Phosphatidylserine Vesicles Research Articles

Cholesterol and cholesterol bilayer domains inhibit binding of alpha-crystallin to the membranes made of the major phospholipids of eye lens fiber cell plasma membranes

The concentration of α-crystallin decreases in the eye lens cytoplasm, with a corresponding increase in membrane-bound α-crystallin during cataract formation. The eye lens's fiber cell plasma membrane consists of extremely high cholesterol (Chol) content, forming cholesterol bilayer domains (CBDs) within the membrane. The role of high Chol content in the lens membrane is unclear. Here, we applied the continuous-wave electron paramagnetic resonance spin-labeling method to probe the role of Chol and CBDs on α-crystallin binding to membranes made of four major phospholipids (PLs) of the eye lens, i.e., phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylethanolamine (PE). Small unilamellar vesicles (SUVs) of PC, SM*, and PS with 0, 23, 33, 50, and 60 mol% Chol and PE* with 0, 9, and 33 mol% Chol were prepared using the rapid solvent exchange method followed by probe-tip sonication. The 1 mol% CSL spin-labels used during SUVs preparation distribute uniformly within the Chol/PL membrane, enabling the investigation of Chol and CBDs' role on α-crystallin binding to the membrane. For PC, SM*, and PS membranes, the binding affinity (Ka) and the maximum percentage of membrane surface occupied (MMSO) by α-crystallin decreased with an increase in Chol concentration. The Ka and MMSO became zero at 50 mol% Chol for PC and 60 mol% Chol for SM* membranes, representing that complete inhibition of α-crystallin binding was possible before the formation of CBDs within the PC membrane but only after the formation of CBDs within the SM* membrane. The Ka and MMSO did not reach zero even at 60 mol% Chol in the PS membrane, representing CBDs at this Chol concentration were not sufficient for complete inhibition of α-crystallin binding to the PS membrane. Both the Ka and MMSO were zero at 0, 9, and 33 mol% Chol in the PE* membrane, representing no binding of α-crystallin to the PE* membrane with and without Chol. The mobility parameter profiles decreased with an increase in α-crystallin binding to the membranes; however, the decrease was more pronounced for the membrane with lower Chol concentration. These results imply that the membranes become more immobilized near the headgroup regions with an increase in α-crystallin binding; however, the Chol antagonizes the capacity of α-crystallin to decrease the mobility near the headgroup regions of the membranes. The maximum splitting profiles remained the same with an increase in α-crystallin concentration, but there was an increase in the maximum splitting with an increase in the Chol concentration in the membranes. It implies that membrane order near the headgroup regions does not change with an increase in α-crystallin concentration but increases with an increase in Chol concentration in the membrane. Based on our data, we hypothesize that the Chol and CBDs decrease hydrophobicity (increase polarity) near the membrane surface, inhibiting the hydrophobic binding of α-crystallin to the membranes. Thus, our data suggest that Chol and CBDs play a positive physiological role by preventing α-crystallin binding to lens membranes and possibly protecting against cataract formation and progression.

Procoagulant Activity of Skeletal Muscle Myosin Is Not Caused By Contaminating Phosphatidylserine-Vesicles

