Binding Of Liposomes Research Articles

Retention of E-selectin functionalized liposome fanny packs on Jurkat cells following invasion through collagen

Circulating immune cells are an appealing candidate to serve as carriers of therapeutic cargo via nanoparticles conjugated to their surface, for several reasons: these cells are highly migratory and can squeeze through small pores of diameter smaller than their resting size; they are easily accessible in the peripheral blood via minimally invasive IV injection of particles, or can be harvested, processed ex vivo, and reintroduced to the body; they are adept at traveling through the circulation with minimal destruction and thus have access to various tissue beds of the body; and immune cells have built-in signal transduction machinery which allows them to actively engage in chemotaxis and home to regions of the tissue containing tumors, invading microorganisms, or injuries in need of wound healing. In this study, we sought to examine and quantify the degree to which nanoscale liposomes, functionalized with E-selectin adhesion receptor, could bind to a model T cell line and remain on the surface of the cells as they migrate through collagen gels of varying density in a transwell cell migration chamber. It is demonstrated that physiological levels of fluid shear stress are necessary to achieve optimal binding of the E-selectin liposomes to the cell surface as expected, and that CD3/CD28 antibody activation of the T cells was not necessary for effective liposome binding. Nanoscale liposomes were successfully conveyed by the migrating cells across a layer of rat tail type 1 collagen gel ranging in composition from 1 to 3 mg/mL. The relative fraction of liposomes carried through the collagen decreased at higher collagen density, likely due to the expected decrease in average pore size, and increased fiber content in the gels. Taken together, these results support the idea that T cells could be an effective cellular carrier of therapeutic molecules either attached to the surface of nanoscale liposomes or encapsulated within their interior.

Exploring lipid-protein interactions in plant membranes.

Once regarded as mere membrane building blocks, lipids are now recognized as diverse and intricate players that mold the functions, identities, and responses of cellular membranes. Although the interactions of lipids with integral and peripheral membrane proteins are crucial for their localization, activity, and function, how proteins bind lipids is still far from being thoroughly explored. Describing and characterizing these dynamic protein-lipid interactions is thus essential to understanding the membrane-associated processes. Here we review the current range of experimental techniques employed to study plant protein-lipid interactions, integrating various methods. We summarize the principles, advantages, and limitations of classical in vitro biochemical approaches, including protein-lipid overlays and various liposome binding assays, and complement them with in vivo microscopic techniques centered around the use of genetically encoded lipid sensors and pharmacological or genetic membrane lipid manipulation tools. We also highlight several emerging techniques still awaiting their advancement into plant membrane research and emphasize the need to use complementary experimental strategies as key for elucidating the mechanistic roles of protein-lipid interactions in plant cell biology.

Synthesis and Validation of TRIFAPYs as a Family of Transfection Agents for Therapeutic Oligonucleotides.

Transfection agents play a crucial role in facilitating the uptake of nucleic acids into eukaryotic cells offering potential therapeutic solutions for genetic disorders. However, progress in this field needs the development of improved systems that guarantee efficient transfection. Here, we describe the synthesis of a set of chemical delivery agents (TRIFAPYs) containing alkyl chains of different lengths based on the 1,3,5-tris[(4-alkyloxy-1pyridinio)methyl]benzene tribromide structure. Their delivery properties for therapeutic oligonucleotides were evaluated using PolyPurine Reverse Hoogsteen hairpins (PPRHs) as a silencing tool. The binding of liposomes to PPRHs was evaluated by retardation assays in agarose gels. The complexes had a size of 125 nm as determined by DLS, forming well-defined concentrical vesicles as visualized by Cryo-TEM. The prostate cancer cell line PC-3 was used to study the internalization of the nanoparticles by fluorescence microscopy and flow cytometry. The mechanism of entrance involved in the cellular uptake was mainly by clathrin-mediated endocytosis. Cytotoxicity analyses determined the intrinsic toxicity caused by each TRIFAPY and the effect on cell viability upon transfection of a specific PPRH (HpsPr-C) directed against the antiapoptotic target survivin. TRIFAPYs C12-C18 were selected to expand these studies in the breast cancer cell line SKBR-3 opening the usage of TRIFAPYs for both sexes and, in the hCMEC/D3 cell line, as a model for the blood-brain barrier. The mRNA levels of survivin decreased, while apoptosis levels increased upon the transfection of HpsPr-C with these TRIFAPYs in PC-3 cells. Therefore, TRIFAPYs can be considered novel lipid-based vehicles for the delivery of therapeutic oligonucleotides.

