Phosphatidylserine Headgroups Research Articles

Lipid-Protein Nanodiscs Offer New Perspectives for Structural and Functional Studies of Water-Soluble Membrane-Active Peptides

Lipid-protein nanodiscs (LPNs) are nanoscaled fragments of a lipid bilayer stabilized in solution by the apolipoprotein or a special membrane scaffold protein (MSP). In this work, the applicability of LPN-based membrane mimetics in the investigation of water-soluble membrane-active peptides was studied. It was shown that a pore-forming antimicrobial peptide arenicin-2 from marine lugworm (charge of +6) disintegrates LPNs containing both zwitterionic phosphatidylcholine (PC) and anionic phosphatidylglycerol (PG) lipids. In contrast, the spider toxin VSTx1 (charge of +3), a modifier of Kv channel gating, effectively binds to the LPNs containing anionic lipids (POPC/DOPG, 3 : 1) and does not cause their disruption. VSTx1 has a lower affinity to LPNs containing zwitterionic lipids (POPC), and it weakly interacts with the protein component of nanodiscs, MSP (charge of -6). The neurotoxin II (NTII, charge of +4) from cobra venom, an inhibitor of the nicotinic acetylcholine receptor, shows a comparatively low affinity to LPNs containing anionic lipids (POPC/DOPG, 3 : 1 or POPC/DOPS, 4 : 1), and it does not bind to LPNs/POPC. The obtained data show that NTII interacts with the LPN/POPC/DOPS surface in several orientations, and that the exchange process among complexes with different topologies proceeds fast on the NMR timescale. Only one of the possible NTII orientations allows for the previously proposed specific interaction between the toxin and the polar head group of phosphatidylserine from the receptor environment (Lesovoy et al., Biophys. J. 2009. V. 97. № 7. P. 2089-2097). These results indicate that LPNs can be used in structural and functional studies of water-soluble membrane-active peptides (probably except pore-forming ones) and in studies of the molecular mechanisms of peptide-membrane interaction.

Tissue Factor Residues That Putatively Interact with Membrane Phospholipids

Blood clotting is initiated by the two-subunit enzyme consisting of the plasma protease, factor VIIa (the catalytic subunit), bound to the integral membrane protein, tissue factor (the regulatory subunit). Molecular dynamics simulations have predicted that certain residues in the tissue factor ectodomain interact with phosphatidylserine headgroups to ensure optimal positioning of the tissue factor/factor VIIa complex relative to its membrane-bound protein substrates, factors IX and X. In this study, we individually mutated to alanine all the putative phosphatidylserine-interactive residues in the tissue factor ectodomain and measured their effects on tissue factor cofactor function (activation of factors IX and X by tissue factor/factor VIIa, and clotting of plasma). Some tissue factor mutants exhibited decreased activity in all three assays, with the most profound defects observed from mutations in or near the flexible loop from Lys159 to Gly164. The decreased activity of all of these tissue factor mutants could be partially or completely overcome by increasing the phosphatidylserine content of tissue factor-liposomes. Additionally, yeast surface display was used to screen a random library of tissue factor mutants for enhanced factor VIIa binding. Surprisingly, mutations at a single amino acid (Lys165) predominated, with the Lys165→Glu mutant exhibiting a 3-fold enhancement in factor VIIa binding affinity. Our studies reveal the functional contributions of residues in the C-terminal half of the tissue factor ectodomain that are implicated in interacting with phosphatidylserine headgroups to enhance tissue factor cofactor activity, possibly by allosterically modulating the conformation of the adjacent substrate-binding exosite region of tissue factor.

