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The term extracellular vesicles (EVs) has been recommended to describe various membrane-bound vesicles secreted by most living cells and found in various biological fluids. They gained growing interest as mediators of cell-cell communication and for their roles in different patho-physiological processes. In addition, they were recently considered as disease biomarkers and new drug delivery systems. However, it is still difficult to link a biological function to a specific EV population among the heterogenous EV mixture secreted in the extracellular space due to limitations of optimal isolation methods. EV classification according to their size as small (\textless200 nm) and large (\textgreater200 nm) vesicles is also completed by the identification of selected proteins, nucleic acids and lipids. In this review, we summarized briefly knowledge about the composition and role of EV lipids that received less attention compared to their protein and nucleic acid content. Lipids are not only essential structural components of EVs, but can give important information on their biogenesis. Especially, we discussed our recent data showing the utility of bis(monoacylglycero)phosphate (BMP), a specific endolysosomal lipid marker, that could sign the endosomal origin of small EVs, classically named as exosomes.

Supplementary Figure 1 from Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism

Supplementary Figure 2 from Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism

Supplementary Figure 4 from Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism

Supplementary Figure 3 from Novel Lipogenic Enzyme ELOVL7 Is Involved in Prostate Cancer Growth through Saturated Long-Chain Fatty Acid Metabolism

International audience; Bis(monoacylglycero)phosphate (BMP), also known as lysobisphosphatidic acid (LBPA), is a phospholipid specifically enriched in the late endosome-lysosome compartment playing a crucial role for the fate of endocytosed components. Due to its presence in extracellular fluids during diseases associated with endolysosomal dysfunction, it is considered as a possible biomarker of disorders such as genetic lysosomal storage diseases and cationic amphiphilic drug-induced phospholipidosis. However, there is no true validation of this biomarker in human studies, nor a clear identification of the carrier of this endolysosome-specific lipid in biofluids. The present study demonstrates that in absence of any sign of renal failure, BMP, especially all docosahexaenoyl containing species, are significantly increased in the urine of patients treated with the antiarrhythmic drug amiodarone. Such urinary BMP increase could reflect a generalized drug-induced perturbation of the endolysosome compartment as observed in vitro with amiodarone-treated human macrophages. Noteworthy, BMP was associated with extracellular vesicles (EVs) isolated from human urines and extracellular medium of human embryonic kidney HEK293 cells and co-localizing with classical EV protein markers CD63 and ALIX. In the context of drug-induced endolysosomal dysfunction, increased BMP-rich EV release could be useful to remove excess of undigested material. This first human pilot study not only reveals BMP as a urinary biomarker of amiodarone-induced endolysosomal dysfunction, but also highlights its utility to prove the endosomal origin of EVs, also named as exosomes. This peculiar lipid already known as a canonical late endosome-lysosome marker, may be thus considered as a new lipid marker of urinary exosomes.

SummaryEpidermal growth factor receptor (EGFR) activates major cell signaling pathways that regulate various cell responses. Its dimerization and clustering coupled with its lateral mobility are critical for EGFR function, but the contribution of the plasma membrane environment to EGFR function is unknown. Here we show, using single-molecule analysis, that EGFR mobility and clustering are altered by the depletion of cholesterol or sphingomyelin, major lipids of membrane subdomains, causing significant changes in EGFR signaling. When cholesterol was depleted, the subdomain boundary in EGFR diffusion disappeared, the fraction of EGFR pre-dimers was increased, and the ligand-induced phosphorylation of EGFR was enhanced. In addition, the depletion of either lipid prevented the formation of immobile clusters after EGF association and decreased the phosphorylation of downstream proteins. Our results revealed that cholesterol plays dichotomous roles in the signaling pathway of EGFR and that clustering in the membrane subdomains is critical for EGFR signal transduction.