Kinetics of brain gangliosides binding to myelin-associated glycoprotein from fluctuation analysis of single liposome binding events

Kinetics of brain gangliosides binding to myelin-associated glycoprotein from fluctuation analysis of single liposome binding events

Structural flexibility of apolipoprotein E-derived arginine-rich peptides improves their cell penetration capability

Amphipathic arginine-rich peptide, A2-17, exhibits moderate perturbation of lipid membranes and the highest cell penetration among its structural isomers. We investigated the direct cell-membrane penetration mechanism of the A2-17 peptide while focusing on structural flexibility. We designed conformationally constrained versions of A2-17, stapled (StpA2-17) and stitched (StchA2-17), whose α-helical conformations were stabilized by chemical crosslinking. Circular dichroism confirmed that StpA2-17 and StchA2-17 had higher α-helix content than A2-17 in aqueous solution. Upon liposome binding, only A2-17 exhibited a coil-to-helix transition. Confocal microscopy revealed that A2-17 had higher cell penetration efficiency than StpA2-17, whereas StchA2-17 remained on the cell membrane without cell penetration. Although the tryptophan fluorescence analysis suggested that A2-17 and its analogs had similar membrane-insertion positions between the interface and hydrophobic core, StchA2-17 exhibited a higher membrane affinity than A2-17 or StpA2-17. Atomic force microscopy demonstrated that A2-17 reduced the mechanical rigidity of liposomes to a greater extent than StpA2-17 and StchA2-17. Finally, electrophysiological analysis showed that A2-17 induced a higher charge influx through transient pores in a planer lipid bilayer than StpA2-17 and StchA2-17. These findings indicate that structural flexibility, which enables diverse conformations of A2-17, leads to a membrane perturbation mode that contributes to cell membrane penetration.

Membrane binding of endocytic myosin-1s is inhibited by a class of ankyrin repeat proteins.

Myosin-1s are monomeric actin-based motors that function at membranes. Myo1 is the single myosin-1 isoform in Schizosaccharomyces pombe that works redundantly with Wsp1-Vrp1 to activate the Arp2/3 complex for endocytosis. Here, we identified Ank1 as an uncharacterized cytoplasmic Myo1 binding partner. We found that in ank1Δ cells, Myo1 dramatically redistributed from endocytic patches to decorate the entire plasma membrane and endocytosis was defective. Biochemical analysis and structural predictions suggested that the Ank1 ankyrin repeats bind the Myo1 lever arm and the Ank1 acidic tail binds the Myo1 TH1 domain to prevent TH1-dependent Myo1 membrane binding. Indeed, Ank1 overexpression precluded Myo1 membrane localization and recombinant Ank1 reduced purified Myo1 liposome binding in vitro. Based on biochemical and cell biological analyses, we propose budding yeast Ank1 and human OSTF1 are functional Ank1 orthologs and that cytoplasmic sequestration by small ankyrin repeat proteins is a conserved mechanism regulating myosin-1s in endocytosis.

Protein Corona Attenuates the Targeting of Antitumor Sialyl Lewis X-Decorated Liposomes to Vascular Endothelial Cells under Flow Conditions.

Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLeX) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLeX conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome-cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLeX content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells.

Tuning Targeted Liposome Avidity to Cells via Lipid Phase Separation.

The addition of both cell-targeting moieties and polyethylene glycol (PEG) to nanoparticle (NP) drug delivery systems is a standard approach to improve the biodistribution, specificity, and uptake of therapeutic cargo. The spatial presentation of these molecules affects avidity of the NP to target cells in part through an interplay between the local ligand concentration and the steric hindrance imposed by PEG molecules. Here, we show that lipid phase separation in nanoparticles can modulate liposome avidity by changing the proximity of PEG and targeting protein molecules on a nanoparticle surface. Using lipid-anchored nickel-nitrilotriacetic acid (Ni-NTA) as a model ligand, we demonstrate that the attachment of lipid anchored Ni-NTA and PEG molecules to distinct lipid domains in nanoparticles can enhance liposome binding to cancer cells by increasing ligand clustering and reducing steric hindrance. We then use this technique to enhance the binding of RGD-modified liposomes, which can bind to integrins overexpressed on many cancer cells. These results demonstrate the potential of lipid phase separation to modulate the spatial presentation of targeting and shielding molecules on lipid nanocarriers, offering a powerful tool to enhance the efficacy of NP drug delivery systems.

Liposome functionalized reduced graphene oxide for rapid electrochemical sensing of bacteria.