Skeletal muscle myosin (SkM) can bind factor (F)Xa and FVa, thereby providing a surface that promotes thrombin generation by the prothrombinase complex (FXa:FVa:Ca++) that cleaves prothrombin. A recent BLOOD paper (Novakovic & Gilbert, 2020) asserted that this activity of SkM preparations is entirely due to contaminating phosphatidylserine (PS)-containing phospholipid vesicles in SkM preparations because annexin V and lactadherin neutralized the ability of SkM to enhance prothrombin activation. However, annexin V and lactadherin are certainly not monospecific for binding PS as they are globular proteins that can bind other lipids and many proteins. Without any PS measurements or use of any reagents specific for PS (e.g., monoclonal (mAb) anti-PS antibodies), that report, in a gross overinterpretation of its incomplete data, dismissed any direct role for myosin for SkM's procoagulant activity. When we previously observed that annexin V is inhibitory of SkM's support for prothrombinase activity, we sent a sample of SkM (Cytoskeletal Inc) to Avanti Polar Lipids for quantitation of the PS content based on liquid chromatography-mass spectrometry. That analysis showed that only a small amount of PS was present in the SkM, approximately 0.90 µmol PS per 40. µmol SkM. This amount of PS present in SkM preparations is not enough to explain SkM's procoagulant activity. For example, standardized purified prothrombinase reaction mixture assays show that 10 nM SkM enables formation of 3 nmol thrombin/min while 0.22 nM PS (in 1.1 nM phosphatidylcholine (PC)(80%)/PS(20%) vesicles) enables formation of only 0.4 nmol thrombin/min (Figure 1A). We directly assessed the role of contaminating PS for SkM's prothrombinase support using the well characterized anti-PS mAb 11.31 (aka mAb PGN632). When the ability of mAb 11.31 to inhibit prothrombinase enhancement by SkM or, in controls, by PC/PS vesicles was determined, the data showed that, in controls, mAb 11.31 at 1.0 nM severely inhibited PC/PS vesicle's enhancement of prothrombinase by > 90% (Figure 1B). The dose-response gave an inhibitory IC50 value of 0.2 nM which is near this mAb's reported Kd of 0.17-0.35 nM for PS, establishing the potent ability of this anti-PS mAb to neutralize PS procoagulant activity. However, there was no substantial inhibition of SkM's enhancement of prothrombinase by the anti-PS mAb 11.31 at up to 1 nM mAb 11.31 (Figure 1B). This indicates that contaminating, PS-containing vesicles are not a significant factor for SkM-dependent enhancement of prothrombin activation by the SkM preparation -- in direct contradiction of the assertion of Novakovic & Gilbert (BLOOD 2020). That recent report was correct that annexin V inhibits the ability of SkM to enhance prothrombinase as well as the ability of PC/PS vesicles to do so (Figure 1B). So it was the overinterpretation of the annexin V and lactadherin data as well as the failure to provide any direct measurement of PS that were problematic in that report. The observation that annexin V and lactadherin inhibit SkM's enhancement of prothrombinase merits further studies to understand what may be their mechanistic influences. Another informative test for an essential role for PS in SkM's enhancement of prothrombinase involves the use of FXa that lacks its gamma-carboxyglutamic acid (Gla) domain because this N-terminal domain of FXa is required for FXa binding to PS-containing phospholipid vesicles. Data in Figure 1A show that SkM, but not PS-containing PL vesicles, supports the prothrombinase activity of des-Gla Domain (DG)-FXa which lacks its Gla domain. Dose-response data show that, in prothrombinase assays, DG-FXa has only 1% activity in the presence of PC/PS vesicles but has 25-35% activity in presence of SkM (Figure 1A). These data indicate that SkM's procoagulant activity does not absolutely require the Gla domain of FXa whereas PS-containing vesicles do require the Gla domain of FXa for significant activity. These data prove that the recent assertion that PS vesicle contamination, not myosin, explains SkM's procoagulant activity represents an overinterpretation of annexin V and lactadherin data and is simply wrong. In conclusion, both new data here, i.e., PS content of SkM preparations and data for effects of anti-PS mAb 11.31 (Figure 1B) and previous data (Deguchi et al, Blood 2016 and J Biol Chem, 2019), affirm that the myosin protein is a key factor for SkM's ability to enhance prothrombin activation. Figure 1 Disclosures No relevant conflicts of interest to declare.

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Hydrogen‐Deuterium Exchange Reveals Distinct Activation States of PLCβ2 BY G‐Proteins

