Pulmonary surfactant proteins A and D recognize lipid ligands on Mycoplasma pneumoniae and markedly augment the innate immune response to the organism.
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Asthma↗
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Results for “lipid ligands”
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A quantitative electrophoretic migration shift assay for analyzing the specific binding of proteins to lipid ligands in vesicles or micelles.
We present a new assay for analyzing the specific binding of proteins to lipid ligands contained within vesicles or micelles. This assay, referred to as the electrophoretic migration shift assay, was developed using a model system composed of cholera toxin and of its physiological receptor, monosialoganglioside GM1. Using polyacrylamide gel electrophoresis in non-denaturing conditions, the migration of toxin components known to interact with GM1 was retarded when GM1 was present in either lipid vesicles or micelles. This effect was specific, as the migration of proteins not interacting with GM1 was not modified. The localization of retarded proteins and of lipids on gels was further determined by autoradiography. The stoichiometry of binding between cholera toxin and GM1 was determined, giving a value of five GM1 per one pentameric assembly of cholera toxin B-subunits, in agreement with previous studies. The general applicability of this assay was further established using both streptavidin and annexin V together with specific lipid ligands. This assay is fast, simple, quantitative, and requires only microgram quantities of protein.
Annexin A5↗
[Study of lipid ligands in serum albumin in sportsmen using thin layer chromatography].
Lipid composition and the ability of serum albumin to bind lipid components and products of their peroxidation were investigated in cyclists of different rating during moderate workloads. Ratios of subfractions of phospholipids and other lipid metabolites in blood serum albumin were determined by the method of thin-layer chromatography. Diene conjugates and diene ketones were found to be actively formed in plasma of highly trained cyclists in whom a high lipid capacity of albumin was associated with its low affinity for lipid hydroperoxides.
Adolescent↗
[The characteristics of lipid ligand binding by serum albumin in athletes].
The ability of serum albumin to bind lipids, fatty acids and products of their peroxidation was studied in well trained sportsmen during a competitive period. Detected quantitative and qualitative alterations point to a protein involvement in metabolic changes in an adaptation to work loads.
Bicycling↗
Tear lipocalins bind a broad array of lipid ligands.
To identify the native ligands of tear lipocalins, tear proteins were separated by size exclusion chromatography and the lipid content in the major protein fractions identified. Lipids extracted from native tears and purified tear lipocalins comigrated with fatty acids, fatty alcohols, phospholipids, glycolipids, and cholesterol on thin layer chromatograms. Abundant stearic and palmitic acids as well as cholesterol, and lesser amounts of lauric acid were specifically identified in extracts of purified lipocalins by gas chromatography-mass spectroscopy. A preliminary study of the ligand-protein interaction was carried out using nitroxide spin-labeled lipids.
Carrier Proteins↗
Conformationally constrained analogues of diacylglycerol (DAG). 23. Hydrophobic ligand-protein interactions versus ligand-lipid interactions of DAG-lactones with protein kinase C (PK-C).
The constrained glycerol backbone of DAG-lactones, when combined with highly branched alkyl chains, has engendered a series of DAG-lactone ligands capable of binding protein kinase C (PK-C) with affinities that approximate those of phorbol esters. These branched chains not only appear to be involved in making important hydrophobic contacts with the protein (specific interactions) but also provide adequate lipophilicity to facilitate partitioning into the lipid-rich membrane environment (nonspecific interactions). With the idea of minimizing the nonspecific interactions without reducing lipophilicity, the present work explores the strategy of relocating lipophilicity from the side chain to the lactone "core". Such a transfer of lipophilicity, exemplified by compounds 1 and 3, was conceived to allow the new hydrophobic groups on the lactone to engage in specific hydrophobic contacts inside the binding pocket without any expectation of interfering with the hydrogen-bonding network of the DAG-lactone pharmacophore. Surprisingly, both (E)-3 and (Z)-3 showed a significant decrease in binding affinity. From the molecular docking studies performed with the new ligands, we conclude that the binding pocket of the C1 domain of PK-C is sterically restricted and prevents the methyl groups at the C-3 position of the lactone from engaging in productive hydrophobic contacts with the receptor.
Binding Sites↗
IgG autoantibodies against beta2-glycoprotein I complexed with a lipid ligand derived from oxidized low-density lipoprotein are associated with arterial thrombosis in antiphospholipid syndrome.
