Tracking sphingosine metabolism and transport in sphingolipidoses: NPC1 deficiency as a test case.

The late endosomal/lysosomal compartment (LE/LY) plays a key role in sphingolipid breakdown, with the last degradative step catalyzed by acid ceramidase. The released sphingosine can be converted to ceramide in the ER and transported by ceramide transfer protein (CERT) to the Golgi for conversion to sphingomyelin. The mechanism by which sphingosine exits LE/LY is unknown but Niemann-Pick C1 protein (NPC1) has been suggested to be involved. Here, we used sphingomyelin, ceramide and sphingosine labeled with [(3)H] in carbon-3 of the sphingosine backbone and targeted them to LE/LY in low-density lipoprotein (LDL) particles. These probes traced LE/LY sphingolipid degradation and recycling as suggested by (1) accumulation of [(3)H]-sphingomyelin-derived [(3)H]-ceramide and depletion of [(3)H]-sphingosine upon acid ceramidase depletion, and (2) accumulation of [(3)H]-sphingosine-derived [(3)H]-ceramide and attenuation of [(3)H]-sphingomyelin synthesis upon CERT depletion. NPC1 silencing did not result in the accumulation of [(3)H]-sphingosine derived from [(3)H]-sphingomyelin/LDL or [(3)H]-ceramide/LDL. Additional evidence against NPC1 playing a significant role in LE/LY sphingosine export was obtained in experiments using the [(3)H]-sphingolipids or a fluorescent sphingosine derivative in NPC1 knock-out cells. Instead, NPC1-deficient cells displayed an increased affinity for sphingosine independently of protein-mediated lipid transport. This likely contributes to the increased sphingosine content of NPC1 cells.

FTY720 analogues as sphingosine kinase 1 inhibitors: enzyme inhibition kinetics, allosterism, proteasomal degradation, and actin rearrangement in MCF-7 breast cancer cells.

Sphingosine kinase 1 (SK1) catalyzes the conversion of sphingosine to the bioactive lipid sphingosine 1-phosphate. We have previously demonstrated that FTY720 and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 activity. Here, we show that (S)-FTY720 vinylphosphonate binds to a putative allosteric site in SK1 contingent on formation of the enzyme-sphingosine complex. We report that SK1 is an oligomeric protein (minimally a dimer) containing noncooperative catalytic sites and that the allosteric site exerts an autoinhibition of the catalytic site. A model is proposed in which (S)-FTY720 vinylphosphonate binding to and stabilization of the allosteric site might enhance the autoinhibitory effect on SK1 activity. Further evidence for the existence of allosteric site(s) in SK1 was demonstrated by data showing that two new FTY720 analogues (a conjugate of sphingosine with a fluorophore and (S)-FTY720 regioisomer) increased SK1 activity, suggesting relief of autoinhibition of SK1 activity. Comparisons with the SK1 inhibitor, SKi or siRNA knockdown of SK1 indicated that (S)-FTY720 vinylphosphonate and FTY720 behave as typical SK1 inhibitors in preventing sphingosine 1-phosphate-stimulated rearrangement of actin in MCF-7 cells. These findings are discussed in relation to the anticancer properties of SK1 inhibitors.

FTY720 stimulates 27-hydroxycholesterol production and confers atheroprotective effects in human primary macrophages.

RATIONALE: The synthetic sphingosine analog FTY720 is undergoing clinical trials as an immunomodulatory compound, acting primarily via sphingosine 1-phosphate receptor activation. Sphingolipid and cholesterol homeostasis are closely connected but whether FTY720 affects atherogenesis in humans is not known. OBJECTIVE: We examined the effects of FTY720 on the processing of scavenged lipoprotein cholesterol in human primary monocyte-derived macrophages. METHODS AND RESULTS: FTY720 did not affect cholesterol uptake but inhibited its delivery to the endoplasmic reticulum, reducing cellular free cholesterol cytotoxicity. This was accompanied by increased levels of Niemann-Pick C1 protein (NPC1) and ATP-binding cassette transporter (ABC)A1 proteins and increased efflux of endosomal cholesterol to apolipoprotein A-I. These effects were not dependent on sphingosine 1-phosphate receptor activation. Instead, FTY720 stimulated the production of 27-hydroxycholesterol, an endogenous ligand of the liver X receptor, leading to liver X receptor-induced upregulation of ABCA1. Fluorescently labeled FTY720 was targeted to late endosomes, and the FTY720-induced upregulation of ABCA1 was NPC1-dependent, but the endosomal exit of FTY720 itself was not. CONCLUSIONS: We conclude that FTY720 decreases cholesterol toxicity in primary human macrophages by reducing the delivery of scavenged lipoprotein cholesterol to the endoplasmic reticulum and facilitating its release to physiological extracellular acceptors. Furthermore, FTY720 stimulates 27-hydroxycholesterol production, providing an explanation for the atheroprotective effects and identifying a novel mechanism by which FTY720 modulates signaling.