Membrane Regulation and Signal Transduction by Annexin A5

Annexins are an abundant family of proteins well known for their Ca2+-dependent membrane binding ability. Though they've been implicated in numerous membrane processes their method of action and signal integration is largely unknown. Here we propose a mechanism for annexin to integrate information regarding Ca2+ concentration and resting distribution of lipid and cholesterol in one leaflet, and transduce that information across the bilayer. In a system poised near random distribution, such as lipids of the inner leaflet of the plasma membrane, order can be imposed through even weak interactions. We've previously shown annexin's weak Ca2+-independent membrane binding acts to create a cooperative Ca2+ binding response. In this work we demonstrate not only annexin's ability to respond to and integrate information of membrane composition; but also its ability change and regulate that distribution. Protein interactions with membranes can induce membrane order and domain formation if the protein has specificity for individual lipids distributed non-ideally, or even randomly throughout the membrane. A protein that preferentially binds a free component of the membrane alters the amount of that component within other membrane domains. Annexin perturbs membrane distribution through its preferential binding and sequestration of phosphatidylserine headgroups (PS). Cholesterol perturbs phospholipid physical properties and preferentially forms complexes with PS. Then the local sequestering of PC-cholesterol complexes, and annexin-induced membrane domain formation upon Ca2+ influx, can act as the driving force for cholesterol redistribution across the bilayer. Using ITC as well as fluorescent probes such as DHE (cholesterol analogue) and carboxyfluorescein encapsulating LUVs. We show annexin's binding of cholesterol-PS complexes induces differential Ca2+ binding sites, and redistributes cholesterol throughout the bilayer without disrupting membrane permeability. Together this suggests that annexin has the capacity to redistribute cholesterol across the bilayer in a calcium-ion and membrane composition dependent manner.

Dynamics of Neurotoxin II Provides Adaptivity of its Functional Sites

The details of protein internal motions could be of crucial importance to understanding its function. Such a details can't be directly obtained by conventional NMR methods: the set of NMR structures and relaxation order parameters do not provide motional models. One of the viable approaches is the analysis of molecular dynamics trajectories verified by NMR structural and relaxation data sets. The technique was implemented to the peripheral protein Neurotoxin II (NTII). High-quality experimental structures of 13С/15N labeled NTII were used as starting points for a set of MD trajectories covering a total of 7 microseconds. 3JHNHA, 3JHAHB and 3JHBHG coupling constants together with backbone and sidechain 15N-/13C-relaxation rates, heteronuclear NOEs and chemical shifts were back-calculated from MD trajectories and found to be in a good agreement with experimental data. The analysis of MD trajectories revealed a number of highly flexible protein regions with locally correlated motions in the nanosecond time scale. Two of the regions, as was shown previously, provide NTII binding to the environment of the Nicotinic Acetylcholine Receptor (nAChR) and to the receptor itself. Flexibility of protein binding sites could lead to their adaptivity favoring binding to target molecules. In the case of NTII two flexible binding sites of different targeting (membrane binding site and nAChR inhibition site) act successively. High flexibility of the binding site of NTII provides its adaptivity to the specific interaction with the lipid head groups of phosphatidylserine in the environment of nAChR enabling the membrane catalysis mechanism for NTII/nAChR interaction. Then high flexibility and, accordingly, adaptivity of the nAChR inhibition site of NTII plays its role in the final step of the membrane catalysis - high affinity binding of NTII to nAChR.

Synthesis of biomembrane-mimic polymers with various phospholipid head groups

Lipid bilayers in biomembranes consist of diverse phospholipids, including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) with various compositions according to the cell and tissue types. We synthesized biomembrane-mimic polymers, poly(2-methacryloyloxyethyl phosphoric acid) (PMPA), poly(2-methacryloyloxyethyl phosphorylethanolamine) (PMPE), and poly(2-methacryloyloxyethyl phosphorylserine) (PMPS), with PA, PE, and PS head groups, respectively. PA monomer was synthesized from 2-hydroxyethyl methacrylate (HEMA) and dimethyl chlorophosphate (DCP). PE and PS monomers were synthesized from N-tert-butoxycarbonyl (tBoc) protected ethanolamine and serine through the reaction with 2-chloro-2-oxo-1,3,2-dioxaphospholane (COP). Each biomembrane-mimic polymer was successfully synthesized by atom transfer radical polymerization (ATRP) from the monomer. The molecular weight distributions of PMPA, PMPE, and PMPS were analyzed by gel permeation chromatography (GPC) and in vitro cytotoxicity was also examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. The new biomembrane-mimic polymers could be used to prepare a polymeric platform that mimic a cell- or tissue-specific membrane for future applications in biomedical fields such as tissue engineering or bioimplants.