G‐protein coupled receptors (GPCRs) are an important class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G‐proteins. One major effector of G proteins is phospholipase C (PLC), which hydrolyzes Phosphatidylinositol 4,5 bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1,4,5, trisphosphate (IP3). PLCβ2 and PLCβ3 isoforms are directly activated by binding Gαq, Gβγ or both. The mechanism of activation of PLCβ2 by Gβγ is unknown, and the binding site(s) for Gβγ on PLCβ remain controversial.Here we use hydrogen‐deuterium exchange mass spectrometry (HDXMS) to understand the conformational dynamics of PLCβ and to answer open questions in the field concerning the mechanism of activation of PLC by Gβγ and Gαq. For these experiments, several complexes were examined with HDXMS: PLCβ2 alone, PLCβ2+ PIP2/Phosphoethanolamine (PE)/Phosphatidylserine (PS) vesicles, or PLCβ2+ PIP2/PE/PS vesicles plus Gβγ or Gαq‐AlF4. Incubation of PLCβ2 with PIP2/PE/PS vesicles strongly protects regions in the C‐terminus, confirming that this region is involved in membrane binding. In the presence of Gαq‐AlF4, PLCβ2 shows protection from deuterium exchange in the helix‐loop‐helix region of the proximal C‐terminal domain (CTD) and the EF‐hand domain on PLCβ2 that exactly correspond to regions shown to interact with Gαq in the Gαq‐PLCβ3 crystal structure. These data validate that our approach accurately reports bona fide protein‐protein and protein‐lipid interactions. Incubation of Gβγ with PLCβ2 does not reveal any regions of strong protection, but rather causes strong increases in deuterium exchange in the of the CTD of PLCβ2, indicating that the CTD of PLC undergoes a large conformational rearrangement upon Gβγ binding. This increase in deuterium exchange of the CTD of PLC is not seen upon Gαq activation. This indicates that there are distinct conformational states of PLCβ induced by Gαq and Gβγ interactions, suggesting that Gαq and Gβγ use different mechanisms for activation of PLCβ. To determine the mechanism of activation of PLCβ, we used a purified reconstitution system of PLC activity. In vitro reconstitution experiments show that CTD‐deleted PLCβ2 exhibits increased activity in response to Gβγ. Additionally, the distal CTD of PLCβ inhibits CTD‐deleted PLCβ2 activity in both lipid‐dependent and lipid‐independent reconstitution system. Taken together, these results suggest that Gβγ activates PLCβ by breaking an autoinhibitory interaction of the distal CTD with the core enzyme of PLC.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PS Membrane Asymmetry Influences the Folding and Insertion of a Transmembrane Helix

The plasma membrane (PM) has an asymmetric distribution of lipids between the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is actively sequestered to the inner leaflet of the PM but can redistribute to the outer leaflet when the cell is damaged or at the onset of apoptosis. However, membranes contain also proteins. Marginally hydrophobic membrane proteins contain acidic residues in their transmembrane sequence and can experience topological transitions after membrane association. The pH low insertion peptide (pHLIP), which undergoes a topological reorientation and inserts into the membrane at low pH, as its name implies, is a well-characterized model for studying these transitions. Although it is known that the inclusion of PS in symmetric vesicles affects the membrane insertion process of pHLIP by lowering the pH midpoint of insertion, it is unclear how PS asymmetry affects these topological transitions. Here, we studied pHLIP's topology using freely-floating asymmetric phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We found that the protocol to create asymmetric vesicles had to be modified due to the inclusion of PS. We used Annexin V labeled with an Alexa 568 fluorophore as a new way of quantifying PS asymmetry. For pHLIP, membrane insertion was affected by the surface charge difference between bilayer leaflets because of the asymmetric distribution of charged lipids. We thus conclude that lipid asymmetry can have consequences for the behavior of membrane-associated proteins. A corollary is that model studies using symmetric bilayers to mimic the PM may fail to capture important details of protein-membrane interactions.

Interfacial charges drive the organization of supported lipid membranes and their interaction with nanoparticles.

In this work, we investigated the interaction of cationic gold nanoparticles (AuNPs) with an anionic solid-supported lipid bilayer (SSLB) prepared via the spontaneous fusion of vesicles of phosphatidylserine (DPPS) and phosphatidylcholine (DPPC) on SiO2. We combined sum frequency generation (SFG) spectroscopy at the SSLBs interfaces with electrophoretic light scattering at the vesicles/liquid interfaces, and we provided further insight into the formation of organized DPPS-DPPC films on SiO2 and their interaction with NPs. We found that there is a critical threshold of the relative vesicles/substrate interfacial zeta potentials, beyond which the conformational organization of SSLBs failed. Moreover, we also demonstrated that the presence of anionic DPPS lipids in model mixed DPPS-DPPC membranes accelerated the interaction rate with cationic AuNPs.

The localization of α-synuclein in the process of differentiation of human erythroid cells.