Sphingomyelin (SM) is a mammalian lipid mainly distributed in the outer leaflet of the plasma membrane (PM). We show that peripheral myelin protein 2 (PMP2), a member of the fatty-acid-binding protein (FABP) family, can localize at the PM and controls the transbilayer distribution of SM. Genetic screening with genome-wide small hairpin RNA libraries identifies PMP2 as a protein involved in the transbilayer movement of SM. A biochemical assay demonstrates that PMP2 is a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P

Viruses, including the novel coronavirus SARS-CoV-2, redirect infected cell metabolism to their own purposes. After binding to its receptor angiotensin-converting enzyme 2 (ACE2) on the cell surface, the SARS-CoV-2 is taken up by receptor-mediated endocytosis ending in the acidic endolysosomal compartment. The virus hijacks the endosomal machinery leading to fusion of viral and endosomal membranes and release of the viral RNA into the cytosol. This mini-review specifically highlights the membrane lipid organization of the endosomal system focusing on the unconventional and late endosome/lysosome-specific phospholipid, bis(monoacylglycero)phosphate (BMP). BMP is enriched in alveolar macrophages of lung, one of the target tissue of SARS-CoV-2. This review details the BMP structure, its unsaturated fatty acid composition and fusogenic properties that are essential for the highly dynamic formation of the intraluminal vesicles inside the endosomes. Interestingly, BMP is necessary for infection and replication of enveloped RNA virus such as SARS-CoV-1 and Dengue virus. We also emphasize the role of BMP in lipid sorting and degradation, especially cholesterol transport in cooperation with Niemann Pick type C proteins (NPC 1 and 2) and with some oxysterol-binding protein (OSBP)-related proteins (ORPs) as well as in sphingolipid degradation. Interestingly, numerous virus infection required NPC1 as well as ORPs along the endocytic pathway. Furthermore, BMP content is increased during pathological endosomal lipid accumulation in various lysosomal storage disorders. This is particularly important knowing the high percentage of patients with metabolic disorders among the SARS-CoV-2 infected patients presenting severe forms of COVID-19., Highlights • Host cell cholesterol is required for efficient viral infection. • NPC and ORPs regulate cholesterol exit from endolysosomes and viral infection. • Endosomal phospholipid BMP interacts with sterol transport proteins NPC and ORPs. • BMP by controlling cholesterol trafficking could be involved in virus infection. • BMP could be a potential therapeutic target for COVID-19.

International audience; We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

International audience; Bis(Monoacylglycero) Phosphate (BMP) is a unique phospholipid localized in late endosomes, a critical cellular compartment in low density lipoprotein (LDL)-cholesterol metabolism. In previous work, we demonstrated the important role of BMP in the regulation of macrophage cholesterol homeostasis. BMP exerts a protective role against the pro-apoptotic effect of oxidized LDL (oxLDL) by reducing the production of deleterious oxysterols. As the intracellular sterol traffic in macrophages is in part regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we investigated the role of ORP11, localized at the Golgi-late endosomes interface, in the BMP-mediated protection from oxLDL/oxysterol cytotoxicity. Stably silencing of ORP11 in mouse RAW264.7 macrophages via a shRNA lentiviruses system had no effect on BMP production. However, ORP11 knockdown abrogated the protective action of BMP against oxLDL induced apoptosis. In oxLDL treated control cells, BMP enrichment was associated with reduced generation of 7-oxysterols, while these oxysterol species were abundant in the ORP11 knock-down cells. Of note, BMP enrichment in ORP11 knock-down cells was associated with a drastic increase in free cholesterol and linked to a decrease of cholesterol efflux. The expression of ATP-binding cassette-transporter G1 (ABCG1) was also reduced in the ORP11 knock-down cells. These observations demonstrate a cooperative function of OPR11 and BMP, in intracellular cholesterol trafficking in cultured mac-rophages. We suggest that BMP favors the egress of cholesterol from late endosomes via an ORP11-dependent mechanism, resulting in a reduced production of cytotoxic 7-oxysterols.

International audience; Oxidized LDL (OxLDL) that are enriched in products of lipid peroxidation including oxysterols have been shown to induce cellular oxidative stress and cytotoxicity therefore accelerating atheroma plaque formation. Upon oxLDL exposure of THP-1 macrophages, intracellular oxidation of LDL derived-cholesterol as well as endogenous cholesterol was increased. The oxysterols intracellularly produced were efficiently exported to HDL whereas apolipoprotein A1 was inefficient. These findings prompted us to investigate the consequences of modification of HDL by oxidation and glycation as observed in type 2 diabetes with respect to oxysterol and cholesterol efflux. We show that efflux of oxysterols was significantly impaired after in vitro oxidation and glycoxidation of HDL whereas glycation alone had no impact. Cholesterol efflux was only slightly decreased by oxHDL or glycoxidized HDL and not changed with glycated HDL. The defect of HDL towards oxysterol efflux was also observed with HDL isolated from diabetic subjects as compared to healthy controls. These findings support a deleterious cellular retention of oxysterols due to dysfunctional HDL in type 2 diabetes.