Lipid Flip-Flop in Binary Membranes Composed of Phosphatidylserine and Phosphatidylcholine

The kinetics and thermodynamics of lipid flip-flop in bilayers composed of 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were studied using sum-frequency vibrational spectroscopy. The kinetics of DSPC and DPPS flip-flop were examined as a function of temperature and bilayer composition. The rate of DSPC flip-flop did not exhibit any significant dependence on bilayer composition while the rate of DPPS flip-flop was inversely dependent on the mole fraction of DPPS. The transition-state thermodynamics for DSPC and DPPS lipids in these mixed bilayers were determined in order to identify the energetic impact of the phosphatidylserine headgroup on lipid flip-flop. The thermodynamics for the DSPC component remained statistically identical to bilayers composed entirely of DSPC. The activation energy for the DPPS component showed a linear correlation with the mole fraction of DPPS for all bilayer compositions. The enthalpy and entropy for DPPS flip-flop did not increase linearly with the fraction of DPPS but did directly correlate with the molecular area. The DPPS component also exhibited enthalpy-entropy compensation which suggests that lipid hydration may play a significant role in membrane dynamics.

Factor VII and Protein C Are Phosphatidic Acid-Binding Proteins

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Structural Properties of Model Phosphatidylcholine Flippases

Lipid translocation from one lipid bilayer leaflet to the other, termed flip-flop, is required for the distribution of newly synthesized phospholipids during membrane biogenesis. However, a dedicated biogenic lipid flippase has not yet been identified. Here, we show that the efficiency by which model transmembrane peptides facilitate flip of reporter lipids with different headgroups critically depends on their content of helix-destabilizing residues, the charge state of polar flanking residues, and the composition of the host membrane. In particular, increased backbone dynamics of the transmembrane helix relates to its increased ability to flip lipids with phosphatidylcholine and phosphatidylserine headgroups, whereas a more rigid helix favors phosphatidylethanolamine flip. Further, the transmembrane domains of many SNARE protein subtypes share essential features with the dynamic model peptides. Indeed, recombinant SNAREs possess significant lipid flippase activity.

An in vivo molecular imaging probe 18F-Annexin B1 for apoptosis detection by PET/CT: preparation and preliminary evaluation

There is an increasing need to develop non-invasive molecular imaging strategies for visualizing and quantifying apoptosis status of diseases (especially for cancer) for diagnosis and monitoring treatment response. Since externalization of phosphatidylserine (PS) is one of the early molecular events during apoptosis, Annexin B1 (AnxB1), a member of Annexins family with high affinity toward the head group of PS, could be a potential positron emission tomography (PET) imaging probe for imaging cell death process after labeled by positron-emitting nuclides, such as (18)F. In the present study, we investigated a novel PET probe, (18)F-labeled Annexin B1 ((18)F-AnxB1), for apoptosis imaging. (18)F-AnxB1 was prepared reliably by conjugating AnxB1 with a (18)F-tag, N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB), in a radiolabeling yield of about 20 % within 40 min. The in vitro binding of (18)F-AnxB1 with apoptotic cells induced by anti-Fas antibody showed twofold increase compared to those without treatment, confirmed by flow cytometric analysis with AnxV-FITC/PI staining. Stability tests demonstrated (18)F-AnxB1 was rather stable in vitro and in vivo without degradation. The serial (18)F-AnxB1 PET/CT scans in healthy rats outlined its biodistribution and pharmacokinetics, indicating a rapid renal clearance and predominant accumulation into kidney and bladder at 2 h p.i. (18)F-AnxB1 PET/CT imaging was successfully applied to visualize in vivo apoptosis sites in tumor induced by chemotherapy and in kidney simulated by ischemia-reperfusion injury. The high-contrast images were obtained at 2 h p.i. to delineate apoptotic tumor. Apoptotic region could be still identified by (18)F-AnxB1 PET 4 h p.i., despite the high probe retention in kidneys. In summary, we have developed (18)F-AnxB1 as a PS-specific PET probe for the apoptosis detection and quantification which could have broad applications from disease diagnosis to treatment monitoring, especially in the cases of cancer.