Although the neuronal protein α-synuclein (α-syn) is thought to play a central role in the pathogenesis of Parkinson's disease (PD), its physiological function remains unknown. It is known that α-syn is also abundantly expressed in erythrocytes. However, its role in erythrocytes is also unknown. In the present study, we investigated the localization of α-syn in human erythroblasts and erythrocytes. Protein expression of α-syn increased during terminal differentiation of erythroblasts (from day 7 to day 13), whereas its mRNA level peaked at day 11. α-syn was detected in the nucleus, and was also seen in the cytoplasm and at the plasma membrane after day 11. In erythroblasts undergoing nucleus extrusion (day 13), α-syn was detected at the periphery of the nucleus. Interestingly, we found that recombinant α-syn binds to trypsinized inside-out vesicles of erythrocytes and phosphatidylserine (PS) liposomes. The dissociation constants for binding to PS/phosphatidylcholine (PC) liposomes of N-terminally acetylated (NAc) α-syn was lower than that of non NAc α-syn. This suggests that N-terminal acetylation plays a significant functional role. The results of the present study collectively suggest that α-syn is involved in the enucleation of erythroblasts and the stabilization of erythroid membranes.

Hydrogen‐Deuterium exchange reveals distinct activation states of PLCβ2 by G‐proteins

G‐protein coupled receptors (GPCRs) are an important class of cell surface receptors that transduce extracellular stimuli to intracellular signaling events via G‐proteins. One major effector of G proteins is phospholipase C (PLC), which hydrolyzes Phosphatidylinositol 4,5 bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1,4,5, trisphosphate (IP3). PLCβ2 and PLCβ3 isoforms are directly activated by binding Gαq, Gβγ or both. The mechanism of activation of PLCβ2 by Gβγ is unknown, and the binding site(s) for Gβγ on PLCβ remain controversial.Here, we use hydrogen‐deuterium exchange mass spectrometry (HDXMS) to understand the conformational dynamics of PLCβ and to answer open questions in the field concerning the mechanism of activation of PLC by Gβγ and Gαq. For these experiments, several complexes were examined with HDXMS: PLCβ2 alone, PLCβ2+ PIP2/Phosphoethanolamine (PE)/Phosphatidylserine (PS) vesicles, or PLCβ2+ PIP2/PE/PS vesicles plus Gβγ or Gαq‐AlF4. Incubation of PLCβ2 with PIP2/PE/PS vesicles strongly protects regions in the C‐terminus, confirming that this region is involved in membrane binding. In the presence of Gαq‐AlF4, PLCβ2 shows protection from deuterium exchange in the helix‐loop‐helix region of the proximal C‐terminal domain (CTD) and the EF‐hand domain on PLCβ2 that exactly correspond to regions shown to interact with Gαq in the Gαq‐PLCβ3 crystal structure. These data validate that our approach accurately reports bona fide protein‐protein and protein‐lipid interactions. Incubation of Gβγ with PLCβ2 does not reveal any regions of strong protection, but rather causes strong increases in deuterium exchange in the of the CTD of PLCβ2, indicating that the CTD of PLC undergoes a large conformational rearrangement upon Gβγ binding. This increase in deuterium exchange of the CTD of PLC is not seen upon Gαq activation. This indicates that there are distinct conformational states of PLCβ induced by Gαq and Gβγ interactions, suggesting that Gαq and Gβγ use different mechanisms for activation of PLCβ. Finally, these data suggest a mechanism for activation of PLC by Gβγ, where Gβγ binding to PLC breaks an autoinhibitory interaction involving the CTD of PLC and either the membrane or another portion of PLCβ2.Support or Funding InformationThis work is supported by NIH R01 GM081772 to AVS.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A cationic, C-terminal patch and structural rearrangements in Ebola virus matrix VP40 protein control its interactions with phosphatidylserine

Ebola virus (EBOV) is a filamentous lipid-enveloped virus that causes hemorrhagic fever with a high fatality rate. Viral protein 40 (VP40) is the major EBOV matrix protein and regulates viral budding from the plasma membrane. VP40 is a transformer/morpheein that can structurally rearrange its native homodimer into either a hexameric filament that facilitates viral budding or an RNA-binding octameric ring that regulates viral transcription. VP40 associates with plasma-membrane lipids such as phosphatidylserine (PS), and this association is critical to budding from the host cell. However, it is poorly understood how different VP40 structures interact with PS, what essential residues are involved in this association, and whether VP40 has true selectivity for PS among different glycerophospholipid headgroups. In this study, we used lipid-binding assays, MD simulations, and cellular imaging to investigate the molecular basis of VP40-PS interactions and to determine whether different VP40 structures (i.e. monomer, dimer, and octamer) can interact with PS-containing membranes. Results from quantitative analysis indicated that VP40 associates with PS vesicles via a cationic patch in the C-terminal domain (Lys224, 225 and Lys274, 275). Substitutions of these residues with alanine reduced PS-vesicle binding by >40-fold and abrogated VP40 localization to the plasma membrane. Dimeric VP40 had 2-fold greater affinity for PS-containing membranes than the monomer, whereas binding of the VP40 octameric ring was reduced by nearly 10-fold. Taken together, these results suggest the different VP40 structures known to form in the viral life cycle harbor different affinities for PS-containing membranes.