Pathogenic bacteria represent a severe threat to global public health, particularly with the growing rate of antibiotic resistance, and, therefore, indicate a critical need for developing efficient sensing platforms. Liposome-based sensors are collocating interest due to their intrinsic fusogenic ability to fuse with the outer membrane of bacteria. However, the lack of a conducting property limits their applicability for developing biosensing platforms. In this study, we report conjugation of liposomes with reduced graphene oxide (rGO) for fabricating a rapid and sensitive biosensor for electrochemical detection of Escherichia coli (E. coli). The large surface area of rGO facilitated binding of liposomes with their surface, and the intrinsic electrical and biocompatible properties assisted electrochemical sensing of bacteria. The electrochemical response of the liposome and the rGO-liposome coated electrode shows nonconducting and conducting characteristics, respectively. A significant change in the peak current of differential pulse voltammetry with the gradual variation of bacterial density in the electrolyte was observed for the glassy carbon electrode rGO-liposome (GCE-L-rGO) surface only. The detection sensitivity of GCE-L-rGO sensors was ∼26 μA/106 cells per ml of electrolyte for varying cell densities from 3 × 103 to 3 × 104 cells/ml. The proposed sensing technique can serve as an alternative to conventional methodologies for rapid and in situ detection of bacterial load in different samples, laying the foundation for new applications in clinical diagnostics.

Specific Binding and Endocytosis of Liposomes to HEK293T Cells via Myrisoylated Pre-S1 Peptide Bound to Sodium Taurocholate Cotransporting Polypeptide.

(1) Background: Sodium taurocholate cotransporting polypeptide (NTCP) functions as a key receptor for the hepatitis B virus (HBV) infection. Analyzing HBV and NTCP interaction is an important issue not only for basic research but also for the development of anti-HBV therapeutics. We developed here a novel model system to analyze the interaction of NTCP with liposomes instead of HBV. (2) Methods: Liposomal binding and endocytosis through NTCP in HEK293T cells were achieved by serial treatments of HEL293T cells transiently expressing NTCP-green fluorescence protein (GFP) fusion protein with a synthetic biotinylated pre-S1 peptide (Myr47-Bio) and streptavidin (SA) complex (i.e., Myr47-Bio+SA) followed by biotinylated liposomes. By this procedure, binding of [biotinylated liposomes]-[Myr47-Bio+SA]-[NTCP-GFP] was formed. (3) Results: Using this model system, we found that liposomal binding to NTCP on the cell surface via Myr47-Bio+SA was far more efficient than that to scavenger receptor class B type 1 (SR-B1). Furthermore, liposomes bound to cell surface NTCP via Myr47-Bio+SA were endocytosed into cells after cells were cultured at 37 °C. However, this endocytosis was suppressed by 4 °C or cytochalasin B treatment. (4) Conclusions: This model system will be useful for not only analyzing HBV entry mechanisms but also screening substances to prevent HBV infection.

Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

Protein corona mediated liposomal drug delivery for bacterial infection management

Liposomes have been widely investigated as a class of promising antibiotic delivery systems for the treatment of life-threatening bacterial infections. However, the inevitable formation of protein corona on the liposomal surface can heavily impact in vivo performance. A better understanding of the effects of protein corona on liposomal behavior can significantly improve antibacterial liposomal drug development. Here, the critical role of protein corona in mediating liposome-bacteria interactions was elucidated. Adsorption of negatively charged protein on cationic liposome weakened electrostatic attraction-enhanced liposomal binding to the bacteria. Cumulative complement deposition on anionic liposome composed of phosphatidylglycerol (DSPG sLip) contributed to a superior binding affinity of DSPG sLip to planktonic bacteria and biofilms, which was exploited to enhance bacteria-targeted drug delivery. In both S. aureus-related osteomyelitis and pneumonia mice models, DSPG sLip was demonstrated as a promising antibiotic nanocarrier for managing MRSA infection, indicating the benefits of lipid composition-based protein corona modulation in liposomal antibiotic delivery for bacterial infection treatment.

Characterization of PDL1 enhanced siRNA/albumin liposome for effective therapeutic function in lung cancer.

The applications of liposomes are limited due to poor structural stability and short drug circulation time. This study aims to build an albumin-based liposomal delivery system to provide strategies for tumor specificity, efficient gene delivery and effective release of albumin liposomes. In this study, siRNA loaded PDL1-targeted albumin liposome was constructed for the treatment of lung cancer and its function was evaluated. Physical parameters such as particle size, potential and infrared spectrum were detected and microscopic morphology was observed by electron microscopy to detect the binding and uptake capacity of albumin liposome with cells. The optimal preparation process and binding ratio of PDL1-targeted albumin liposome/siRNA complex were determined. The constructed siRNA loaded PDL1-targeted albumin liposomes has low toxicity, high loading rate and tumor cell targeted gene therapy ability. Moreover, it increased T cell activation and down-regulated siRNA expression, effectively realizing the inhibition of lung cancer cells. The results showed that the PDL1-targeted albumin liposome could be used as a high efficient delivery vector of siRNA, and was a high efficient and safe nano vector for tumor targeted gene therapy.