International audience; Sphingomyelin (SM) is a major sphingolipid in mammalian cells whereas its analog, ceramide phosphoethanolamine (CPE) is found in trace amounts in mammalian cells and in larger amounts in invertebrates such as insect cells like Drosophila melanogaster. To visualize endogenous SM or CPE, we need specific probes able to recognize the chemical structure of the lipid, rather than its physical property. A limited number of proteins is known to specifically and strongly bind SM or CPE. These proteins are either toxins produced by non-mammalian organisms, subunits or fragments of toxins or a protein that has similar structure to a toxin. These proteins labeled with small fluorophore (e.g. Alexa Fluor) or conjugated to fluorescent proteins (e.g. mCherry) or other types of markers (e.g. I-125, maltose-binding protein) are used to detect SM or CPE. Here we summarize the characteristics of specific SM-binding proteins, lysenin and equinatoxin II; CPE- and SM/cholesterol (Chol) binding aegerolysin proteins, pleurotolysin A(2), ostreolysin and erylysin A and SM/Chol-binding protein, nakanori. Then we give examples of their applications including their limitations related not only to their lipid specificity and binding constants, but also to the lipid organization in the membrane.

This mini-review presents recent advances in the regulation of the membrane transbilayer movement (or flip-flop) of diacylglycerol (DAG), a key intermediate in lipid metabolism and a second messenger in lipid-mediated signaling. Despite progresses in lipid biophysics and imaging, little is known about the DAG dynamics across the two leaflets of the plasma membrane in living cells. Previous model membrane studies with DAG analogs demonstrated their fast flip-flop suggesting that DAG is evenly distributed between the two leaflets of the plasma membrane. However, recent molecular dynamics simulations indicate that DAG transbilayer movement depends on the lipid environment surrounding the lipid, i.e. DAG flips more slowly across a more ordered "lipid raft-like" bilayer (enriched in sphingomyelin/cholesterol) than across a more fluid bilayer (composed of unsaturated glycerophospholipids). Furthermore using the yellow fluorescent protein-tagged C1AB domain from protein kinase C-γ (EYFP-C1AB) that selectively binds DAG, we recently proved that the sphingomyelin (SM) content in the plasma membrane outer leaflet regulates DAG transbilayer movement in Madin-Darby canine kidney cells treated with bacterial phosphatidylcholine-specific phospholipase C. The dose-dependent inhibition of DAG flip-flop by SM could be reproduced in model membranes using fluorescent short chain DAG analog. Regulation of DAG transbilayer movement by the outer leaflet SM content is expected to modify the downstream recruitment of C1-domain containing effectors, thus bringing new insights on the role of DAG dynamics in cell pathophysiology.

Lipids play an essential role in the structure of the endosomal membranes as well as in their dynamic rearrangement during the transport of internalized cargoes along the endocytic pathway. In this review, we discuss the function of endosomal lipids mainly in mammalian cells, focusing on two well-known components of the lipid rafts, sphingomyelin and cholesterol, as well as on three anionic phospholipids, phosphatidylserine, polyphosphoinositides and the atypical phospholipid, bis(monoacylglycero)phosphate/lysobisphosphatidic acid. We detail the structure, metabolism, distribution and role of these lipids in the endosome system as well as their importance in pathological conditions where modification of the endosomal membrane flow can lead to various diseases such as lipid-storage diseases, myopathies and neuropathies.

International audience; Hormone-sensitive acute steroid biosynthesis requires trafficking of cholesterol from intracellular sources to the inner mitochondrial membrane. The precise location of the intracellular cholesterol and its transport mechanism are uncertain. Perfringolysin O, produced by Clostridium perfringens, binds cholesterol. Its fourth domain (D4) retains cholesterol-binding properties but not cytotoxicity. We transfected steroidogenic MA-10 cells of mouse Leydig cell tumors with the mCherry-D4 plasmid. Tagged D4 with fluorescent proteins enabled us to track cholesterol. The staining was primarily localized to the inner leaflet of the plasma membrane and was partially released upon treatment with dibutyryl-cAMP (Bt2cAMP), a cAMP analog. Inhibitors of cholesterol import into mitochondria blocked steroidogenesis and prevented release of D4 (and presumably cholesterol) from the plasma membrane. We conclude that the bulk of the steroidogenic pool of cholesterol, mobilized by Bt2cAMP for acute steroidogenesis, originates from the plasma membrane. Treatment of the cells with steroid metabolites, 22(R)-hydroxycholesterol and pregnenolone, also reduced D4 release from the plasma membrane, perhaps evidence for a feedback effect of elevated steroid formation on cholesterol release. Interestingly, D4 staining was localized to endosomes during Bt2cAMP stimulation suggesting that these organelles are on the route of cholesterol trafficking from the plasma membrane to mitochondria. Finally, D4 was expressed in primary rat Leydig cells with a lentivirus and was released from the plasma membrane following Bt2cAMP treatment. We conclude that the plasma membrane is the source of cholesterol for steroidogenesis in these cells as well as in MA-10 cells.