Membrane Lipid Asymmetry, a Key Property of the Mammalian Plasma Membrane, Alters the Insertion of the pHLIP Peptide

The plasma membrane has an asymmetric distribution of lipids between the inner and outer leaflets of the bilayer. Two types of phospholipid asymmetry can occur between the leaflets: head group asymmetry and acyl chain asymmetry. One lipid head group of interest is phosphatidylserine (PS), which carries a net negative charge. In healthy cells, PS is actively sequestered to the inner leaflet of the membrane but can redistribute to the outer leaflet in situations such as apoptosis and cancer. Not only do membranes contain lipids, they also contain proteins. Marginally hydrophobic membrane proteins can contain acidic residues in their transmembrane sequence and can have topological transitions after membrane association. What is unclear is how lipid asymmetry affects these topological transitions. Here, we propose to use the pH low insertion peptide (pHLIP) as a model to study topological transitions in relation to membrane head group asymmetry in free floating asymmetric phosphatidylcholine (PC)/PS vesicles. Based on environmental pH, pHLIP undergoes a topological reorientation and inserts into the membrane. The inclusion of PS in symmetric vesicles affects the insertion process by lowering the pH midpoint of insertio n. However, we do not know how PS asymmetry will affect insertion. We propose that having an asymmetric distribution of PS (more in the inner leaflet) will affect the insertion of pHLIP. Fluorescence spectroscopy data show that the pH midpoint of pHLIP insertion into asymmetr ic vesicles is higher compared to symmetric vesicles containing PS. Acrylamide quenching data show that the two tryptophans in pHLIP are more solvent exposed in the presence of asymmetry. We hypothesize that pHLIP insertion is affected by a change in the transmembrane potential induced by the asymmetric distribution of charged lipids between the leaflets.

PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes

PTEN is one of the most frequent mutated tumor suppressor proteins in human cancer. This project investigates how PTEN interacts with lipid model membranes of different composition and morphology. We hypothesize that PTEN binds synergistically to the plasma membrane (PM) by interacting with phosphatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic lipids. While the currently accepted model is that PTEN's C2 domain interacts with phosphatidylserine (PS), we hypothesize that the C2 domain can interact with any anionic lipid, as long as this lipid has a sufficiently high local concentration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched domains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting that PI is a better second binding partner than PS. We are using stopped-flow fluorescence spectrophotometry as an ensemble technique that provides us with information about the association and dissociation kinetics of PTEN when interacting with lipid model membranes of different composition. Using this technique we have found that PTEN binds to pure PS and PI vesicles with a similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composition. From these data, we determine the PTEN diffusion coefficient, hopping frequency and dwell times. In the aggregate, this allows us to describe in detail PTEN binding mechanisms with differently composed lipid model membranes.

Membrane Deformation Induced by Cation Binding to Negatively Charged Phospholipids

The divalent cation Ca2+ plays a critical role in many cell signaling and membrane trafficking pathways, including exocytosis and membrane fusion. Ca2+ signal transduction is commonly thought to occur through interaction with protein partners; however, it may also interact with negatively charged phospholipids within the plasma membrane leading to changes in lipid behavior.Here we show that an asymmetric concentration of Ca2+ ions across the membrane can generate tubules or protrusions from membranes containing phosphatidylserine (PS) or phosphatidylinositol-4,5-bisphosphate (PIP2). We transferred single vesicles with controlled membrane tension into Ca2+ solution and observed the formation of inward tubules, indicating negative curvature generation by Ca2+. The Ca2+-induced membrane deformation is inhibited by high tension or low cation binding, in a similar manner as we have previously observed for BAR domain proteins. We propose that the membrane deformation is caused by Ca2+-induced clustering of PS and PIP2 creating negative curvature. In confirmation of this we find that Mg2+, which is known to only weakly cluster PS and PIP2, only generates curvature at relatively high concentrations and low tension. Additionally, for PS vesicles the membrane deformation requires high Ca2+ concentration (a few millimolar), while PIP2 vesicles, which are more sensitive to Ca2+-induced clustering, are deformed at lower Ca2+ concentrations. The presence of salt ions (Na+) also influences Ca2+-induced membrane curvature. High salt content can reduce Ca2+-induced curvature by shielding Ca2+ ions from the membrane. An asymmetric salt concentration can influence membrane structure and either enhance or oppose Ca2+-induced membrane curvature. The observed Ca2+-induced membrane deformation represents a new mechanism for Ca2+ signaling events. This mechanism is likely to be especially important for the role of Ca2+ in exocytosis and membrane fusion events.

Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy

Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain’s reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells.

Role of Hydrogen Bonding and Polyanion Composition in the Formation of Lipid Bilayers on Top of Polyelectrolyte Multilayers.

The self-assembly of mixed vesicles of zwitterionic phosphatidylcholine (PC) and anionic phosphatidylserine (PS) phospholipids on top of polyelectrolyte multilayers (PEMs) of poly(allylamine hydrochloride) (PAH), as a polycation, and polystyrenesulfonate (PSS), as a polyanion, is investigated as a function of the vesicle composition by means of the quartz crystal microbalance with dissipation (QCM-D), cryo-transmission electron microscopy (Cryo-TEM), atomic force microscopy (AFM), and atomic force spectroscopy (AFS). Vesicles with molar percentages of PS between 50% and 70% result in the formation of lipid bilayers on top of the PEMs. Vesicles with over 50% of PC or over 80% of PS do not assembly into bilayers. AFS studies performed with a PAH-modified cantilever approaching and retracting from the lipid assemblies reveal that the main interaction between PAH and the lipids takes place through hydrogen bonding between the amine groups of PAH and the carboxylate and phosphate groups of PS and with the phosphate groups of PC. The interaction of PAH with PS is much stronger than with PC. AFS measurements on assemblies with 50% PC and 50% PS revealed similar adhesion forces to pure PS assemblies, but the PAH chains can reorganize much better on the lipids as a consequence of the presence of PC. QCM-D experiments show that vesicles with a lipid composition of 50% PC and 50% PS do not form bilayers if PSS is replaced by alginate (Alg) or poly(acrylic acid) (PAA).

Phosphatidylserine Vesicles Enable Efficient En Bloc Transmission of Enteroviruses

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Dynamic Studies of the Tumour Suppressor Protien PTEN Binding to Membranes Composed PI(4,5)P2 and Various Anionic Lipids

The tumor suppressor gene, Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) encodes a protein that is involved in many human cancers. It binds to the plasma membrane to dephosphorylate the 3-position of phosphatidylinositol-3,4,5-trisphosphate producing phosphatidylinositol-4,5-bisphosphate (PI[4,5]P2). PTEN is an agonist to PI 3-kinase, thereby inhibiting the PI3K/Akt signaling pathway and controlling cell proliferation and survival. PTEN membrane association depends strongly on the composition and lateral distribution of the lipids in the membrane. Using stopped flow kinetic and steady state fluorescence experiments, we characterize the different steps associated with PTEN membrane association and dissociation.We find that PTEN membrane association is governed by the nature of the anionic lipids in the model membrane and their lateral distribution. It has been found previously that PTEN interacts synergistically with phosphatidylserine (PS) and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). When comparing the kinetics of PTEN binding to PC/PS (30%) and PC/PI(4,5)P2 (5%) vesicles, we find a faster kon value and slower koff rate constant for the PI(4,5)P2 containing vesicles. When both PS and PI(4,5)P2 are present in the vesicles, we find the fastest association rate constants. In model membrane systems, PS and PI(4,5)P2 show only limited co-localization. Therefore, the question arises whether PS in biological membranes is the second binding partner, in addition to PI(4,5)P2, of PTEN. We hypothesize that phosphatidylinositol (PI), which forms domains with PI(4,5)P2, may replace PS in at least some physiological scenarios as the second binding partner. We find that PTEN binding to PI vesicles is as strong as PTEN binding to PS vesicles, confirming the hypothesis that PTEN binding to PS or PI is non-specific electrostatic. This finding is a major paradigm shift in the PTEN field.