Vaccine co-display of CSP and Pfs230 on liposomes targeting two Plasmodium falciparum differentiation stages

A vaccine targeting multiple stages of the Plasmodium falciparum parasite life cycle is desirable. The sporozoite surface Circumsporozoite Protein (CSP) is the target of leading anti-infective P. falciparum pre-erythrocytic vaccines. Pfs230, a sexual-stage P. falciparum surface protein, is currently in trials as the basis for a transmission-blocking vaccine, which inhibits parasite development in the mosquito vector. Here, recombinant full-length CSP and a Pfs230 fragment (Pfs230D1+) are co-displayed on immunogenic liposomes to induce immunity against both infection and transmission. Liposomes contain cobalt-porphyrin phospholipid (CoPoP), monophosphoryl lipid A and QS-21, and rapidly bind His-tagged CSP and Pfs230D1+ upon admixture to form bivalent particles that maintain reactivity with conformational monoclonal antibodies. Use of multicolor fluorophore-labeled antigens reveals liposome binding upon admixture, stability in serum and enhanced uptake in murine macrophages in vitro. Bivalent liposomes induce humoral and cellular responses against both CSP and Pfs230D1+. Vaccine-induced antibodies reduce parasite numbers in mosquito midguts in a standard membrane feeding assay. Mice immunized with liposome-displayed antigens or that passively receive antibodies from immunized rabbits have reduced parasite liver burden following challenge with transgenic sporozoites expressing P. falciparum CSP.

Aptamer-conjugated nanoliposomes containing COL1A1 siRNA sensitize CRC cells to conventional chemotherapeutic drugs

COL1A1 is an important extracellular matrix component that is associated with poor prognosis in cancers. In this study, As1411 aptamer-conjugated liposomes were used for targeted siRNA delivery against the COL1A1 gene in colorectal cancer (CRC) cells. Cationic liposomes were synthesized and siRNA loading and conjugation of aptamer were confirmed by gel shift assay and spectrophotometry method. Release of siRNA from liposomes was assessed using dialysis method. Binding and uptake of aptamer-conjugated liposomes to and into cancer cells was assessed by fluorescence microscopy and flowcytometry. Gene expression was evaluated using qRT-PCR. Cell viability, chemosensitivity and apoptosis were determined by MTT assay and Annexin/PI kit. Cellular studies showed that liposomal transfer of COL1A1 siRNA into HCT116 and HEK293 cells significantly reduced the expression level of corresponding gen and cell viability, and significantly increased the sensitivity to chemotherapy drugs while free siRNA had no such effects. Aptamer conjugation was associated with increased cellular effects in HCT116 cells, but not in HEK293 cells. Our study revealed that delivery of COL1A1 siRNA via AS1411-targeted liposomes is a promising therapeutic approach to overcome treatment resistance in CRC.

