crucial for the proper cellular targeting of myo1c, our results suggest that it is unlikely that this connection is a suitable anchor for force generation.

The Effect of Hydrophilic Substitutions and Anionic Lipids upon the Transverse Positioning of the Transmembrane Helix of the ErbB2 (neu) Protein Incorporated into Model Membrane Vesicles

The sequence of the transmembrane (TM) helix of ErbB2, a member of the epidermal growth factor receptor (ErbB) family, can influence its activity. In this report, the sequence and lipid dependence of the transverse position of a model-membrane-inserted peptides containing the ErbB2 TM helix and some of the juxtamembrane (JM) residues were studied. For the ErbB2 TM helix inserted into phosphatidylcholine vesicles, the activating V664E mutation was found to induce a transverse shift involving the movement of the E residue toward the membrane surface. This shortened the effective length of the TM-spanning portion of the sequence. The transverse shift was observed with the E664 residue in both the uncharged and charged states, but the extent of the shift was larger when the E residue was charged. When a series of hydrophilic residues was substituted for V664, the resulting transverse shifts at pH 7.0 decreased in the order D,H>E>Q>K>G>V. Except for His, this order is strongly correlated to that reported for the degree to which these substitutions induce cellular transformation when introduced into full-length ErbB2. To examine the effect of lipid on transverse shift, we studied the uncharged V664Q mutation. The presence of 20% of the anionic lipid DOPS (dioleoylphosphatidylserine) in the model membrane vesicles, which introduces a physiologically relevant level of anionic lipid, did not affect the degree of transverse shift. However, in the case of a peptide containing a V674Q substitution, in which the Q is closer to the C-terminus of the ErbB2 TM helix than the N-terminus, transverse shift was suppressed in vesicles containing 20% DOPS. This suggests that the interaction of the cationic JM residues flanking the C-terminus of the ErbB2 TM helix interact with anionic lipids to anchor the C-terminal end of the TM helix. This anchoring site may act as a pivot that amplifies transverse movements of the ErbB2 TM segment to induce a large swinging-type motion in the extracellular domain of the protein, affecting ErbB2 activity. Interactions interrupting C-terminal JM residue association with anionic lipid might partly impact ErbB2 activity by disrupting this pivoting.

A Molecular Dynamics Study on the Binding and Interaction of the Amyloid-Beta (1-42) Peptide with Phospholipid Bilayers

The Amyloid-Beta (A-Beta) peptide is a key aggregate species in Alzheimer's disease. While aspects of the A-Beta peptide aggregation pathway have previously been elucidated, the initial conversion of monomer peptides into an oligomer during aggregation is not clearly understood. One potential mediator of these early stages of aggregation is interactions of A-Beta with cell membranes, particularly anionic cell membranes. We use unconstrained and umbrella sampling molecular dynamics simulations to investigate interactions between the 42-amino acid A-Beta peptide and model bilayers consisting of zwitterionic dipalmitoylphophatidylcholine (DPPC) lipids and anionic dioleoylphosphatidylserine (DOPS) lipids. From this work, we determine that A-Beta binds to the surface of DPPC and DOPS bilayers over the small length scales used in simulations. Our results also support the hypothesis that the charge on the bilayer surface and on the peptide affects both the free energy of peptide-membrane binding and the distribution of the peptide on the bilayer surface. Finally, no significant secondary structure change is observed in the peptide during the timescales used in these simulations. This result may indicate all-atom simulation times are too short to observe secondary structure changes in this system or that structure change during the oligomerization process requires peptide-peptide interactions. Our work demonstrates that interactions between the A-Beta peptide and lipid bilayers promote a peptide distribution on the bilayer surface that is prone to peptide-peptide interactions, which can influence the propensity of A-Beta to aggregate into higher order structures.

Interaction Between Amyloid- β (1–42) Peptide and Phospholipid Bilayers: A Molecular Dynamics Study

The amyloid- β (A β) peptide is a key aggregate species in Alzheimer's disease. Although important aspects of A β peptide aggregation are understood, the initial stage of aggregation from monomer to oligomer is still not clear. One potential mediator of this early aggregation process is interactions of A β with anionic cell membranes. We used unconstrained and umbrella sampling molecular dynamics simulations to investigate interactions between the 42-amino acid A β peptide and model bilayers of zwitterionic dipalmitoylphosphatidylcholine (DPPC) lipids and anionic dioleoylphosphatidylserine (DOPS) lipids. Using these methods, we determined that A β is attracted to the surface of DPPC and DOPS bilayers over the small length scales used in these simulations. We also found supporting evidence that the charge on both the bilayer surface and the peptide affects the free energy of binding of the peptide to the bilayer surface and the distribution of the peptide on the bilayer surface. Our work demonstrates that interactions between the A β peptide and lipid bilayer promotes a peptide distribution on the bilayer surface that is prone to peptide-peptide interactions, which can influence the propensity of A β to aggregate into higher-order structures.

Isolation of Stable (αβ)4-Tetraprotomer from Na+/K+-ATPase Solubilized in the Presence of Short-Chain Fatty Acids

Previously, it was demonstrated that acetate anions increase the higher oligomer (H), consuming (alphabeta) 2-diprotomer (D) and alphabeta-protomer (P) of solubilized dog kidney Na (+)/K (+)-ATPase [ Kobayashi, T. et al. (2007) J. Biochem. 142, 157-173 ]. Presently, short-chain fatty acids, such as propionate (Prop) and butyrate, have been substituted effectively for acetate. The molecular weight of 6.01 x 10 (5) for H and quantitative Na (+)/K (+)-dependent interconversion among H, D, and P showed that H was an (alphabeta) 4-tetraprotomer (T). T was optimally isolated from the enzyme solubilized in aqueous 40 mM K (+)Prop at pH 5.6 by gel chromatography performed at 0 degrees C with elution buffer containing synthetic dioleoyl phosphatidylserine (PS). K 0.5 values of K (+)-congeners constituting K (+)Prop for the maximal amount of T were NH 4 (+) >> Rb (+) congruent with K (+) > Tl (+), while Na (+) had no effect. The oligomers of T, D, and P were simultaneously assayed for ATPase upon elution from the gel column, resulting in a specific activity ratio of 1:2:2. The activity of the chromatographically isolated T increased with an increasing dioleoyl PS, giving a saturated activity of 2.38 units/mg at pH 5.6 and 25 degrees C, and the active enzyme chromatography of T showed 34% dissociation into D by exposing it at 25 degrees C. On the basis of these data, the specific ATPase activities of T, D, and P were concluded to be 32, 65, and 65 units/mg, respectively, under the conventionally optimal conditions of pH 7.3 and 37 degrees C, suggesting an equivalence to a fully active enzyme for D and P but half activity for T. The physiological significance of the stable form of T remains to be investigated.