dioleoyl-phosphatidylethanolamine and 1-2-dioleoylphosphatidylserine

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

Topics: Blood Coagulation; Bradykinin; Calcium; Cations, Divalent; Factor IX; Factor XI; Factor XII; Fibrin; Humans; Kallikreins; Kinetics; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding; Thrombin

Complexin-1 (Cpx) and α-synuclein (α-Syn) are involved in neurotransmitter release through an interaction with synaptic vesicles (SVs). Recent studies demonstrated that Cpx and α-Syn preferentially associate with highly curved membranes, like SVs, to correctly position them for fusion. Here, based on recent experimental results, to further propose a possible explanation for this mechanism, we performed in silico simulations probing interactions between Cpx or α-Syn and membranes of varying curvature. We found that the preferential association is attributed to smaller, curved membranes containing more packing defects that expose hydrophobic acyl tails, which may favorably interact with hydrophobic residues of Cpx and α-Syn. The number of membrane defects is proportional to the curvature and the size can be regulated by cholesterol.

Topics: Adaptor Proteins, Vesicular Transport; alpha-Synuclein; Cholesterol; Hydrogen Bonding; Lipid Bilayers; Molecular Dynamics Simulation; Nerve Tissue Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding; Synaptic Vesicles

Quantum dots increasingly gain popularity for in vivo applications. However, their delivery and accumulation into cells can be challenging and there is still lack of detailed information. Thereby, the application of advanced fluorescence techniques can expand the portfolio of useful parameters for a more comprehensive evaluation. Here, we encapsulated hydrophilic quantum dots into liposomes for studying cellular uptake of these so-called lipodots into living cells. First, we investigated photophysical properties of free quantum dots and lipodots observing changes in the fluorescence decay time and translational diffusion behaviour. In comparison to empty liposomes, lipodots exhibited an altered zeta potential, whereas their hydrodynamic size did not change. Fluorescence lifetime imaging microscopy (FLIM) and fluorescence correlation spectroscopy (FCS), both combined with two-photon excitation (2P), were used to investigate the interaction behaviour of lipodots with an insect epithelial tissue. In contrast to the application of free quantum dots, their successful delivery into the cytosol of salivary gland duct cells could be observed when applying lipodots. Lipodots with different lipid compositions and surface charges did not result in considerable differences in the intracellular labelling pattern, luminescence decay time and diffusion behaviour. However, quantum dot degradation after intracellular accumulation could be assumed from reduced luminescence decay times and blue-shifted luminescence signals. In addition to single diffusing quantum dots, possible intracellular clustering of quantum dots could be assumed from increased diffusion times. Thus, by using a simple and manageable liposome carrier system, 2P-FLIM and 2P-FCS recording protocols could be tested, which are promising for investigating the fate of quantum dots during cellular interaction.

Topics: Animals; Cadmium; Diffusion; Epithelial Cells; Humans; Hydrophobic and Hydrophilic Interactions; Liposomes; Luminescent Measurements; Male; Microscopy, Fluorescence; Periplaneta; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Quantum Dots; Salivary Glands; Selenium; Sulfides; Zinc Compounds

Amyloid fibrillation causes serious neurodegenerative diseases and amyloidosis; however, the detailed mechanisms by which the structural states of precursor proteins in a lipid membrane-associated environment contribute to amyloidogenesis still remains to be elucidated. We examined the relationship between structural states of intrinsically-disordered wild-type and mutant α-synuclein (αSN) and amyloidogenesis on two-types of model membranes. Highly-unstructured wild-type αSN (αSN

Topics: alpha-Synuclein; Amyloid; Dose-Response Relationship, Drug; Dynamic Light Scattering; Humans; Membrane Lipids; Models, Chemical; Nuclear Magnetic Resonance, Biomolecular; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Binding; Protein Conformation; Sequence Deletion; Unilamellar Liposomes

The fusion of biological membranes may require splayed lipids whose tails transiently visit the headgroup region of the bilayer, a scenario suggested by molecular dynamics simulations. Here, we examined the lipid splay hypothesis experimentally by relating liposome fusion and lipid splay induced by model transmembrane domains (TMDs). Our results reveal that a conformationally flexible transmembrane helix promotes outer leaflet mixing and lipid splay more strongly than a conformationally rigid one. The lipid dependence of basal as well as of TMD-driven lipid mixing and splay suggests that the cone-shaped phosphatidylethanolamine stimulates basal fusion via enhancing lipid splay and that the negatively charged phosphatidylserine inhibits fusion via electrostatic repulsion. Phosphatidylserine also strongly differentiates basal and helix-driven fusion, which is related to its preferred interaction with the conformationally more flexible transmembrane helix. Thus, the contribution of a transmembrane helix to membrane fusion appears to depend on lipid binding, which results in lipid splay.

Topics: Lipid Bilayers; Liposomes; Membrane Fusion; Peptides; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines

The study of ion channel activity and the screening of possible inhibitor molecules require reliable methods for production of active channel proteins, their insertion into artificial membranes and for the measurement of their activity. Here we report on cell-free expression of soluble and active K

Topics: Elapid Venoms; Escherichia coli; Fluorescent Dyes; Gene Expression; Genetic Vectors; Humans; Isoxazoles; Kv1.1 Potassium Channel; Kv1.3 Potassium Channel; Membrane Potentials; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Proteolipids; Recombinant Proteins; Subcellular Fractions; Valinomycin

The enzymatic lipolysis of complex natural lipoproteic assemblies such as milk fat globules is central in neonatal digestion. This process first requires the rapid adsorption of a lipolytic enzyme, gastric lipase, onto the membrane enveloping the triglyceride substrate before the onset of catalytic activity. The interactions governing lipase adsorption onto this complex lipid/water interface are not fully elucidated. This study was designed to unravel the interactions of recombinant dog gastric lipase (rDGL) with model monolayers presenting liquid-liquid phase coexistence and mimicking the outer leaflet of the milk fat globule membrane. Combining biophysical tools (ellipsometry, tensiometry and atomic force microscopy), it was evidenced that rDGL partitions toward liquid expanded phase and at phase boundaries. rDGL gets adsorbed at several levels of insertion suggesting molecular cooperation that may favor insertion and strongly impacts on the lipid phase lateral organization. The addition of phosphatidylserine, negatively charged, reinforced adsorption; hence besides hydrophobic interactions and as further investigated through surface potential modeling, rDGL adsorption is favored by electrostatic interactions.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Adsorption; Animals; Cattle; Dogs; Glycolipids; Glycoproteins; Lipase; Lipid Droplets; Microscopy, Atomic Force; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Recombinant Proteins; Static Electricity; Stomach; Surface Tension; Unilamellar Liposomes; Water

Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca(2+)-binding C2 domains that tether membranes via interactions with anionic phospholipids. It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them. As quantitative information on membrane gaps is key to understanding general cellular mechanisms, including the role of syt-1, we developed a fluorescence-lifetime based inter-membrane distance ruler using membrane-anchored DNAs of various lengths as calibration standards. Wild-type and mutant data provide evidence that full-length syt-1 indeed regulates membrane gaps: without Ca(2+), syt-1 maintains membranes at distances of ~7-8 nm. Activation with 100 μM Ca(2+) decreases the distance to ~5 nm by binding the C2 domains to opposing membranes, respectively. These values reveal that activated syt-1 adjusts membrane distances to the level that promotes SNARE complex assembly.

Topics: Animals; Calcium; Carboxylic Acids; Cations, Divalent; Cholesterol; Fluorescent Dyes; Gene Expression; Microscopy, Fluorescence; Oligonucleotides; Phosphatidylethanolamines; Phosphatidylserines; Presynaptic Terminals; Proteolipids; Rats; Recombinant Proteins; SNARE Proteins; Synaptic Vesicles; Synaptotagmin I; Unilamellar Liposomes

It has recently been reported that N-ethylmaleimide-sensitive fusion ATPase (NSF) can fuse protein-free liposomes containing substantial amounts of 1,2-dioleoylphosphatidylserine (DOPS) and 1, 2-dioleoyl-phosphatidyl-ethanolamine (DOPE) (Otter-Nilsson et al., 1999). The authors impart physiological significance to this observation and propose to re-conceptualize the general role of NSF in fusion processes. We can confirm that isolated NSF can fuse liposomes of the specified composition. However, this activity of NSF is resistant to inactivation by N-ethylmaleimide and does not depend on the presence of alpha-SNAP (soluble NSF-attachment protein). Moreover, under the same conditions, either alpha-SNAP, other proteins apparently unrelated to vesicular transport (glyceraldehyde-3-phosphate dehydrogenase or lactic dehydrogenase) or even 3 mM magnesium ions can also cause lipid mixing. In contrast, neither NSF nor the other proteins nor magnesium had any significant fusogenic activity with liposomes composed of a biologically occurring mixture of lipids. A straightforward explanation is that the lipid composition chosen as optimal for NSF favors non-specific fusion because it is physically unstable when formed into liposomes. A variety of minor perturbations could then trigger coalescence.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Carrier Proteins; Ethylmaleimide; Glyceraldehyde-3-Phosphate Dehydrogenases; Golgi Apparatus; L-Lactate Dehydrogenase; Lipid Metabolism; Liposomes; Magnesium; Membrane Fusion; Membrane Proteins; N-Ethylmaleimide-Sensitive Proteins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Rats; SNARE Proteins; Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; Thermodynamics; Vesicular Transport Proteins