Copper binding affinity of the C2B domain of synaptotagmin-1 and its potential role in the nonclassical secretion of acidic fibroblast growth factor

Fibroblast growth factor 1 (FGF1) is a heparin-binding proangiogenic protein. FGF1 lacks the conventional N-terminal signal peptide required for secretion through the endoplasmic reticulum (ER)–Golgi secretory pathway. FGF1 is released through a Cu2+-mediated nonclassical secretion pathway. The secretion of FGF1 involves the formation of a Cu2+-mediated multiprotein release complex (MRC) including FGF1, S100A13 (a calcium-binding protein) and p40 synaptotagmin (Syt1). It is believed that the binding of Cu2+ to the C2B domain is important for the release of FGF1 into the extracellular medium. In this study, using a variety of biophysical studies, Cu2+ and lipid interactions of the C2B domain of Syt1 were characterized. Isothermal titration calorimetry (ITC) experiments reveal that the C2B domain binds to Cu2+ in a biphasic manner involving an initial endothermic and a subsequent exothermic phase. Fluorescence energy transfer experiments using Tb3+ show that there are two Cu2+-binding pockets on the C2B domain, and one of these is also a Ca2+-binding site. Lipid-binding studies using ITC demonstrate that the C2B domain preferentially binds to small unilamellar vesicles of phosphatidyl serine (PS). Results of the differential scanning calorimetry and limited trypsin digestion experiments suggest that the C2B domain is marginally destabilized upon binding to PS vesicles. These results, for the first time, suggest that the main role of the C2B domain of Syt1 is to serve as an anchor for the FGF1 MRC on the membrane bilayer. In addition, the binding of the C2B domain to the lipid bilayer is shown to significantly decrease the binding affinity of the protein to Cu2+. The study provides valuable insights on the sequence of structural events that occur in the nonclassical secretion of FGF1.

Thrombin-Stimulated Platelets Have Functional Binding Sites For Factor VIIIa That Are Distinct From Phosphatidylserine

BackgroundFactor VIII (fVIII) functions as a co-factor for factor IXa on the membranes of stimulated platelets. Binding sites for fVIII(a) are expressed at two levels; thrombin induces 3,000 – 20,000 sites/platelet while the combination of collagen and thrombin or A28137 induce >50,000 sites/platelet. HypothesisWe hypothesized that binding sites for fVIII(a) on thrombin-stimulated platelets, are distinct from phosphatidylserine (PS), while those on maximally stimulated platelets are predominantly PS-containing sites. Corollaries were 1) that epitopes on fVIII interact with the non-PS sites and 2) that a macromolecule or a macromolecule complex comprises the binding sites on thrombin-stimulated platelets. MethodsPlatelets were purified on a density gradient and binding of fluorescein-labeled fVIII (fVIII-fluor) was measured by flow cytometry using a Becton Dickinson LSR-Fortessa flow cytometer. Factor VIII activity was measured in a discontinuous factor Xase assay using extruded phospholipid vesicles of composition PS:PE:PC 4:20:76 or platelets as the membrane source. Oligomeric fibrin was immobilized by incubating thrombin, 1 u/ml, with fibrinogen, 10 µg/ml for 10 min without mixing prior to addition of 59D8-Superose beads. Binding of fVIII-4 Ala to platelets was measured in complex with Alexa-488 labeled mAb GMA-8021, against the A2 domain. Polyphosphate was size-fractionated and recombinant PPX-MBD produced as previously described. ResultsLactadherin, a phosphatidyl-L-serine-binding protein, competed for 97% of factor VIII-fluorescein (fVIII-fluor) binding sites on A23187-stimulated platelets but only 30% of binding sites on thrombin-stimulated platelets. Unlabeled fVIII competed with fVIII-fluor for all binding sites. A fVIII C2 domain mutant, with no measurable phospholipid binding - M2199A/F2200A/L2251A/L2252A (fVIII-4Ala) bound to only 3,000 – 5,000 sites on platelets stimulated with A23187 but to a similar number on thrombin-stimulated platelets with a KDof 7 nM. These data indicate that non-PS sites are dominant on thrombin-stimulated platelets but that PS-containing sites comprise at least 95% of sites on A23187-stimulated platelets.We evaluated a panel of mAb's against the fVIII-C2 domain for platelet-specific inhibition of binding and function. mAb's ESH4 and I54, with overlapping epitopes, blocked binding of fVIII to thrombin-stimulated platelets but only decreased affinity for PS-containing membranes. In 1-stage and 2-stage commercial aPTT assays ESH4 inhibited 28-33% of fVIII activity. In contrast, ESH4 inhibited 80% of fVIII activity on thrombin-stimulated platelets. mAb's ESH8 and G99, with partially overlapping epitopes, decreased the affinity of fVIII-fluor for thrombin-stimulated platelets approx. 70% but had no effect on phospholipid binding. ESH8 inhibited 58 ± 8% of fVIII activity on thrombin-stimulated platelets but did not decrease activity supported by phospholipid vesicles.Because oligomeric fibrin is required for expression of most fVIII binding sites on thrombin-stimulated platelets (Phillips et al 2004; JTH 2:1806) we hypothesized that oligomeric, platelet-bound fibrin is a constituent of fVIII binding sites. fVIII-fluor bound to fibrin monomers and oligomers immobilized on mAb 59D8-Superose, detected in solution by flow cytometry. Binding was enhanced by mixing polyphosphate (polyP) with fibrinogen prior to thrombin, with a maximum gain in affinity at 0.1 µM elemental phosphorous. The apparent affinity of fibrin-polyP for fVIII-fluor was 2-12 nM, based on competition studies with unlabeled fVIII. Like binding to platelets, specific binding of fVIII to fibrin-polyP was blocked by mAb's ESH4, I54 and diminished by ESH8, and G99. Thrombin-stimulated platelets, but not resting platelets, exhibited bound polyP, as detected by PPX-MBP, specific for polyP. Thus, bound polyP is present on thrombin-stimulated platelets under conditions that lead to binding of oligomeric fibrin. ConclusionsThese data indicate that thrombin-stimulated platelets bind fVIII via a non-PS binding site and that the binding is mediated by epitopes that have greater functional importance on platelets than on phospholipid vesicles. Platelet-bound oligomeric fibrin with polyP is a candidate for the non-PS binding site. These findings have clinical relevance to detection of inhibitory antibodies against fVIII. Disclosures:No relevant conflicts of interest to declare.