We recently reported [J. Lipid Res. 42 (2001), 697; 43 (2002), 1486; 44 (2003), 716] that [beta2-glycoprotein I (beta2GPI) forms complexes with oxidized LDL (oxLDL) and autoantibodies against these complexes are present in patients with SLE and antiphospholipid syndrome (APS). The relationship of beta2GPI/oxLDL complexes and IgG autoantibodies against beta2GPI complexed with oxLig-1 (an oxLDL-derived ligand) with clinical manifestations of APS was studied in 150 APS and SLE patients. The beta2GPI/oxLDL levels of APS patients were similar to those of SLE patients without APS, but they were significantly higher than healthy individuals. There was no difference in the complex levels among the patients with arterial, venous thrombosis, or pregnancy morbidity. IgG anti-beta2GPI/oxLig-1 levels of APS were significantly higher than those of SLE without APS and healthy individuals. Further, antibody levels of APS patients with arterial thrombosis were significantly higher than those patients with venous thrombosis and pregnancy morbidity. Thus, oxidation of LDL leads the complex formation with beta2GPI in SLE and APS patients. In contrast, anti-beta2GPI/oxLig-1 autoantibodies were generated only in APS and were strongly associated with arterial thrombosis. These results suggest that autoantibodies against beta2GPI/oxLDL complexes are etiologically important in the development of atherosclerosis in APS.
Adolescent↗
Resonance energy transfer study using a rhenium metal-ligand lipid conjugate as the donor in a model membrane.
We measured steady state and time-resolved resonance energy transfer between donors and acceptors in model membranes. The donor was a long lifetime rhenium-lipid complex, which displayed a mean lifetime of 1 microsecond and lifetime components as long as 3 microseconds in the labeled DOPC membranes. The transfer efficiencies were found to be substantially larger than those predicted without consideration of lateral diffusion. The larger transfer efficiencies are consistent with a mutual lateral diffusion coefficient in the membrane near 2 x 10(-8) cm2/s. These results demonstrate that lateral diffusion in membranes can be detected with microsecond lipid probes.
Energy Transfer↗
[Therapeutic effect of diammonium glycyrrhizinate lipid ligand on carbon-tetrarchloride induced fatty livers in rats].
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Animals↗
Diversity in lipid A binding ligands: comparison of lipid A monoclonal antibodies with rBPI23.
1. Mabs with a high affinity for free lipid A do not bind when it is covalently linked, i.e. in the form of LPS. 2. Lipid A-binding Mabs may be divided into three categories: I. Monoreactive Mabs that bind to the hydrophillic backbone of lipid A II. Polyreactive Kdo Mabs III. Polyreactive Mabs that bind by hydrophobic interactions 3. rBPI23 binds either free or covalently linked lipid A.
Animals↗
Murine lipid phosphate phosphohydrolase-3 acts as a cell-associated integrin ligand.
Lipid phosphate phosphohydrolase-3 (LPP3) is a cell surface protein that exhibits ectoenzyme activity. Previously, we identified human LPP3 in a functional assay of angiogenesis and showed that the Arg-Gly-Asp (RGD) motif in the proposed second extracellular domain interacts with a subset of integrins to mediate cell-cell adhesion. In contrast to the RGD domain of human LPP3, murine Lpp3 contains a variant sequence, Arg-Gly-Glu (RGE). Whether the RGE motif of murine Lpp3 mediates cell-cell interaction has not been studied. In this report, we test the hypothesis that the cell adhesion function of the LPP3 protein is conserved across mouse and human. A glutathione S-transferase (GST) fusion protein of the proposed second extracellular loop of the murine Lpp3 sequence (GST-mLpp3-RGE) promoted attachment of cells in a long-term cell adhesion assay. GST-mLpp3-RGE interacted with alpha(5)beta(1) and alpha(v)beta(3) integrins in a solid-phase ELISA, while a mutant control, GST-hLPP3-RAD, did not. Long-term adhesion of endothelial cells to GST-mLpp3-RGE induced phosphorylation of FAK, SHC, and CAS, whereas adhesion to GST-hLPP3-RAD failed to do so. Upon long-term adhesion both the GST-hLPP3-RGD and GST-mLpp3-RGE substrates bound to the alpha(5)beta(1) integrin of FRT-alpha(5)(+) cells, an interaction that was inhibited by an anti-alpha(5) integrin antibody. In addition, a cell aggregation assay showed that the intact mLpp3-RGE protein interacts with alpha(5)beta(1) and alpha(v)beta(3) integrins expressed by adjacent cells, an interaction that can be blocked by GRGDSP peptides and anti-LPP3-RGD antibodies. These data, together with the known importance of integrins in angiogenesis, provide a mechanism for the function of LPP3 in cell-cell interactions in both human and mouse.
Amino Acid Sequence↗
Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands.