Targeted Cancer Immunotherapy by Nanoparticle T Cell Engagers

IntroductionAcute myeloid leukemia (AML) is the most common type of leukemia. Emerging immunotherapies such as chimeric antigen receptor T (CAR‐T) cells and T cell engagers (TCEs), have the potential to enhance T cell recognition and response against cancers, but come with serious limitations such as short half‐life, complex production, high cost, and inability to customize for another antigen. To overcome these limitations, we previously developed a nanoparticle‐based T cell engagers (nanoTCEs) technology that has a longer half‐life (60h vs 2h for TCEs), is convenient to make, easily customizable, and robustly activates T cells for cancer killing. In this study, we hypothesized that nanoTCEs targeting CD33/CD3 would effectively activate T cell response in an AML disease model.Material and MethodsLiposomes were produced via the thin‐film hydration method with cholesterol, DPPC, and DSPE‐PEG200 at a molar ratio of 30:65:5 respectively (Figure 1A). CD33/CD3 nanoTCEs were created by conjugating anti‐CD3 and anti‐CD33 monoclonal antibodies (mAbs) on liposome surface with streptavidin‐biotin chemistry. Liposome binding to human T cells and AML cell lines was performed with fluorescently labeled nanoTCEs for 2 hours. The activation of T cell subsets and killing of AML cell lines was analyzed by flow cytometry after 4 days in a 3D culture model. For in vivo efficacy, immunodeficient NCG mice were inoculated with AML cell line and received Isotype/CD3 or CD33/CD3 nanoTCEs injections (0.5 mg/mouse) weekly and monitored for tumor progression and survival.ResultsAML cell lines (K052, MOLM‐14. NOMO‐1, and THP‐1) exhibited high expression of CD33 compared to controls. CD33/CD3 nanoTCEs bound to human T cells and AML cell lines with higher specificity than isotype controls. In addition, the CD33/CD3 nanoTCEs induced increased activation of both CD4+ and CD8+ T cells (Figure 1B, 1C) and achieved greater AML killing compared to the untreated and Isotype/CD3 groups (Figure 1D). Lastly, our in vivo results showed that weekly CD33/CD3 nanoTCE injections extended survival and decreased tumor progression of AML bearing mice compared to Isotype/CD3 nanoTCEs treated mice. By day 66 of the in vivo study, none of the Isotype/CD3 cohort was alive, while 100% of the CD33/CD3 cohort remained living (Figure 2).Discussion and ConclusionThis study shows that nanoTCEs are a promising novel platform for immunotherapy in AML. CD33/CD3 nanoTCEs demonstrated specific engagement of T cells to AML cells, which induced T cell activation and AML cell killing both in vitro and in vivo. It not only circumvented the limitations of traditional T cell engagers, but also presented numerous advantages such as short production time, low cost, and superior pharmacokinetic profile. Moreover, this technology is highly customizable and can be tailored to target multiple cancer antigens simultaneously. The current findings provide a strong preclinical basis for future first‐in‐human clinical trials of CD33/CD3 nanoTCEs as a novel AML immunotherapy.

Filament organization of the bacterial actin MreB is dependent on the nucleotide state.

MreB, the bacterial ancestor of eukaryotic actin, is responsible for shape in most rod-shaped bacteria. Despite belonging to the actin family, the relevance of nucleotide-driven polymerization dynamics for MreB function is unclear. Here, we provide insights into the effect of nucleotide state on membrane binding of Spiroplasma citri MreB5 (ScMreB5). Filaments of ScMreB5WT and an ATPase-deficient mutant, ScMreB5E134A, assemble independently of the nucleotide state. However, capture of the filament dynamics revealed that efficient filament formation and organization through lateral interactions are affected in ScMreB5E134A. Hence, the catalytic glutamate functions as a switch, (a) by sensing the ATP-bound state for filament assembly and (b) by assisting hydrolysis, thereby potentially triggering disassembly, as observed in other actins. Glu134 mutation and the bound nucleotide exhibit an allosteric effect on membrane binding, as observed from the differential liposome binding. We suggest that the conserved ATP-dependent polymerization and disassembly upon ATP hydrolysis among actins has been repurposed in MreBs for modulating filament organization on the membrane.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Synaptic vesicle binding of α-synuclein is modulated by β- and γ-synucleins.

SUMMARYα-synuclein, β-synuclein, and γ-synuclein are abundantly expressed proteins in the vertebrate nervous system. α-synuclein functions in neurotransmitter release by binding to and clustering synaptic vesicles and chaperoning SNARE-complex assembly. Pathologically, aggregates originating from soluble pools of α-synuclein are deposited into Lewy bodies in Parkinson’s disease and related synucleinopathies. The functions of β-synuclein and γ-synuclein in presynaptic terminals remain poorly studied. Using in vitro liposome binding studies, circular dichroism spectroscopy, immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments on isolated synaptic vesicles in combination with subcellular fractionation of brains from synuclein mouse models, we show that β-synuclein and γ-synuclein have a reduced affinity toward synaptic vesicles compared with α-synuclein, and that heteromerization of β-synuclein or γ-synuclein with α-synuclein results in reduced synaptic vesicle binding of α-synuclein in a concentration-dependent manner. Our data suggest that β-synuclein and γ-synuclein are modulators of synaptic vesicle binding of α-synuclein and thereby reduce α-synuclein’s physiological activity at the neuronal synapse.

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: Role of a conserved aromatic cage.

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the α clade and the β clade. Most α clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in β clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_βIB1). A structural comparison of available structures of phospholipases D (PLDs) reveals a conserved aromatic cage in the α clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the α clade, Loxosceles intermedia αIA1 (Li_αIA) and Loxosceles laeta αIII1 (Ll_αIII1), and one from the β clade, St_βIB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_βIB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the α clade PLDs. Here, we confirmed that the i-face of α and β clade PLDs is involved in their binding to choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the α clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.