International audience; In this mini-review, we summarize current knowledge about the lipid-binding characteristics of two types of toxins used to visualize the membrane distribution of phosphoethanolamine-containing lipid species: the glycerophospholipid, phosphatidylethanolamine (PE) and the sphingolipid, ceramide phosphoethanolamine (CPE). The lantibiotic cinnamycin and the structurally-related peptide duramycin produced by some Gram-positive bacteria were among the first toxins characterized by their specificity for PE which is widely present in animal kingdoms from bacteria to mammals. These toxins promoted their binding to PE-containing membranes by changing membrane curvature and by inducing transbilayer lipid movement. The recognition of the conical shape and negative curvature adopted by the PE species within the membrane, is important to understand how lipid-peptide interaction can occur. Three mushroom-derived proteins belonging to the aegerolysin family, pleurotolysin A2, ostreolysin and erylysin A were recently described as efficient tools to visualize the membrane distribution of CPE which is found in trace amounts in mammalian cells but in higher amounts in some developmental stages of lower eukaryotes like Trypanosoma and in invertebrates such as Drosophila. The recent development of lantibiotic-based PE-specific and aegerolysin-based CPE-specific probes is useful to visualize and specify the role of these lipids in various pathophysiological events such as cell division, apoptosis, tumor vasculature and parasite developmental stages.

Diacylglycerol (DAG) is a key component in lipid metabolism and signaling. Previous model membrane studies using DAG analogs suggest their rapid membrane transbilayer movement. However, little is known about the DAG distribution and dynamics in cell membranes. Using live-cell fluorescence microscopy, we monitored the transbilayer movement of DAG with the yellow fluorescent protein-tagged C1AB domain from protein kinase C-γ (EYFP-C1AB), which selectively binds DAG. When HeLa cells were treated with Bacillus cereus phospholipase C (Bc-PLC) to produce DAG on the outer leaflet of the plasma membrane, intracellularly expressed EYFP-C1AB probe accumulated at the plasma membrane, indicating the transbilayer movement of the outer leaflet DAG to the inner leaflet. This Bc-PLC-induced translocation of EYFP-C1AB probe to the plasma membrane was not observed in the sphingolipid-enriched plasma membrane of Madin-Darby canine kidney cells, but was recovered after cell treatment with sphingomyelinase or preincubation with an inhibitor of sphingolipid biosynthesis. The inhibitory effect of sphingomyelin (SM) on the transbilayer movement of DAG was reproduced in model membranes using a fluorescent short-chain DAG analog. These results demonstrate that the SM content on the outer leaflet regulates the transbilayer movement of DAG in the plasma membrane, thus providing new insights into the dynamics of DAG in cell pathophysiology.

International audience; Previous work in our laboratory demonstrated that in-utero exposure to a mixture of the phytoestrogen Genistein (GEN), and plasticizer DEHP, induces short- and long-term alterations in testicular gene and protein expression different from individual exposures. These studies identified fetal and adult Leydig cells as sensitive targets for low dose endocrine disruptor (ED) mixtures. To further investigate the direct effects and mechanisms of toxicity of GEN and DEHP, MA-10 mouse tumor Leydig cells were exposed in-vitro to varying concentrations of GEN and MEHP, the principal bioactive metabolite of DEHP. Combined 10muM GEN+10muM MEHP had a stimulatory effect on basal progesterone production. Consistent with increased androgenicity, the mRNA of steroidogenic and cholesterol mediators Star, Cyp11a, Srb1 and Hsl, as well as upstream orphan nuclear receptors Nr2f2 and Sf1 were all significantly increased uniquely in the mixture treatment group. Insl3, a sensitive marker of Leydig endocrine disruption and cell function, was significantly decreased by combined GEN+MEHP. Lipid analysis by high-performance thin layer chromatography demonstrated the ability of combined 10muM combined GEN+MEHP, but not individual exposures, to increase levels of several neutral lipids and phospholipid classes, indicating a generalized deregulation of lipid homeostasis. Further investigation by qPCR analysis revealed a concomitant increase in cholesterol (Hmgcoa) and phospholipid (Srebp1c, Fasn) mediator mRNAs, suggesting the possible involvement of upstream LXRalpha agonism. These results suggest a deregulation of MA-10 Leydig function in response to a combination of GEN+MEHP. We propose a working model for GEN+MEHP doses relevant to human exposure involving LXR agonism and activation of other transcription factors. Taken more broadly, this research highlights the importance of assessing the impact of ED mixtures in multiple toxicological models across a range of environmentally relevant doses.