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.

Structural basis of the strict phospholipid binding specificity of the pleckstrin homology domain of human evectin-2

Evectin-2 is a recycling endosomal protein involved in retrograde transport. Its primary sequence contains an N-terminal pleckstrin homology (PH) domain and a C-terminal hydrophobic region. The PH domain of evectin-2 can specifically bind phosphatidylserine, which is enriched in recycling endosomes, and plays an essential role in retrograde transport from recycling endosomes to the trans-Golgi network. The structure of human evectin-2 PH domain in complex with O-phospho-L-serine has recently been reported and demonstrates how the head group of phosphatidylserine is recognized. However, it was not possible to elucidate from the structure why evectin-2 cannot bind phosphatidic acid or phosphatidylethanolamine, which share a common moiety with phosphatidylserine. Here, the crystal structure at 1.75 Å resolution of an apo form of human evectin-2 PH domain, in which the ligand-binding site is free from crystal packing and is thus appropriate for comparison with the structure of the complex, is reported. Comparison between the structures of the apo form and the O-phospho-L-serine complex revealed ligand-induced conformational change evoked by interaction between the carboxyl moiety of the head group of phosphatidylserine and the main-chain N atom of Thr14. This structural change effectively explains the strict ligand specificity of the PH domain of human evectin-2.

Imaging of blood plasma coagulation at supported lipid membranes

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO2 and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems.

Snake Cytotoxins Bind to Membranes via Interactions with Phosphatidylserine Head Groups of Lipids

The major representatives of Elapidae snake venom, cytotoxins (CTs), share similar three-fingered fold and exert diverse range of biological activities against various cell types. CT-induced cell death starts from the membrane recognition process, whose molecular details remain unclear. It is known, however, that the presence of anionic lipids in cell membranes is one of the important factors determining CT-membrane binding. In this work, we therefore investigated specific interactions between one of the most abundant of such lipids, phosphatidylserine (PS), and CT 4 of Naja kaouthia using a combined, experimental and modeling, approach. It was shown that incorporation of PS into zwitterionic liposomes greatly increased the membrane-damaging activity of CT 4 measured by the release of the liposome-entrapped calcein fluorescent dye. The CT-induced leakage rate depends on the PS concentration with a maximum at approximately 20% PS. Interestingly, the effects observed for PS were much more pronounced than those measured for another anionic lipid, sulfatide. To delineate the potential PS binding sites on CT 4 and estimate their relative affinities, a series of computer simulations was performed for the systems containing the head group of PS and different spatial models of CT 4 in aqueous solution and in an implicit membrane. This was done using an original hybrid computational protocol implementing docking, Monte Carlo and molecular dynamics simulations. As a result, at least three putative PS-binding sites with different affinities to PS molecule were delineated. Being located in different parts of the CT molecule, these anion-binding sites can potentially facilitate and modulate the multi-step process of the toxin insertion into lipid bilayers. This feature together with the diverse binding affinities of the sites to a wide variety of anionic targets on the membrane surface appears to be functionally meaningful and may adjust CT action against different types of cells.

Atomic view of calcium-induced clustering of phosphatidylserine in mixed lipid bilayers.

Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca(2+) ions. While Ca(2+) is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca(2+) on the structure and dynamics of lipid bilayers. Ca(2+)-induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing (13)C,(15)N-labeled PS demonstrate that Ca(2+) ions promote two major PS headgroup conformations, which are well resolved in two-dimensional (13)C-(13)C, (15)N-(13)C, and (31)P-(13)C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca(2+) provide an atomic view of the conformational effects underlying the observed spectra.

Charachterizing Structure and Dynamics of Calcium-Induced Clusters of Phosphatidylserine in Mixed Lipid Bilayers

The cellular membrane plays a key role in the regulation and activation of peripheral membrane proteins. For instance, the enzymatic activity of several coagulation factors and signaling proteins is regulated by their specific binding to negatively charged regions of the cellular membrane that are rich in anionic lipids such as phosphatidylserine (PS). The lipid composition of the membrane and the ionic content of the immediate solution significantly modify structural properties of the bilayer surface. In particular, calcium-induced clustering of PS lipids has been suggested to modulate membrane-protein interactions. We employ our novel highly mobile membrane mimetic (HMMM) model combined with molecular dynamics simulations to investigate structural and dynamic properties determining these interactions. The HMMM model, while preserving full representation of the lipid head groups that are required for detailed characterization of specific interactions, provides 1-2 orders of magnitude speed up in lipid mobility. Extended simulations with HMMM systems including anionic (POPS), zwitterionic phosphatydilcholine (POPC), or POPS/POPC binary mixtures provide a detailed view of structural changes that occur due to lipid-lipid and lipid-ion interactions, specifically those that drive PS clustering. Simulations revealed a diverse set of PC-PS-Ca2+ microdomains of consistent geometry. In particular, we observed 2PC:2PS:Ca2+:water and 2PS:Ca2+:3water stoichiometries. Ca2+ ions interact with phosphate groups of PC and PS as well as with the carboxy groups of PS. Interestingly, unimolecular chelation of Ca2+ by the same PS head group is often observed within the clusters. In contrast to monovalent Na+, the presence of divalent Ca2+ shows its long-lived coordination with lipid head groups that modulates their orientation and leads to formation of PS clusters. Prior to the development of the HMMM method these observations were out of reach of atomistic simulations.

Structural Insights Into How Clotting Proteins with GLA Domains Bind to Membrane Surfaces.

AbstractAbstract 1141Most 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. In spite of their importance, a clear picture of how GLA domains bind to the membrane interface has yet to emerge. A further intriguing aspect of the membrane's role in blood coagulation is that certain phospholipids, most notably phosphatidylethanolamine (PE), strongly synergize with PS to promote clotting reactions. The mechanisms of this synergy, and of PE's contribution to GLA domain binding, are poorly understood – although a number of hypotheses have been put forward. We now propose a new hypothesis to explain GLA domain binding to membranes, which we term the ABC (Anything But Choline) hypothesis; it invokes two main types of protein-phospholipid interactions: a single 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. We have utilized liposomes and nanoscale phospholipid bilayers (Nanodiscs) in studies employing a series of techniques including solid-state NMR (SSNMR) and surface plasmon resonance (SPR) to address the mechanism of GLA domain-membrane interactions. We provide direct evidence in favor of the ABC hypothesis for GLA domain binding to membrane surfaces. Using SSNMR, we demonstrate that two distinct PS headgroup conformations are induced by binding of calcium ions, and that a third, novel PS headgroup conformation is induced when the prothrombin GLA domain engages the membrane. SPR studies have allowed for the determination of thermodynamic profiles of GLA domains interacting with phospholipid bilayers containing PS and/or PE, providing further insights to the mechanisms of GLA domain-membrane interactions. Disclosures:No relevant conflicts of interest to declare.

