Bis(monoacylglycero)phosphate regulates oxysterol binding protein-related protein 11 dependent sterol trafficking

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 macrophages. 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.Peer reviewe

Protein probes to visualize sphingomyelin and ceramide phosphoethanolamine

International audienceSphingomyelin (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

Probing phosphoethanolamine-containing lipids in membranes with duramycin/cinnamycin and aegerolysin proteins

International audienceIn 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

Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin

International audienceLysenin, which is an earthworm toxin, strongly binds to sphingomyelin (SM). Lysenin oligomerizes on SM-rich domains and can induce cell death by forming pores in the membrane. In this review, the assembly of lysenin on SM-containing membranes is discussed mostly on the basis of the information gained by atomic force microscopy (AFM). AFM data show that lysenin assembles into a hexagonal close packed (hcp) structure by rapid reorganization of its oligomers on an SM/cholesterol membrane. In case of a phase-separated membrane of SM, lysenin induces phase mixing as a result of pore formation in SM-rich domains, and consequently its hcp assembly covers the entire membrane. Besides the lytic action, lysenin is important as an SM marker and its pore has the potential to be used as a biosensor in the future. These points are also highlighted in this review

Bis(monoacylglycero)phosphate, an important actor in the host endocytic machinery hijacked by SARS-CoV-2 and related viruses

International audienceViruses, 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

Pore-forming toxins: Properties, diversity, and uses as tools to image sphingomyelin and ceramide phosphoethanolamine

International audiencePore-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

Plasma Membrane Origin of the Steroidogenic Pool of Cholesterol Used in Hormone-induced Acute Steroid Formation in Leydig Cells

International audienceHormone-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

PMP2/FABP8 induces PI(4,5)P2-dependent transbilayer reorganization of sphingomyelin in the plasma membrane

International audienceSphingomyelin (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)P2)-binding protein. PMP2 induces the tubulation of model membranes in a PI(4,5)P2-dependent manner, accompanied by the modification of the transbilayer membrane distribution of lipids. In the PM of PMP2-overexpressing cells, inner-leaflet SM is increased whereas outer-leaflet SM is reduced. PMP2 is a causative protein of Charcot-Marie-Tooth disease (CMT). A mutation in PMP2 associated with CMT increases its affinity for PI(4,5)P2, inducing membrane tubulation and the subsequent transbilayer movement of lipids

Stimulatory effects of combined endocrine disruptors on MA-10 Leydig cell steroid production and lipid homeostasis

International audiencePrevious 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

In vitro oxidized HDL and HDL from type 2 diabetes patients have reduced ability to efflux oxysterols from THP-1 macrophages

International audienceOxidized 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