BACKGROUND: . Plant nonspecific lipid-transfer proteins (nsLTPs) bind a variety of very different lipids in vitro, including phospholipids, glycolipids, fatty acids and acyl coenzyme As. In this study we have determined the structure of a nsLTP complexed with palmitoyl coenzyme A (PCoA) in order to further our understanding of the structural mechanism of the broad specificity of these proteins and its relation to the function of nsLTPs in vivo. RESULTS: . 1H and 13C nuclear magnetic resonance spectroscopy (NMR) have been used to study the complex between a nsLTP isolated from barley seeds (bLTP) and the ligand PCoA. The resonances of 97% of the 1H atoms were assigned for the complexed bLTP and nearly all of the resonances were assigned in the bound PCoA ligand. The palmitoyl chain of the ligand was uniformly 13C-labelled allowing the two ends of the hydrocarbon chain to be assigned. The comparison of a subset of 20 calculated structures to an average structure showed root mean square deviations of 1.89 +/- 0.19 for all C, N, O, P and S atoms of the entire complex and of 0.57 +/- 0.09 for the peptide backbone atoms of the four alpha helices of the complexed bLTP. The four-helix topology of the uncomplexed bLTP is maintained in the complexed form of the protein. The bLTP only binds the hydrophobic parts of PCoA with the rest of the ligand remaining exposed to the solvent. The palmitoyl chain moiety of the ligand is placed in the interior of the protein and bent in a U-shape. This part of the ligand is completely buried within a hydrophobic pocket of the protein. CONCLUSIONS: . A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.
Amino Acid Sequence↗
Membrane-protein interactions in cell signaling and membrane trafficking.
Research in the past decade has revealed that many cytosolic proteins are recruited to different cellular membranes to form protein-protein and lipid-protein interactions during cell signaling and membrane trafficking. Membrane recruitment of these peripheral proteins is mediated by a growing number of modular membrane-targeting domains, including C1, C2, PH, FYVE, PX, ENTH, ANTH, BAR, FERM, and tubby domains, that recognize specific lipid molecules in the membranes. Structural studies of these membrane-targeting domains demonstrate how they specifically recognize their cognate lipid ligands. However, the mechanisms by which these domains and their host proteins are recruited to and interact with various cell membranes are only beginning to unravel with recent computational studies, in vitro membrane binding studies using model membranes, and cellular translocation studies using fluorescent protein-tagged proteins. This review summarizes the recent progress in our understanding of how the kinetics and energetics of membrane-protein interactions are regulated during the cellular membrane targeting and activation of peripheral proteins.
Animals↗
Protein lipid overlay assay.
The Protein Lipid Overlay (PLO) assay enables the identification of the lipid ligands with which lipid binding proteins interact. This assay also provides qualitative information on the relative affinity with which a protein binds to a lipid. In the PLO assay, serial dilutions of different lipids are spotted onto a nitrocellulose membrane to which they attach. These membranes are then incubated with a lipid binding protein possessing an epitope tag. The membranes are washed and the protein, still bound to the membrane by virtue of its interaction with lipid(s), is detected by immunoblotting with an antibody recognizing the epitope tag. This procedure requires only a few micrograms of protein and is quicker and cheaper to perform than other methods that have been developed to assess protein-lipid interactions. The reagents required for the PLO assay are readily available from commercial sources and the assay can be performed in any laboratory, even by those with no prior expertise in this area.
Biological Transport↗
Inhibition of cytoskeletal rearrangement by botulinum C2 toxin amplifies ligand-evoked lipid mediator generation in human neutrophils.
Botulinum C2 toxin, a binary toxin that ADP-ribosylates nonmuscle G-actin, was used as a selective tool to evaluate the role of actin-dependent cytoskeletal rearrangement in ligand-evoked lipid mediator generation. Human neutrophils (PMN) were preincubated with varying concentrations of the toxin for 30 min. Lipoxygenase products of arachidonic acid were measured by chromatographic techniques in the presence of exogenous arachidonic acid to probe PMN 5-lipoxygenase activity. Formation of platelet-activating factor (PAF) was assayed by the bioincorporation of [3H]acetate. Stimulation was performed with the soluble chemotactic ligands formyl-methionyl-leucyl-phenylalanine (FMLP) and PAF, as well as opsonized zymosan. PMN pretreatment with C2 toxin in the range between 200/400 and 800/1600 ng/ml C2I/II caused a dose-dependent suppression of the basal F-actin content and of stimulus-induced actin assembly. Phosphoinositide hydrolysis (measured as liberated inositol phosphates) and PAF generation in response to FMLP and exogenous PAF were markedly increased at these toxin doses. Minor C2 toxin concentrations (range, approximately 25/50 to 200/400 ng/ml C2I/II) were sufficient to amplify stimulus-induced formation of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraenoic acid (5-HETE). With increasing toxin doses, leukotriene generation declined and 5-HETE became the predominant metabolite. In contrast to the soluble ligands, the zymosan-effected generation of PAF and leukotrienes was dose-dependently inhibited by C2 toxin concentrations of greater than 200/400 ng/ml, paralleled by a loss of motile and phagocytotic functions in these cells. We conclude that selective inhibition of actin assembly amplifies PAF and 5-lipoxygenase product formation in response to soluble chemoattractants with distinct dose dependences. The augmentation of PAF generation may be linked to amplified second messenger levels at higher doses of C2 toxin, whereas the sensitivity of the 5-lipoxygenase metabolism to low concentrations may indicate toxin effect on a small, functionally specified, actin pool. The present data support an important role of cytoskeletal rearrangement in temporal and/or spatial limitation of chemoattractant-evoked PMN activation.