The inhibitor of phagocytosis, O-phospho- l-serine, suppresses Müller glia proliferation and cone cell regeneration in the light-damaged zebrafish retina

The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Müller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Müller glia are known to phagocytose apoptotic retinal cells during development, we tested if Müller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24 h of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Müller glia, although Müller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP) fl1, resulted in some Müller glia containing both TUNEL signal and EGFP, which indicated that this subset of Müller glia engulfed apoptotic photoreceptor cell bodies. To determine if phagocytosis induced the Müller glial proliferative response in the light-damaged retina, we utilized O-phospho- l-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Müller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, l-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist ( RS)-α-Methylserine- O-phosphate (MSOP) or the specific group I antagonist, l-2-amino-3-phophonopropanoic acid (L-AP3) affected Müller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Müller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Müller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Müller glia proliferation in the light-damaged retina.

Histidine 14 Modulates Membrane Binding and Neurotoxicity of the Alzheimer's Disease Amyloid-β Peptide

Amyloid-beta peptide (Abeta) toxicity is thought to be responsible for the neurodegeneration associated with Alzheimer's disease. While the mechanism(s) that modulate this toxicity are still widely debated, it has previously been demonstrated that modifications to the three histidine residues (6, 13, and 14) of Abeta are able to modulate the toxicity. Therefore to further elucidate the potential role of the histidine (H) residues in Abeta toxicity, we synthesized Abeta peptides with single alanine substitutions for each of the three histidine residues and ascertained how these substitutions affect peptide aggregation, metal binding, redox chemistry, and cell membrane interactions, factors which have previously been shown to modulate Abeta toxicity. Abeta{42} H13A and Abeta{42} H6A modified peptides were able to induce significant cell toxicity in primary cortical cell cultures at levels similar to the wild-type peptide. However, Abeta{42} H14A did not induce any measurable toxicity in the same cultures. This lack of toxicity correlated with the inability of the Abeta{42} H14A to bind to cell membranes. The interaction of Abeta with cell membranes has previously been shown to be dependent on electrostatic interactions between Abeta and the negatively charged head group of phosphatidylserine. Our data suggests that it is the imidazole sidechain of histidine 14 that modulates this interaction and strategies inhibiting this interaction may have therapeutic potential for Alzheimer's disease.

Specific Membrane Binding of Neurotoxin II Can Facilitate Its Delivery to Acetylcholine Receptor

The action of three-finger snake α-neurotoxins at their targets, nicotinic acetylcholine receptors (nAChR), is widely studied because of its biological and pharmacological relevance. Most such studies deal only with ligands and receptor models; however, for many ligand/receptor systems the membrane environment may affect ligand binding. In this work we focused on binding of short-chain α-neurotoxin II (NTII) from Naja oxiana to the native-like lipid bilayer, and the possible role played by the membrane in delivering the toxin to nAChR. Experimental (NMR and mutagenesis) and molecular modeling (molecular-dynamics simulation) studies revealed a specific interaction of the toxin molecule with the phosphatidylserine headgroup of lipids, resulting in the proper topology of NTII on lipid bilayers favoring the attack of nAChR. Analysis of short-chain α-neurotoxins showed that most of them possess a high positive charge and sequence homology in the lipid-binding motif of NTII, implying that interaction with the membrane surrounding nAChR may be common for the toxin family.

Cisplatin interaction with phosphatidylserine bilayer studied by solid-state NMR spectroscopy

Cisplatin [cis-diamminedichloridoplatinum(II)] is used in chemotherapy where platinum-DNA adducts initiate tumor cell death. It is possible that side effects such as neurotoxicity and cellular cisplatin resistance can be due to interaction of cisplatin with lipids and the phospholipid bilayer. In this study, (13)C, (31)P, and (15)N solid-state NMR spectra of 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayers, POPS bilayers with 10 mol% cisplatin, and POPS bilayers with 30 mol% cisplatin were acquired. In addition, (15)N{(31)P} rotational echo double resonance spectra of POPS bilayers with 30 mol% cisplatin were acquired. The data demonstrate that the serine head group of phosphatidylserine binds to the aquated form of cisplatin and that cisplatin release of ammine takes place. It appears that the cisplatin release of ammine is followed by another cisplatin-POPS complex formation, possibly with cisplatin binding to one of the oxygen atoms of the POPS phosphate moiety.