Leishmania amazonensis exhibits phosphatidylserine-dependent procoagulant activity, a process that is counteracted by sandfly saliva

Leishmania parasites expose phosphatidylserine (PS) on their surface, a process that has been associated with regulation of host's immune responses. In this study we demonstrate that PS exposure by metacyclic promastigotes of Leishmania amazonensis favours blood coagulation. L. amazonensis accelerates in vitro coagulation of human plasma. In addition, L. amazonensis supports the assembly of the prothrombinase complex, thus promoting thrombin formation. This process was reversed by annexin V which blocks PS binding sites. During blood meal, Lutzomyia longipalpis sandfly inject saliva in the bite site, which has a series of pharmacologically active compounds that inhibit blood coagulation. Since saliva and parasites are co-injected in the host during natural transmission, we evaluated the anticoagulant properties of sandfly saliva in counteracting the procoagulant activity of L. amazonensis . Lu. longipalpis saliva reverses plasma clotting promoted by promastigotes. It also inhibits thrombin formation by the prothrombinase complex assembled either in phosphatidylcholine (PC)/PS vesicles or in L. amazonensis . Sandfly saliva inhibits factor X activation by the intrinsic tenase complex assembled on PC/PS vesicles and blocks factor Xa catalytic activity. Altogether our results show that metacyclic promastigotes of L. amazonensis are procoagulant due to PS exposure. Notably, this effect is efficiently counteracted by sandfly saliva.

Turn‐On Fluorescence Detection of Apoptotic Cells Using a Zinc(II)‐Dipicolylamine‐Functionalized Poly(diacetylene) Liposome

A liposome-based fluorescence sensing system for apoptotic cells has been developed from stimuli-responsive poly(diacetylene)-liposomes for the first time. The combination of the liposome components, a phosphatidylserine-binding Zn(II)-dipicolylamine component and an alcohol-terminated component in the ratio of 2:1, has led to an efficient detection system for apoptotic cells, as demonstrated by confocal fluorescence microscopy and FACS analysis. The liposome shows a color change from blue to reddish purple and emits fluorescence in the turn-on mode upon interaction with phosphatidylserine. The present system thus avoids the washing steps required for "always-on"-type sensing systems.