International audience; Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8β-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8β-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8β-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.

The 1602 cm–1 Raman signature, which we call the “Raman spectroscopic signature of life” in yeasts, is a marker Raman band for cell metabolic activity. Despite the established fact that its intensity sensitively reflects the metabolic status of the cell, its molecular origin remained unclear. In this work, we propose ergosterol as the major contributor of the 1602 cm–1 Raman signature. The theoretical isotope shift calculation for ergosterol agreed with previous observations. Furthermore, experiments showed that the Raman spectrum of ergosterol corresponds very well with the depleting spectral component in yeast that behaves together with the 1602 cm–1 signature when the cells are under stress. This work implies that the 1602 cm–1 Raman signature could serve as an intrinsic ergosterol marker in yeasts for the study of sterol metabolism in vivo and in a label-free manner, which could not be done by any other techniques at the current stage. (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP(2)) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.

Ceramides play a crucial role in divergent signaling events, including differentiation, senescence, proliferation, and apoptosis. Ceramides are a minor lipid component in terms of content; thus, highly sensitive detection is required for accurate quantification. The recently developed isobaric tags for relative and absolute quantitation (iTRAQ) method enables a precise comparison of both protein and aminophospholipids. However, iTRAQ tagging had not been applied to the determination of sphingolipids. Here we report a method for the simultaneous measurement of multiple ceramide and monohexosylceramide samples using iTRAQ tags. Samples were hydrolyzed with sphingolipid ceramide N-deacylase (SCDase) to expose the free amino group of the sphingolipids, to which the N-hydroxysuccinimide group of iTRAQ reagent was conjugated. The reaction was performed in the presence of a cleavable detergent, 3-[3-(1,1-bisalkyloxyethyl)pyridine-1-yl]propane-1-sulfonate (PPS) to both improve the hydrolysis and ensure the accuracy of the mass spectrometry analysis performed after iTRAQ labeling. This method was successfully applied to the profiling of ceramides and monohexosylceramides in sphingomyelinase-treated Madin Darby canine kidney (MDCK) cells and apoptotic Jurkat cells.

Free cholesterol accumulation in the liver is relevant to the pathogenesis of nonalcoholic steatohepatitis. Acute free cholesterol accumulation induced the fusion of LDs, followed by degradation of the coat protein of LDs, perilipin 2, and association of apolipoprotein 100 to LDs in Rab18-dependent manner., Dysregulated hepatic cholesterol homeostasis with free cholesterol accumulation in the liver is relevant to the pathogenesis of nonalcoholic steatohepatitis, contributing to the chronicity of liver toxicity. Here we examined the effect of free cholesterol accumulation on the morphology and biochemical properties of lipid droplets (LDs) in cultured hepatocytes. Acute free cholesterol accumulation induced the fusion of LDs, followed by degradation of the coat protein of LDs, perilipin 2 (PLIN2; also called adipophilin or adipose differentiation–related protein), and association of apolipoprotein B 100 (ApoB 100) to LDs. The degradation of PLIN2 was inhibited by inhibitors of ubiquitination, autophagy, and protein synthesis. The results indicate that association of ApoB 100 with LDs is dependent on the activity of low–molecular weight GTP-binding protein Rab18 and highlight the role of LDs as targets of free cholesterol toxicity in hepatocytes.

The hormone-sensitive and rate-limiting step in steroid biosynthesis is the movement of cholesterol from intracellular sources to the inner mitochondrial membrane (IMM). Despite the numerous studie...

International audience; Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.

Lysobisphosphatidic acid (LBPA) is highly accumulated in specific domains of the late endosome and is involved in the biogenesis and function of this organelle. Little is known about the biosynthesis and metabolism of this lipid. We examined its FA composition and the incorporation of exogenous FA into LBPA in the human monocytic leukemia cell line THP-1. The LBPA FA composition in THP-1 cells exhibits an elevated amount of oleic acid (18:1n-9) and enrichment of PUFA, especially DHA (22:6n-3). DHA supplemented to the medium was efficiently incorporated into LBPA. In contrast, arachidonic acid (20:4n-6) was hardly esterified to LBPA under the same experimental conditions. The turnover of DHA in LBPA was similar to that in other phospholipids. Specific incorporation of DHA into LBPA was also observed in baby hamster kidney fibroblasts, although LBPA in these cells contains very low endogenous levels of DHA in normal growth conditions. Our resuIts, together with published observations, suggest that the specific incorporation of DHA into LBPA is a common phenomenon in mammalian cells. The physiological significance of DHA-enriched LBPA is discussed.

We previously described enterophilin-1 (Ent-1), a new intestinal protein bearing an extended leucine zipper and a B30.2 domain. Ent-1 expression is associated with growth arrest and enterocyte differentiation. To investigate the importance of Ent-1 in the differentiation, we performed a yeast two-hybrid screening. We identified sorting nexin 1 (SNX1) as a novel partner of Ent-1 and confirmed the specificity of interaction by co-immunoprecipitation experiments in mammalian cells. SNX1 is associated with endosomal membranes and triggers the endosome-to-lysosome pathway of epidermal growth factor receptor (EGFR). We observe by immunofluorescence microscopy that Ent-1 and SNX1 are co-localized on vesicular and tubulovesicular structures, which are different from early endosome antigen 1-containing endosomes. By gel filtration chromatography, we show that Ent-1 and SNX1 co-eluted in macromolecular complexes containing part of EGFR. Furthermore, overexpressed Ent-1 decreases cell surface EGFR. Ent-1 and SNX1 co-overexpression strongly extends EGFR diminution, indicating a synergetic effect of both proteins on cell surface EGFR removal. Interestingly, the increase of endogenous Ent-1 expression correlates with the decrease of EGFR during spontaneous differentiation of Caco-2 cells. We thus propose a role of Ent-1 in the trafficking of EGFR to down-regulate intestinal mitogenic signals, highlighting the mechanisms of cell growth arrest associated with enterocytic differentiation.

For the past decade evidence has gathered for the implication of lysosomes in the development of programmed cell death. Recent data advocate for dual roles of heat-shock protein 70.1 (Hsp70.1) not only as a molecular chaperone for damaged/aged proteins but also as a guardian of lysosomal integrity. Hsp70.1-mediated lysosomal stabilization is tightly regulated, but its disorder under extreme conditions results in lysosomal rupture to cause cell death. Comparing with the pathological hallmark ‘granulo-vacuolar degenerations’ in Alzheimer’s disease, the postischemic monkey neurons were carefully observed with microscope. Intriguingly, we found very similar change, and identified it as ‘lysosomal vesiculosis’ by electron microscopy. However, the exact molecular and structural impacts of the Hsp70.1 disorder upon the lysosomal membrane are hardly elucidated in the human brain because of the practical and ethical problems. Accordingly, using the monkey brain tissues after in-vivo and in-vitro oxidative stresses, we studied molecular modifications of Hsp70.1 and its counterpart bis(monoacylglycero) phosphate (BMP), because these molecules are closely related to the lysosomal membrane stability by regulating acid sphingomyelinase. It still remains unelucidated whether ischemia/reperfusion can alter composition or amount of BMP in the brain. Regardless of the brain regions studied, the normal monkey brain tissues showed calpain-mediated cleavage of Hsp70.1 after in-vitro oxidative stress. In the CA1 after in-vivo ischemia/reperfusion, we first found that docosahexaenoic and oleic acids in BMP showed a significant decrease on days 1 and 3, compared to non-ischemic controls. Since Hsp70.1- BMP binding is indispensable for activating acid sphingomyelinase and producing ceramide to stabilize lysosomal membrane, dysfunction of Hsp70.1 and BMP presumably causes storage of sphingomyelin and deficiency of ceramide at the lysosomal membrane, leading to its destabilization in the postischemic CA1 neurons. These data, combined together, suggest that lysosomal destabilization induced by calpain-mediated cleavage of carbonylated Hsp70.1 and the concomitant BMP breakdown causes ‘lysosomal vesiculosis’.

Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.

A number of epidemiologic studies have indicated a strong association between dietary fat intake and prostate cancer development, suggesting that lipid metabolism plays some important roles in prostate carcinogenesis and its progression. In this study, through our genome-wide gene expression analysis of clinical prostate cancer cells, we identified a novel lipogenic gene, ELOVL7, coding a possible long-chain fatty acid elongase, as overexpressed in prostate cancer cells. ELOVL7 expression is regulated by the androgen pathway through SREBP1, as well as other lipogenic enzymes. Knockdown of ELOVL7 resulted in drastic attenuation of prostate cancer cell growth, and it is notable that high-fat diet promoted the growth of in vivo tumors of ELOVL7-expressed prostate cancer. In vitro fatty acid elongation assay and fatty acid composition analysis indicated that ELOVL7 was preferentially involved in fatty acid elongation of saturated very-long-chain fatty acids (SVLFA, C20:0∼). Lipid profiles showed that knockdown of ELOVL7 in prostate cancer cells affected SVLFAs in the phospholipids and the neutral lipids, such as cholesterol ester. Focusing on cholesterol ester as a source of de novo steroid synthesis, we show that ELOVL7 affected de novo androgen synthesis in prostate cancer cells. These findings suggest that EVOLV7 could be involved in prostate cancer growth and survival through the metabolism of SVLFAs and their derivatives, could be a key molecule to elucidate the association between fat dietary intake and prostate carcinogenesis, and could also be a promising molecular target for development of new therapeutic or preventive strategies for prostate cancers. [Cancer Res 2009;69(20):8133–40]

Imaging membrane lipid domains to characterize their organization and function has been hindered by the lack of reliable lipid-specific probes. In this paper, we provide detailed methods to investigate, mainly by confocal microscopy, the distribution and dynamics of two components of the "lipid rafts," sphingomyelin (SM) and cholesterol, using two specific lipid probes that have been extensively studied in the laboratory: lysenin, a SM-binding toxin and the fluorescent esters of poly(ethylene glycol) cholesteryl ether (PEG-Chol) that label cholesterol-rich domains. The production of nontoxic forms of lysenin as well as its specific binding behavior have allowed monitoring the distribution and the dynamics of SM-rich domains in living cell membranes. Because of its water-solubility and low toxicity, the fluorescent PEG-Chol can be used to follow the reorganization of cell surface cholesterol-rich domains as well as intracellular cholesterol dynamics in living cells. These probes can thus provide important informations on lipid distribution and traffic in living cell membranes.

Bis(monoacylglycero)phosphate (BMP) is a structural isomer of phosphatidylglycerol that exhibits an unusual sn1:sn1' stereoconfiguration, based on the position of the phosphate moiety on its two glycerol units. Early works have underlined the high concentration of BMP in the lysosomal compartment, especially during some lysosomal storage disorders and drug-induced phospholipidosis. Despite numerous studies, both biosynthetic and degradative pathways of BMP remained not completely elucidated. More recently, BMP has been localized in the internal membranes of late endosomes where it forms specialized lipid domains. Its involvement in both dynamics and lipid/protein sorting functions of late endosomes has started to be documented, especially in the control of cellular cholesterol distribution. BMP also plays an important role in the late endosomal/lysosomal degradative pathway. Another peculiarity of BMP is to be naturally enriched in docosahexaenoic acid and/or to specifically incorporate this fatty acid compared to other polyunsaturated fatty acids, which may confer specific biophysical and functional properties to this phospholipid. This review summarizes and updates our knowledge on BMP with an emphasis on its possible implication in human health and diseases, especially in relation to cholesterol homeostasis.

Bis(monoacylglycero)phosphate (BMP) is a unique lipid enriched in the late endosomes participating in the trafficking of lipids and proteins through this organelle. The de novo biosynthesis of BMP has not been clearly demonstrated. We investigated whether phosphatidylglycerol (PG) and cardiolipin (CL) could serve as precursors of de novo BMP synthesis using two different cellular models: CHO cells deficient in phosphatidylglycerophosphate (PGP) synthase, the enzyme responsible for the first step of PG synthesis; and human lymphoblasts from patients with Barth syndrome (BTHS), characterized by mutations in tafazzin, an enzyme implicated in the deacylation-reacylation cycle of CL. The biosynthesis of both PG and BMP was reduced significantly in the PGP synthase-deficient CHO mutants. Furthermore, overexpression of PGP synthase in the deficient mutants induced an increase of BMP biosynthesis. In contrast to CHO mutants, BMP biosynthesis and its fatty acid composition were not altered in BTHS lymphoblasts. Our results thus suggest that in mammalian cells, PG, but not CL, is a precursor of the de novo biosynthesis of BMP. Despite the decrease of de novo synthesis, the cellular content of BMP remained unchanged in CHO mutants, suggesting that other pathway(s) than de novo biosynthesis are also used for BMP synthesis.

Intestinal cell growth and differentiation are tightly regulated by growth factors and extracellular matrix components along the crypt-villus axis. We previously described enterophilin-1 (Ent-1) as a new intestinal protein associated with growth arrest and enterocyte differentiation. Ent-1 interacted with sorting nexin 1 and decreased cell surface epidermal growth factor receptor. Because beta(1) integrins are mostly found in vivo in the proliferative crypt cells, we investigated the role of Ent-1 in the fate of beta(1) integrin subunits. In undifferentiated intestinal Caco-2 cells, overexpression of Ent-1 induces a marked decrease of alpha(5)beta(1) integrin pools, whereas alpha(2)beta(1) integrin is weakly affected. Conversely, overexpression of sorting nexin 1 has no effect on integrin levels despite its ability to interact with Ent-1. Interestingly, we identified focal adhesion kinase as a new Ent-1 partner using yeast two-hybrid screening and co-precipitation experiments. Furthermore by confocal microscopy, we observed that Ent-1 and beta(1) integrins partly co-localize on vesicular structures, suggesting a role for Ent-1 in integrin trafficking. Because focal adhesion kinase is able to bind both Ent-1 and beta(1) integrins, the kinase might act as a molecular bridge between the two proteins. Altogether, these results support a role of Ent-1 in regulating beta(1) integrin expression that could favor intestinal differentiation.

Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH2-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH2-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein.

A number of epidemiologic studies have indicated a strong association between dietary fat intake and prostate cancer development, suggesting that lipid metabolism plays some important roles in prostate carcinogenesis and its progression. In this study, through our genome-wide gene expression analysis of clinical prostate cancer cells, we identified a novel lipogenic gene, ELOVL7, coding a possible long-chain fatty acid elongase, as overexpressed in prostate cancer cells. ELOVL7 expression is regulated by the androgen pathway through SREBP1, as well as other lipogenic enzymes. Knockdown of ELOVL7 resulted in drastic attenuation of prostate cancer cell growth, and it is notable that high-fat diet promoted the growth of in vivo tumors of ELOVL7-expressed prostate cancer. In vitro fatty acid elongation assay and fatty acid composition analysis indicated that ELOVL7 was preferentially involved in fatty acid elongation of saturated very-long-chain fatty acids (SVLFA, C20:0). Lipid profiles showed that knockdown of ELOVL7 in prostate cancer cells affected SVLFAs in the phospholipids and the neutral lipids, such as cholesterol ester. Focusing on cholesterol ester as a source of de novo steroid synthesis, we show that ELOVL7 affected de novo androgen synthesis in prostate cancer cells. These findings suggest that EVOLV7 could be involved in prostate cancer growth and survival through the metabolism of SVLFAs and their derivatives, could be a key molecule to elucidate the association between fat dietary intake and prostate carcinogenesis, and could also be a promising molecular target for development of new therapeutic or preventive strategies for prostate cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1608.

The release of various fatty acids (FAs) from permeabilized HL-60 cells, predominantly oleic acid (OA) rather than arachidonic acid, was greatly enhanced by GTP-γ-S and vanadate [Tsujishita, Y., Asaoka, Y. and Nishizuka, Y., Proc. Natl. Acad. Sci. USA 91 (1994) 6274–6278]. The present study shows that phospholipase A (A2/A1) activity which cleaves the acyl group from both sn-2 and sn-1 positions of phosphatidylethanolamine (PtdEtn) is increased in HL-60 cells during differentiation to granulocyte-like cells. This enzyme does not require Ca2+ and releases various FAs, preferentially OA from PtdEtn and, to lesser extent, from lysoPtdEtn. Other phospholipids including phosphatidylcholine and phosphatidic acid serve as very poor substrates. Although further studies are necessary to show the direct link of this enzyme activation to receptor stimulation, the results described here imply that this enzyme is responsible for the release of various FAs, particularly OA, from permeabilized HL-60 cells.