Sphingosine interaction with acidic leucine-rich nuclear phosphoprotein-32A (ANP32A) regulates PP2A activity and cyclooxygenase (COX)-2 expression in human endothelial cells.

Sphingolipid metabolites regulate cell fate by acting on specific cellular targets. Although the influence of sphingolipids in cellular signaling has been well recognized, the exact molecular targets and how these targets influence cellular signaling mechanisms remain poorly understood. Toward this goal, we used affinity chromatography coupled with proteomics technology and identified acidic leucine-rich nuclear phosphoprotein-32A (ANP32A), an inhibitor of protein phosphatase 2A (PP2A) as a direct target of sphingosine, N,N'-dimethyl sphingosine (DMS) and phytosphingosine but not dihydrosphingosine or sphingosine 1-phosphate. Treatment of human umbilical vein endothelial cells (HUVEC) with DMS, which is not phosphorylated by sphingosine kinases, led to the activation of PP2A activity. Suppression of ANP32A with siRNA enhanced basal and DMS-activated PP2A activity suggesting that the sphingoid base binds to and relieves the inhibitory action of ANP32A on the PP2A complex. Indeed, DMS relieved the ANP32A-mediated inhibition of PP2A enzyme complex in vitro. Interestingly, DMS treatment induced the p38 stress-activated protein kinase (SAPK) and expression of cyclooxygenase (COX)-2 transcript and protein. Knockdown of ANP32A expression further induced p38 SAPK and COX-2. These data identify ANP32A as a novel molecular target of sphingoid bases that regulates cellular signaling events and inflammatory gene expression.

BODIPY-cholesterol: a new tool to visualize sterol trafficking in living cells and organisms.

Analysis of sterol distribution and transport in living cells has been hampered by the lack of bright, photostable fluorescent sterol derivatives that closely resemble cholesterol. In this study, we employed atomistic simulations and experiments to characterize a cholesterol compound with fluorescent boron dipyrromethene difluoride linked to sterol carbon-24 (BODIPY-cholesterol). This probe packed in the membrane and behaved similarly to cholesterol both in normal and in cholesterol-storage disease cells and with trace amounts allowed the visualization of sterol movement in living systems. Upon injection into the yolk sac, BODIPY-cholesterol did not disturb zebrafish development and was targeted to sterol-enriched brain regions in live fish. We conclude that this new probe closely mimics the membrane partitioning and trafficking of cholesterol and, because of its excellent fluorescent properties, enables the direct monitoring of sterol movement by time-lapse imaging using trace amounts of the probe. This is, to our knowledge, the first cholesterol probe that fulfills these prerequisites.

Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1-phosphate.

Sphingosine 1-phosphate (S1P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S1P from RBC into plasma is a regulated process that does not occur in plasma- or serum-free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non-redundant endogenous triggers for S1P release from RBC. S1P bound to SA and HDL was able to stimulate the S1P(1) receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S1P release, as demonstrated with the anti-S1P antibody Sphingomab. More S1P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S1P release in plasma. In co-cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S1P as a second function of RBC-associated S1P in the absence of SA and HDL and during tight RBC-EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S1P bound to SA and HDL, RBC-associated S1P was significantly incorporated by EC after S1P lyase (SGPL1) inhibition. RBC-associated S1P, therefore, has two functions: (1) It contributes to the cellular pool of SGPL1-sensitive S1P in tissues after transcellular transportation and (2) it helps maintain extracellular S1P levels via SA and HDL independently from SGPL1 activity.

Membrane fluidity and lipid order in ternary giant unilamellar vesicles using a new bodipy-cholesterol derivative.

Cholesterol-rich, liquid-ordered (L(o)) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (L(d)) domains, the number of such analogs with a high affinity for biologically relevant L(o) domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The L(d) phase domains were also labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI-C(12)) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 +/- 0.08 ns, compared with 4.1 +/- 0.4 ns in the presence of DiI-C(12). The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C(12) (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 +/- 0.2 nm. These results also indicate preferential partitioning (K(p) = 1.88) of Bdp-Chol into the L(o) phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence correlation spectroscopy, is (7.4 +/- 0.3) x 10(-8) cm(2)/s and (5.0 +/- 0.2) x 10(-8) cm(2)/s in the L(d) and L(o) phases, respectively. Experimental translational/rotational diffusion coefficient ratios are compared with theoretical predictions using the hydrodynamic model (Saffman-Delbrück). The results suggest that Bdp-Chol is likely to form a complex with other lipid molecules during its macroscopic diffusion in GUV lipid bilayers at room temperature. Our integrated, multiscale results demonstrate the potential of this cholesterol analog for studying lipid-lipid interactions, lipid order, and membrane fluidity of biologically relevant L(o) domains.

Role of ceramide in membrane protein organization investigated by combined AFM and FCS.

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate.

Sphingosine 1-phosphate lyase (SPL) is responsible for the irreversible catabolism of sphingosine 1-phosphate, which signals through five membrane receptors to mediate cell stress responses, angiogenesis, and lymphocyte trafficking. The standard assay for SPL activity utilizes a radioactive dihydrosphingosine 1-phosphate substrate and is expensive and cumbersome. In this study, we describe an SPL assay that employs an omega-labeled BODIPY-sphingosine 1-phosphate substrate, allowing fluorescent product detection by HPLC and incorporating advantages of the BODIPY fluorophore. The major aldehyde product is confirmed by reaction with 2,4-dinitrophenylhydrazine. The SPL-catalyzed reaction is linear over a 30 min time period and yields a K(m) of 35 microM for BODIPY-sphingosine 1-phosphate.

Anticancer activity of a ceramide analog containing a disulfide linkage.

The effects of exogenous short-chain ceramide (1) on the arrest of growth of cancer cells in vitro and induction of apoptosis have been well documented. In the present study, an analog of 1 with a disulfide linkage, N-(4',5'-dithiaheptanoyl)-D-erythro-ceramide (2), was synthesized and found to be significantly more antiproliferative and cytotoxic than 1 in BT549, A549, and DU145 cancer cells. The activity was correlated with a reduction in cellular glutathione (GSH) level. Ceramide analogs with a tri- and tetra-sulfide moiety were also prepared, but they did not deplete cellular GSH levels and did not possess antiproliferative or cytotoxic properties.

Addition of lithiated 5-hydroxymethyl-1,3-dithiane to benzaldehyde: HMPA-controlled trans stereoselectivity.

Addition of 5-substituted dithianyl anions to carbonyl compounds normally produces trans adducts. The presence of a nucleophilic hydroxymethyl group in position 5 dramatically decreases the trans stereoselectivity of the reaction in THF. The trans/cis ratio shows a bell curve dependence on HMPA, fitted to a quantitative model involving a series of equilibrated ion pairs, of which an intermediate contact ion pair possessing three (effective) HMPA molecules yields the trans adduct with much higher stereoselectivity. [structure: see text]

The critical micelle concentrations of lysophosphatidic acid and sphingosylphosphorylcholine.

The critical micelle concentrations (CMC) of lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) were measured by isothermal titration calorimetry. The CMC of LPA decreases with salt concentration and acyl chain length. In water at 25 degrees C, the CMC values of 1-acyl-2-lyso-sn-glycero-3-phosphatidic acid are 1.850, 0.540, 0.082, and 0.346 mM, respectively, when the acyl group is myristoyl, palmitoyl, stearoyl, and oleoyl. The CMC of SPC in 10 mM sodium phosphate buffer, pH 7.4, at 25 degrees C was 0.158 mM, and did not change with an increase in salt concentration.

Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles.

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol.

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehydroergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content.

Synthesis and spectral properties of cholesterol- and FTY720-containing boron dipyrromethene dyes.

Two analogues (1, 2) of free cholesterol and one analogue (3) of the immunosuppressive sphingolipid FTY720 containing a boron dipyrromethene chromophore (BODIPY) were synthesized. The synthetic routes involved preparation of boron dipyrromethene moieties (5, 11), bearing a phenylethynyl group at different positions of the chromophore, and lipids (13, 20) bearing an azido group. The dye was tethered to the lipid via a 1,2,3-triazole in the linker by the click reaction. Analogues derived from 11 [in which an (E)-styrylethynyl moiety is bonded to C-5 of BODIPY] exhibited a marked red shift (approximately 70-80 nm) compared with those derived from 5 (in which a phenylethynyl moiety is bonded to C-8 of BODIPY).

First synthesis of free cholesterol-BODIPY conjugates.

Analogues of cholesterol (compounds 1 and 2) and coprostanol (compound 3) containing the BODIPY fluorophore in the aliphatic tail of the free sterol have been synthesized starting with bisnorcholenic acid, cholenic acid 3beta-acetate, and lithocholic acid, respectively. An ester linkage joining the fluorophore to the sterol nucleus interfered with the ability of the fluorescent sterol to pack with phospholipids in monolayers. However, an analogue in which the linker was devoid of polar atoms exhibited a substantially similar physical behavior to cholesterol in model membranes with respect to localization in raft domains.

Synthesis of photoactivatable analogues of lysophosphatidic acid and covalent labeling of plasma proteins.

[reaction: see text] Lysophosphatidic acids bearing a benzophenone group in either the sn-1 or sn-2 chain of an oleoyl-type ester or oleyl-type ether chain and (32)P in the phosphate group were synthesized. The benzophenone moiety was introduced by selective hydroboration of the double bond of enyne 11 at low temperature, followed by a Suzuki reaction with 4-bromobenzophenone. The key intermediates for the preparation of ester-linked lysophosphatidic acid (LPA) 1 and 3 were obtained in one pot by a modified DIBAL-H reduction of orthoformate intermediate 22. These probes were shown to covalently modify a single protein target in rat plasma containing albumin and several protein targets in rat plasma containing a low level of albumin.

Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization.

Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.

Tracking peptide-membrane interactions: insights from in situ coupled confocal-atomic force microscopy imaging of NAP-22 peptide insertion and assembly.

Elucidating the role that charged membrane proteins play in determining cell membrane structure and dynamics is an area of active study. We have applied in situ correlated atomic force and confocal microscopies to characterize the interaction of the NAP-22 peptide with model membranes prepared as supported planar bilayers containing both liquid-ordered and liquid-disordered domains. Our results demonstrated that the NAP-22 peptide interacts with membranes in a concentration-dependent manner, preferentially inserting into DOPC (ld) domains. While at low peptide concentrations, the NAP-22 peptide formed aggregate-like structures within the ld domains, at high peptide concentrations, it appeared to sequester cholesterol into the ld domains and recruited phosphatidyl-myo-inositol 4,5-bisphosphate by inducing a blending effect that homogenizes the phase-segregated domains into one liquid-ordered domain. This study describes a possible mechanism by which the NAP-22 peptide can affect neuronal morphology.

Different residues mediate recognition of 1-O-oleyllysophosphatidic acid and rosiglitazone in the ligand binding domain of peroxisome proliferator-activated receptor gamma.

Here we showed that a naturally occurring ether analog of lysophosphatidic acid, 1-O-octadecenyl-2-hydroxy-sn-glycero-3-phosphate (AGP), is a high affinity partial agonist of the peroxisome proliferator-activated receptor gamma (PPARgamma). Binding studies using the PPARgamma ligand binding domain showed that [32P]AGP and [3H]rosiglitazone (Rosi) both specifically bind to PPARgamma and compete with each other. [32P]AGP bound PPARgamma with an affinity (Kdapp 60 nm) similar to that of Rosi. However, AGP displaced approximately 40% of bound [3H]Rosi even when applied at a 2000-fold excess. Activation of PPARgamma reporter gene expression by AGP and Rosi showed similar potency, yet AGP-mediated activation was approximately 40% that of Rosi. A complex between AGP and PPARgamma was generated using molecular modeling based on a PPARgamma crystal structure. AGP-interacting residues were compared with Rosi-interacting residues identified within the Rosi-PPARgamma co-crystal complex. These comparisons showed that the two ligands occupy partially overlapping positions but make different hydrogen bonding and ion pairing interactions. Site-specific mutants of PPARgamma were prepared to examine individual ligand binding. H323A and H449A mutants showed reduced binding of Rosi but maintained binding of AGP. In contrast, the R288A showed reduced AGP binding but maintained Rosi binding. Finally, alanine replacement of Tyr-473 abolished binding and activation by Rosi and AGP. These observations indicate that the endogenous lipid mediator AGP is a high affinity ligand of PPARgamma but that it binds via interactions distinct from those involved in Rosi binding. These distinct interactions are likely responsible for the partial PPARgamma agonism of AGP.