Arachidonate 5-Lipoxygenase↗
Ligand and lipid domain stabilization of a membraneous Ca2+-ATPase during hyperthermia.
The susceptibility of the membranous Ca2+-ATPase of sarcoplasmic reticulum to enzymatic inactivation at hyperthermic temperatures was investigated. Inactivation produced a break in the Arrhenius plot at 45-46 degrees C and was accompanied by an increased mobility of spin label, covalently attached to the Ca2+-ATPase. MgADP and MgATP exerted a markedly stabilizing effect on inactivation, both at pH 7.0 and in acidic media. By contrast, high-affinity Ca2+ or Mg2+ binding only moderately stabilized Ca2+-ATPase (inactivation rates were decreased 2-3 times), and this effect was non-additive, i.e., only observed in the absence of the other divalent cation. But withdrawal of K+ and Na+ gave rise to a pronounced destabilization that could be reversed efficiently by high concentrations of Ca2+ or Mg2+. These results are compared with a previous study on detergent solubilized Ca2+-ATPase (Møller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920) which showed the enzyme to be markedly stabilized by Ca2+ as well as by nucleotide. It is concluded that, due to the presence of nucleotide, inactivation of Ca2+-ATPase is not likely to occur during malignant hyperthermia and that the native environment of the lipid bilayer provides stabilization of the membrane-embedded and Ca2+-translocating domain of the Ca2+-ATPase.
Animals↗
Sphingosine 1-phosphate pathway therapeutics: a lipid ligand-receptor paradigm.
New insights have been gained into the therapeutic relevance of the sphingosine 1-phosphate (S1P) pathway, on the basis of reverse pharmacological approaches to defining the mechanism of action of the immunosuppressive agent FTY720. Natural and synthetic sphingosine 1-phosphate receptor agonists can make picomolar interactions with their cognate G-protein-coupled receptors, and provide chemical approaches to defining the contribution of distinct receptor subtypes to pathology, physiology and treatment. The chemistry of S1P receptors and their synthetic ligands present a paradigm for the understanding of lipid-receptor interactions and their contribution to physiology and pathology. These approaches can potentially be extended to a broad, related family of G-protein-coupled receptors that share ligands and ligand similarities.
Animals↗
In vivo effects of CB1 receptor ligands on lipid peroxidation and antioxidant defense systems in the rat brain of healthy and ethanol-treated rats.
In vivo experiments were conducted to study the effects of N-(piperidin-l-yl)-5-(4-chlorophenyl)-1-(2,4-cochlo-rophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR141716A; a potent and selective CB(1)-receptor antagonist) and ara-chidonyl-2-chloroethylamide (ACEA; a selective CB(1)-receptor agonist) on spontaneous lipid peroxidation, glutathione (GSH) level and activities of antioxidant enzymes in rat tissues. Single doses of SR141716A(3 mg/kg, ip) and ACEA(10 mg/kg, ip) had no effect on all indices, studied in the brain, except for a decrease in GSH level by 10 mg/kg of SR141716A. The effects of repeated administration of the CB(1)-receptor ligands (3 mg/kg, ip, once daily for 2 days) on the above indices in the brain and liver of control and ethanol-treated animals were also studied. Two weeks after ethanol exposure, the rats lost weight (by 41%), which correlated with their decreased water and food consumption (by 52% and 33%, respectively). The time of ethanol action was not sufficient to change the biochemical parameters in the brain, except for the lipid peroxidation. However, a decrease in GSH level and superoxide dismutase activity, as well as an increase in lipid peroxidation and glucose-6-phosphate dehydrogenase activity were registered in the liver. The repeated administration of CB(1) receptor ligands restored some of ethanol-induced changes. The present results suggested lack of pro-oxidant activity and potential antioxidant ability of the studied CB(1) receptor ligands, which might contribute to their beneficial effects.
Animals↗