Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility.

Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family.

The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with -oleoyl-galactosylceramide.

Förster Resonance Energy Transfer Study of Cytochrome c-Lipid Interactions.

Specific interactions between a mitochondrial hemoprotein cytochrome c (cyt c) and cardiolipin, a lipid component of mitochondrial membrane, are crucial to electron shuttling and apoptotic activities of this protein. In the present study the Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine as a donor and heme moiety of cyt c as an acceptor was employed to give a quantitative characterization of the protein binding to the model membranes from the mixtures of phosphatidylcholine (PC) with phosphatidylglycerol (PG), phosphatidylserine (PS) or cardiolipin (CL) in different molar ratios. The multiple arrays of the FRET data were globally analyzed in terms of the model of energy transfer in two-dimensional systems combined with the scaled particle adsorption model.

Liposomal formulation of a methotrexate lipophilic prodrug: assessment in tumor cells and mouse T-cell leukemic lymphoma.

In a previous study, a formulation of methotrexate (MTX) incorporated in the lipid bilayer of 100-nm liposomes in the form of diglyceride ester (MTX-DG, lipophilic prodrug) was developed. In this study, first, the interactions of MTX-DG liposomes with various human and mouse tumor cell lines were studied using fluorescence techniques. The liposomes composed of egg phosphatidylcholine (PC)/yeast phosphatidylinositol/MTX-DG, 8:1:1 by mol, were labeled with fluorescent analogs of PC and MTX-DG.

Sphingolipid transfer proteins defined by the GLTP-fold.

Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer 'sandwich' architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation.

Location of novel benzanthrone dyes in model membranes as revealed by resonance energy transfer.

Förster resonance energy transfer (FRET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) as a donor and newly synthesized benzanthrones (referred to here as A8, A6, AM12, AM15 and AM18) as acceptors has been examined to gain insight into molecular level details of the interactions between benzanthrone dyes and model lipid membranes composed of zwitterionic lipid phosphatidylcholine and its mixtures with anionic lipids cardiolipin (CL) and phosphatidylglycerol (PG). FRET data were quantitatively analyzed in terms of the model of energy transfer in two-dimensional systems taking into account the distance dependence of orientation factor. Evidence for A8 location in phospholipid headgroup region has been obtained.

Nanoscale packing differences in sphingomyelin and phosphatidylcholine revealed by BODIPY fluorescence in monolayers: physiological implications.

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites.

Structural insights into lipid-dependent reversible dimerization of human GLTP.

Human glycolipid transfer protein (hsGLTP) forms the prototypical GLTP fold and is characterized by a broad transfer selectivity for glycosphingolipids (GSLs). The GLTP mutation D48V near the `portal entrance' of the glycolipid binding site has recently been shown to enhance selectivity for sulfatides (SFs) containing a long acyl chain. Here, nine novel crystal structures of hsGLTP and the SF-selective mutant complexed with short-acyl-chain monoSF and diSF in different crystal forms are reported in order to elucidate the potential functional roles of lipid-mediated homodimerization.

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine.

Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data.

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds.

The effect of lysozyme amyloid fibrils on cytochrome c-lipid interactions.

Protein polymerization into ordered fibrillar structures (amyloid fibrils) is currently associated with a range of pathological conditions. Recent studies clearly indicate that amyloid cytotoxicity is provoked by a continuum of cross-β-sheet aggregates including mature fibrils. In view of the possible diversity of cytotoxicity mechanisms, the present study addressed the question of whether protein conversion into amyloid fibrils can modify its competitive membrane adsorption behavior.

Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP.

Conformational folding and stability of the HET-C2 glycolipid transfer protein fold: does a molten globule-like state regulate activity?

The glycolipid transfer protein (GLTP) superfamily is defined by the human GLTP fold that represents a novel motif for lipid binding and transfer and for reversible interaction with membranes, i.e., peripheral amphitropic proteins.

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold.

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy.

The intermembrane ceramide transport catalyzed by CERT is sensitive to the lipid environment.

The in vitro activity of the ceramide transporter, CERT has been studied using a fluorescence assay. CERT is responsible for the in vivo non-vesicular trafficking of ceramide between the endoplasmic reticulum and Golgi. In this study we have examined how the membrane environment surrounding the ceramide substrate, the membrane packing density and the membrane charge, are affecting the ceramide transfer activity.

Cytochrome c-lipid interactions: new insights from resonance energy transfer.

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio.

Morphological changes of supported lipid bilayers induced by lysozyme: planar domain formation vs. multilayer stacking.

Total internal reflection fluorescence microscopy (TIRFM) has been utilized to explore the effect of cationic protein lysozyme (Lz) on the morphology of solid-supported lipid bilayers (SLBs) comprised of zwitterionic lipid phosphatidylcholine (PC) and its mixture with anionic lipid cardiolipin (CL). Kinetic TIRFM imaging of different systems revealed subtle interplay between lipid lateral segregation accompanied by exchange of neutral and acidic lipids in the protein-lipid interaction zone, and the formation of lipid multilayer stacks. The switch between these states was shown to be controlled by CL content.

Characterization of the lateral distribution of fluorescent lipid in binary-constituent lipid monolayers by principal component analysis.

Lipid lateral organization in binary-constituent monolayers consisting of fluorescent and nonfluorescent lipids has been investigated by acquiring multiple emission spectra during measurement of each force-area isotherm. The emission spectra reflect BODIPY-labeled lipid surface concentration and lateral mixing with different nonfluorescent lipid species. Using principal component analysis (PCA) each spectrum could be approximated as the linear combination of only two principal vectors.

Structural determination and tryptophan fluorescence of heterokaryon incompatibility C2 protein (HET-C2), a fungal glycolipid transfer protein (GLTP), provide novel insights into glycolipid specificity and membrane interaction by the GLTP fold.

HET-C2 is a fungal protein that transfers glycosphingolipids between membranes and has limited sequence homology with human glycolipid transfer protein (GLTP). The human GLTP fold is unique among lipid binding/transfer proteins, defining the GLTP superfamily. Herein, GLTP fold formation by HET-C2, its glycolipid transfer specificity, and the functional role(s) of its two Trp residues have been investigated.

Microbial alkaloid staurosporine induces formation of nanometer-wide membrane tubular extensions (cytonemes, membrane tethers) in human neutrophils.

In the present work, we demonstrate that microbial alkaloid staurosporine (STS) and Ro 31-8220, structurally related to STS protein kinase C inhibitor, caused development of membrane tubular extensions in human neutrophils upon adhesion to fibronectin-coated substrata. STS-induced tubular extensions interconnected neutrophils in a network and bound serum-opsonized bacteria Salmonella enterica serovar Typhimurium. The diameter of STS-induced extensions varied in the range 160-200 nm.

Nitric oxide-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and hold Salmonella enterica serovar Typhimurium at a distance from the cell surface.

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles.