Anandamide-ceramide interactions in a membrane environment: Molecular dynamic simulations data.

Anandamide is a lipid neurotransmitter that interacts with various plasma membrane lipids. The data here consists of molecular dynamics simulations of anandamide, C18-ceramide and cholesterol performed in vacuo and within a hydrated palmitoyl-oleoyl-phosphatidylcholine (POPC)/cholesterol membrane. Several models of anandamide/cholesterol and anandamide/ceramide complexes are presented. The energy of interaction and the nature of the intermolecular forces involved in each of these complexes are detailed. The impact of water molecules hydrating the POPC/cholesterol membrane for the stability of the anandamide/cholesterol and anandamide/ceramide complexes is also analyzed. From a total number of 1920 water molecules stochatiscally merged with the lipid matrix, 48 were eventually redistributed around the polar head groups of the anandamide/ceramide complex, whereas only 15 reached with the anandamide/cholesterol complex. The interpretation of this dataset is presented in the accompanying article "Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells" [1].

Ceramide binding to anandamide increases its half-life and potentiates its cytotoxicity in human neuroblastoma cells.

Anandamide (AEA) is a ubiquitous lipid that exerts neurotransmitter functions but also controls important biological functions such as proliferation, survival, or programmed cell death. The latter effects are also regulated by ceramide, a lipid enzymatically generated from sphingomyelin hydrolysis by sphingomyelinase. Ceramide has been shown to increase the cellular toxicity of AEA, but the mechanisms controlling this potentiating effect remained unclear. Here we have used a panel of in silico, physicochemical, biochemical and cellular approaches to study the crosstalk between AEA and ceramide apoptotic pathways. Molecular dynamics simulations indicated that AEA and ceramide could form a stable complex in phosphatidylcholine membranes. Consistent with these data, we showed that AEA can specifically insert into ceramide monolayers whereas it did not penetrate into sphingomyelin membranes. Then we have studied the effects of ceramide on AEA-induced toxicity of human neuroblastoma cells. In these experiments, the cells have been either naturally enriched in ceramide by neutral sphingomyelinase pre-incubation or treated with C2-ceramide, a biologically active ceramide analog. Both treatments significantly increased the cytotoxicity of AEA as assessed by the MTS mitochondrial toxicity assay. This effect was correlated with the concomitant accumulation of natural ceramide (or its synthetic analog) and AEA in the cells. A kinetic study of AEA hydrolysis showed that ceramide inhibited the fatty acid amino hydrolase (FAAH) activity in cell extracts. Taken together, these data suggested that ceramide binds to AEA, increases its half-life and potentiates its cytotoxicity. Overall, these mechanisms account for a functional cross-talk between AEA and ceramide apoptotic pathways.

Interaction of Alzheimer's ß-amyloid peptides with cholesterol: mechanistic insights into amyloid pore formation.

Brain cholesterol plays a critical role in Alzheimer's disease and other neurodegenerative diseases. The molecular mechanisms linking cholesterol to neurotoxicity have remained elusive for a long time, but recent data have allowed the identification of functional cholesterol-binding domains in several amyloidogenic proteins involved in neurodegenerative diseases, including Alzheimer's disease. In this review, we analyze the cholesterol binding properties of ß-amyloid (Aß) peptides and the impact of these interactions on amyloid pore formation. We show that although the cholesterol-binding domains of Aß peptides and of transmembrane precursor C99 are partially overlapping, they involve distinct amino acid residues, so that cholesterol has a greater affinity for Aß than for C99. Synthetic 22-35 and 25-35 fragments of Aß retained the ability of the full-length peptide 1-42 to bind cholesterol and to form zinc-sensitive, calcium-permeable amyloid pores in cultured neural cells. Studies with mutant peptides allowed the identification of key residues involved in cholesterol binding and channel formation. Cholesterol promoted the insertion of Aß in the plasma membrane, induced α-helical structuration, and forced the peptide to adopt a tilted topology that favored the oligomerization process. Bexarotene, an amphipathic drug currently considered as a potential candidate medication for the treatment of neurodegenerative diseases, competed with cholesterol for binding to Aß and prevented oligomeric channel formation. These studies indicate that it is possible to prevent the generation of neurotoxic oligomers by targeting the cholesterol-binding domain of Aß peptides. This original strategy could be used for the treatment of Alzheimer's and other neurodegenerative diseases that involve cholesterol-dependent toxic oligomers.

Bexarotene blocks calcium-permeable ion channels formed by neurotoxic Alzheimer's ß-amyloid peptides.

The anticancer drug bexarotene has been shown to restore cognitive functions in animal models of Alzheimer's disease, but its exact mechanism of action remains elusive. In the present report, we have used a combination of molecular, physicochemical, and cellular approaches to elucidate the mechanisms underlying the anti-Alzheimer properties of bexarotene in neural cells. First of all, we noticed that bexarotene shares a structural analogy with cholesterol. We showed that cholesterol and bexarotene compete for the same binding site in the C-terminal region of Alzheimer's ß-amyloid peptide 1-42 (Aß1-42). This common bexarotene/cholesterol binding domain was characterized as a linear motif encompassing amino acid residues 25-35 of Aß1-42. Because cholesterol is involved in the oligomerization of Alzheimer's ß-amyloid peptides into neurotoxic amyloid channels, we studied the capability of bexarotene to interfere with this process. We showed that nanomolar concentrations of bexarotene efficiently prevented the cholesterol-dependent increase of calcium fluxes induced by ß-amyloid peptides Aß1-42 and Aß25-35 in SH-SY5Y cells, suggesting a direct effect of the drug on amyloid channel formation. Molecular dynamics simulations gave structural insights into the role of cholesterol in amyloid channel formation and explained the inhibitory effect of bexarotene. Because it is the first drug that can both inhibit the binding of cholesterol to ß-amyloid peptides and prevent calcium-permeable amyloid pore formation in the plasma membrane of neural cells, bexarotene might be considered as the prototype of a new class of anti-Alzheimer compounds. The experimental approach developed herein can be used as a screening strategy to identify such compounds.

Mechanism of cholesterol-assisted oligomeric channel formation by a short Alzheimer ß-amyloid peptide.

Alzheimer ß-amyloid (Aß) peptides can self-organize into oligomeric ion channels with high neurotoxicity potential. Cholesterol is believed to play a key role in this process, but the molecular mechanisms linking cholesterol and amyloid channel formation have so far remained elusive. Here, we show that the short Aß22-35 peptide, which encompasses the cholesterol-binding domain of Aß, induces a specific increase of Ca(2+) levels in neural cells. This effect is neither observed in calcium-free medium nor in cholesterol-depleted cells, and is inhibited by zinc, a blocker of amyloid channel activity. Double mutations V24G/K28G and N27R/K28R in Aß22-35 modify cholesterol binding and abrogate channel formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α-helical topology of Aß22-35. This facilitates the establishment of an inter-peptide hydrogen bond network involving Asn-27 and Lys-28, a key step in the octamerization of Aß22-35 which proceeds gradually until the formation of a perfect annular channel in a phosphatidylcholine membrane. Overall, these data give mechanistic insights into the role of cholesterol in amyloid channel formation, opening up new therapeutic options for Alzheimer's disease. Aß22-35 peptide, which encompasses the cholesterol binding domain of Aß, induces a specific increase of Ca(2+) level in neural cells. Double mutations V24G/K28G and N27R/K28R modify cholesterol binding and abrogate channels formation. Molecular dynamic simulations suggest that cholesterol induces a tilted α-helical peptide topology facilitating the formation of annular octameric channels, as schematically shown in the graphic (with a hydrogen bond shown in green for two vicinal peptides). Overall, the data give insights into the role of cholesterol in amyloid channel formation and open up new therapeutic options for Alzheimer's disease.

Cholesterol accelerates the binding of Alzheimer's ß-amyloid peptide to ganglioside GM1 through a universal hydrogen-bond-dependent sterol tuning of glycolipid conformation.

Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimer's disease. Yet the molecular mechanisms by which these factors increase Alzheimer's risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimer's beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modeling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes.

Biochemical identification of a linear cholesterol-binding domain within Alzheimer's ß amyloid peptide.

Alzheimer's ß-amyloid (Aß) peptides can self-organize into amyloid pores that may induce acute neurotoxic effects in brain cells. Membrane cholesterol, which regulates Aß production and oligomerization, plays a key role in this process. Although several data suggested that cholesterol could bind to Aß peptides, the molecular mechanisms underlying cholesterol/Aß interactions are mostly unknown. On the basis of docking studies, we identified the linear fragment 22-35 of Aß as a potential cholesterol-binding domain. This domain consists of an atypical concatenation of polar/apolar amino acid residues that was not previously found in cholesterol-binding motifs. Using the Langmuir film balance technique, we showed that synthetic peptides Aß17-40 and Aß22-35, but not Aß1-16, could efficiently penetrate into cholesterol monolayers. The interaction between Aß22-35 and cholesterol was fully saturable and lipid-specific. Single-point mutations of Val-24 and Lys-28 in Aß22-35 prevented cholesterol binding, whereas mutations at residues 29, 33, and 34 had little to no effect. These data were consistent with the in silico identification of Val-24 and Lys-28 as critical residues for cholesterol binding. We conclude that the linear fragment 22-35 of Aß is a functional cholesterol-binding domain that could promote the insertion of ß-amyloid peptides or amyloid pore formation in cholesterol-rich membrane domains.

The NKG2D ligands RAE-1δ and RAE-1ε differ with respect to their receptor affinity, expression profiles and transcriptional regulation.

BACKGROUND: RAE-1 is a ligand of the activating receptor NKG2D expressed by NK cells, NKT, γδT and some CD8(+)T lymphocytes. RAE-1 is overexpressed in tumor cell lines and its expression is induced after viral infection and genotoxic stress. We have recently demonstrated that RAE-1 is expressed in the adult subventricular zone (SVZ) from C57BL/6 mice. RAE-1 is also expressed in vitro by neural stem/progenitor cells (NSPCs) and plays a non-immune role in cell proliferation. The C57BL/6 mouse genome contains two rae-1 genes, rae-1δ and rae-1ε encoding two different proteins. The goals of this study are first to characterize the in vivo and in vitro expression of each gene and secondly to elucidate the mechanisms underlying their respective expression, which are far from known. PRINCIPAL FINDINGS: We observed that Rae-1δ and Rae-1ε transcripts are differentially expressed according to tissues, pathological conditions and cell lines. Embryonic tissue and the adult SVZ mainly expressed Rae-1δ transcripts. The NSPCs derived from the SVZ also mainly expressed RAE-1δ. The interest of this result is especially related to the observation that RAE-1δ is a weak NKG2D ligand compared to RAE-1ε. On the contrary, cell lines expressed either similar levels of RAE-1δ and RAE-1ε proteins or only RAE-1ε. Since the protein expression correlated with the level of transcripts for each rae-1 gene, we postulated that transcriptional regulation is one of the main processes explaining the difference between RAE-1δ and RAE-1ε expression. We indeed identified two different promoter regions for each gene: one mainly involved in the control of rae-1δ gene expression and the other in the control of rae-1ε expression. CONCLUSIONS/SIGNIFICANCE: RAE-1δ and RAE-1ε differ with respect to their function and the control of their expression. Immune function would be mainly exerted by RAE-1ε and non-immune function by RAE-1δ.

Non-lipolytic and lipolytic sequence-related carboxylesterases: a comparative study of the structure-function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase.

To differentiate esterases from lipases at the structure-function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116-123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase-lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.

The food-associated fungal neurotoxin ochratoxin A inhibits the absorption of glutamate by astrocytes through a decrease in cell surface expression of the excitatory amino-acid transporters GLAST and GLT-1.

The food-associated mycotoxin ochratoxin A (OTA) has been demonstrated to be deleterious to numerous cell types including brain cells. Although OTA has been proved to be toxic to astrocytes, no other investigation has been conducted on the impact of OTA on astrocytic functions. In the present study, we evaluated the effect of OTA on one of the major astrocytic functions, i.e. the reabsorption of extracellular glutamate. We found that OTA suppressed glutamate absorption by rat cortical astrocytes with a half inhibitory concentration of 1.3 and 10.1 microM in the absence and presence of fetal calf serum. Although OTA inhibits glutamine synthetase activity, this effect was not involved in OTA-mediated alteration of glutamate absorption since decrease in enzyme activity only occurred at high cytotoxic concentrations of toxin (100 microM). Similarly, alterations in the expression of the excitatory amino-acid transporters were not involved since OTA failed to modify total expression level of GLAST and GLT-1. We found that inhibition of glutamate absorption by OTA was due to a decrease in the expression of GLAST and GLT-1 at the cell surface. We propose that, in addition to being directly toxic to neurons and astrocytes, OTA could also cause the death of brain cells through inhibition of glutamate uptake by astrocytes, leading to the accumulation of extracellular glutamate and ultimately to excitotoxicity.

The first extracellular domain of the tumour stem cell marker CD133 contains an antigenic ganglioside-binding motif.

Prominin 1/CD133 is a marker of transplantable cancer stem cells. We have generated anti-peptide antibodies against a N-terminal epitope of CD133 belonging to a ganglioside-binding domain. The labelling of colon cancer cells with these antibodies was inhibited by various gangliosides including GM1 and GD3, but not GT1b. CD133 immunolabelling progressively decreased to undetectable levels in post-confluent cultures, possibly through ganglioside-mediated epitope masking since the staining was partially recovered after chemical disruption of lipid rafts. We suggest that selected gangliosides could modulate the accessibility of CD133 and regulate cell-cell contacts involving CD133(+) stem cells at the earliest steps of tumour development.

Prediction of glycolipid-binding domains from the amino acid sequence of lipid raft-associated proteins: application to HpaA, a protein involved in the adhesion of Helicobacter pylori to gastrointestinal cells.

Protein-glycolipid interactions mediate the attachment of various pathogens to the host cell surface as well as the association of numerous cellular proteins with lipid rafts. Thus, it is of primary importance to identify the protein domains involved in glycolipid recognition. Using structure similarity searches, we could identify a common glycolipid-binding domain in the three-dimensional structure of several proteins known to interact with lipid rafts. Yet the three-dimensional structure of most raft-targeted proteins is still unknown. In the present study, we have identified a glycolipid-binding domain in the amino acid sequence of a bacterial adhesin (Helicobacter pylori adhesin A, HpaA). The prediction was based on the major properties of the glycolipid-binding domains previously characterized by structural searches. A short (15-mer) synthetic peptide corresponding to this putative glycolipid-binding domain was synthesized, and we studied its interaction with glycolipid monolayers at the air-water interface. The synthetic HpaA peptide recognized LacCer but not Gb3. This glycolipid specificity was in line with that of the whole bacterium. Molecular modeling studies gave some insights into this high selectivity of interaction. It also suggested that Phe147 in HpaA played a key role in LacCer recognition, through sugar-aromatic CH-pi stacking interactions with the hydrophobic side of the galactose ring of LacCer. Correspondingly, the replacement of Phe147 with Ala strongly affected LacCer recognition, whereas substitution with Trp did not. Our method could be used to identify glycolipid-binding domains in microbial and cellular proteins interacting with lipid shells, rafts, and other specialized membrane microdomains.

Cellular isoform of the prion protein PrPc in human intestinal cell lines: genetic polymorphism at codon 129, mRNA quantification and protein detection in lipid rafts.

The cellular isoform of the normal prion protein PrP(c), encoded by the PRNP gene, is expressed in human intestinal epithelial cells where it may represent a potential target for infectious prions. We have sequenced the PRNP gene in Caco-2 and HT-29 parental and clonal cell lines, and found that these cells have a distinct polymorphism at codon 129. HT-29 cells are homozygous Met/Met, whereas Caco-2 cells are heterozygous Met/Val. The 129Val variant was also detected in Caco-2 mRNAs. Real-time PCR quantifications revealed that PrP(c) mRNAs were more expressed in HT-29 cells than in Caco-2 cells. These data were confirmed by studying the expression of PrP(c) in plasma membranes and lipid rafts prepared from these cells. Overall, these results may be important in view of using human intestinal cell lines Caco-2 and HT-29 as cellular in vitro models to study the initial steps of prion propagation after oral inoculation.

Cell penetration properties of maurocalcine, a natural venom peptide active on the intracellular ryanodine receptor.

Maurocalcine (MCa) is a 33-amino acid residue peptide toxin initially isolated from the scorpion Scorpio maurus maurus. Its structural and functional features make it resembling many Cell Penetrating Peptides. In particular, MCa exhibits a characteristic positively charged face that may interact with membrane lipids. External application of MCa is known to produce Ca2+-release from intracellular stores within seconds. MCa binds directly to the skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. The binding sites for MCa have been mapped within the cytoplasmic domain of the ryanodine receptor. In this manuscript, we further investigated how MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa (MCab) was chemically synthesized, coupled to a fluorescent streptavidin indicator (Cy3 or Cy5) and the cell penetration of the entire complex followed by confocal microscopy and FACS analysis. The data provide evidence that MCa allows the penetration of the macro proteic complex and therefore may be used as a vector for the delivery of proteins in the cytoplasm as well as in the nucleus. Using both FACS and confocal analysis, we show that the cell penetration of the fluorescent complex is observed at concentrations as low as 10 nM, is sensitive to membrane potential and is partly inhibited by heparin. We also show that MCa interacts with the disialoganglioside GD3, the most abundant charged lipid in natural membranes. Despite its action on ryanodine receptor, MCa showed no sign of cell toxicity on HEK293 cells suggesting that it may have a wider application range. These data indicate that MCa may cross the plasma membrane directly by cell translocation and has a promising future as a carrier of various drugs and agents of therapeutic, diagnostic and technological value.

The minimal amyloid-forming fragment of the islet amyloid polypeptide is a glycolipid-binding domain.

Several proteins that interact with cell surface glycolipids share a common fold with a solvent-exposed aromatic residue that stacks onto a sugar ring of the glycolipid (CH-pi stacking interaction). Stacking interactions between aromatic residues (pi-pi stacking) also play a pivotal role in the assembly process, including many cases of amyloid fibril formation. We found a structural similarity between a typical glycolipid-binding domain (the V3 loop of HIV-1 gp120) and the minimal amyloid-forming fragment of the human islet amyloid polypeptide, i.e. the octapeptide core module NFGAILSS. In a monolayer assay at the air-water interface, the NFGAILSS peptide specifically interacted with the glycolipid lactosylceramide. The interaction appears to require an aromatic residue, as NLGAILSS was poorly recognized by lactosylceramide, whereas NYGAILSS behaved like NFGAILSS. In addition, we observed that the full-length human islet amyloid polypeptide (1-37) did interact with a monolayer of lactosylceramide, and that the glycolipid film significantly affected the aggregation process of the peptide. As glycolipid-V3 interactions are efficiently inhibited by suramin, a polyaromatic compound, we investigated the effects of suramin on amyloid formation by human islet amyloid polypeptide. We found that suramin inhibited amyloid fibril formation at low concentrations, but dramatically stimulated the process at high concentrations. Taken together, our results indicate that the minimal amyloid-forming fragment of human islet amyloid polypeptide is a glycolipid-binding domain, and provide further experimental support for the role of aromatic pi-pi and CH-pi stacking interactions in the molecular control of the amyloidogenesis process.

Apical uptake and transepithelial transport of sphingosine monomers through intact human intestinal epithelial cells: physicochemical and molecular modeling studies.

The mechanism of absorption of sphingosine was studied in human intestinal epithelial cells Caco-2 and HT-29-D4. The experiments were performed below the critical micellar concentration of sphingosine which was evaluated to 6 microM by surface tension measurements. [3H]Sphingosine uptake was not inhibited by Na+-free conditions, ATP depletion, L-cycloserine or methyl-beta-cyclodextrin, consistent with a passive diffusion mechanism independent of lipid raft integrity. Molecular modeling studies suggested that sphingosine can adopt two distinct conformations: a high-energy "snake-like" conformer in water and an extended low-energy conformer in lipid phases. We propose that the energy stored in the compressed snake-like conformer is transformed into kinetic energy, allowing: (i) the motion of sphingosine through the unstirred water layer bathing the mucosal enterocyte surface, and (ii) its insertion into the enterocyte brush border membrane. Dietary lipids that stabilized the extended sphingosine conformer in mixed micelles (e.g., cholesterol and sphingomyelin) induced a marked inhibition of sphingosine absorption.

Interaction of cholesterol with sphingosine: physicochemical characterization and impact on intestinal absorption.

Molecular associations between sphingomyelin and cholesterol provide a molecular basis for the colocalization of these lipids in plasma membrane microdomains (lipid rafts) and for the inhibitory effect of sphingomyelin on the intestinal absorption of cholesterol. Using surface pressure measurements at the air-water interface, we showed that sphingosine, the common sphingoid backbone of most sphingolipids, formed condensed lipid complexes with cholesterol. Structure-activity relationship studies with long-chain analogs of sphingosine, together with molecular mechanics simulations, were consistent with a specific interaction between sphingosine and the alpha face of cholesterol. The uptake of micellar cholesterol and the effect of sphingosine on cholesterol absorption were studied with two human model intestinal epithelial cell lines, Caco-2 and HT-29-D4. Real-time PCR quantifications of the putative cholesterol transporter Niemann-Pick C1 like 1 (NPC1L1) mRNA revealed that, in these cell lines, the activity of cholesterol transport correlated with the level of NPC1L1 expression. In both cell lines, sphingosine induced a dose-dependent decrease of cholesterol absorption. Yet the effect of sphingosine was more dramatic in Caco-2 cells, which also displayed the highest expression of NPC1L1 mRNA. Altogether, these data suggested that sphingosine interacts specifically with cholesterol and inhibits the intestinal NPC1L1-dependent transport of micellar cholesterol.

The combinatorial extension method reveals a sphingolipid binding domain on pancreatic bile salt-dependent lipase: role in secretion.

Structure similarity searches using a combinatorial extension approach revealed that a protein fold structurally related to the sphingolipid binding domain (SBD) of HIV-1 gp120 (V3 loop) is present on pancreatic bile salt-dependent lipase (BSDL). A synthetic peptide derived from the predicted V3-like domain of BSDL interacted with reconstituted monolayers of sphingolipids such as GalCer and GlcCer. Using Chinese hamster ovary cells stably transfected with the cDNA encoding the rat BSDL (CHO-3B clone) or pancreatic SOJ-6 cells expressing the human BSDL as models, we showed that the enzyme cofractionates with caveolin-1. The secretion of BSDL by CHO-3B cells was inhibited by permeable drugs affecting rafts structure (D609, PDMP, and filipin). Data suggest that the functional interaction between the BSDL SBD and lipid rafts is physiologically relevant and could be essential for sensing the BSDL folding prior to secretion. A tentative model accounting for the phosphorylation-induced dissociation of BSDL from rafts is presented.

The virotoxin model of HIV-1 enteropathy: involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line.

BACKGROUND: Malabsorption and diarrhea are common, serious problems in AIDS patients, and are in part due to the incompletely understood entity HIV enteropathy. Our prior in vitro work has shown that increased transepithelial permeability and glucose malabsorption, similar to HIV enteropathy, are caused by HIV surface protein gp120, although the mechanism remains unclear. RESULTS: We studied the effects of HIV surface protein gp120 on the differentiated intestinal cell line HT-29-D4, specifically the effects on microtubules, transepithelial resistance, and sodium glucose cotransport. gp120 induced extensive microtubule depolymerization, an 80% decrease in transepithelial resistance, and a 70% decrease in sodium-dependent glucose transport, changes closely paralleling those of HIV enteropathy. The effects on transepithelial resistance were used to study potential inhibitors. Neutralizing antibodies to GPR15/Bob but not to CXCR4 (the coreceptor allowing infection with these HIV strains) inhibited these effects. Antibodies to galactosylceramide (GalCer) and a synthetic analog of GalCer also inhibited the gp120-induced changes, suggesting the involvement of GalCer-enriched lipid rafts in gp120 binding to intestinal epithelial cells. CONCLUSION: We conclude that direct HIV infection and gp120-induced cytopathic effects are distinct phenomena. While in vivo confirmation is needed to prove this, gp120 could be a virotoxin significantly contributing to HIV enteropathy.

The mycotoxin deoxynivalenol affects nutrient absorption in human intestinal epithelial cells.

Deoxynivalenol (DON) is a mycotoxin belonging to the tricothecene family that has many toxic effects in animals, including diarrhea and weight loss. Using the human epithelial intestinal cell line HT-29-D4 as an in vitro model, we studied the effect of DON on the uptake of different classes of nutrients, including sugars, amino acids and lipids. At low concentrations (below 10 micro mol/L), DON selectively modulated the activities of intestinal transporters: the D-glucose/D-galactose sodium-dependent transporter (SGLT1) was strongly inhibited by the mycotoxin (50% inhibition at 10 micro mol DON, P < 0.05), followed by the D-fructose transporter GLUT5 (42% inhibition at 10 micro mol/L, P < 0.001), active and passive L-serine transporters (30 and 38% inhibition, respectively, at 10 micro mol/L, P < 0.05). The passive transporters of D-glucose (GLUT) were slightly inhibited by DON (15% inhibition at 1 micro mol/L, P < 0.01), whereas the transport of palmitate was increased by 35% at 10 micro mol/L DON (P < 0.001). In contrast, the uptake of cholesterol was not affected by the mycotoxin. At high concentrations (100 micro mol/L), SGLT1 activity was inhibited by 76% (P < 0.01), whereas the activities of all other transporters were increased. The selective effects of DON on intestinal transporters were mimicked by cycloheximide and deoxycholate, suggesting that inhibition of protein synthesis and induction of apoptosis are the main mechanisms of DON toxicity in intestinal cells.