Exosomal lipids from membrane organization to biomarkers: Focus on an endolysosomal-specific lipid.

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.

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

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.

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

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.

Bis(monoacylglycero)phosphate, a new lipid signature of endosome-derived extracellular vesicles.

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.

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.

Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes.

Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5-10 nm thick slab-like structures.

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

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.

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

Lysenin, 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).

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

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.

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

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.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

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.

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

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.

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

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.

Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis.

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.

Regulation of the transbilayer movement of diacylglycerol in the plasma 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.

Lipid compartmentalization in the endosome system.

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.

Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin-Darby canine kidney cells.

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.

Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum.

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.

On the origin of the 1602 cm-1 Raman band of yeasts; contribution of ergosterol.

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.

A role for sphingomyelin-rich lipid domains in the accumulation of phosphatidylinositol-4,5-bisphosphate to the cleavage furrow during cytokinesis.

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.

Multiplex analysis of sphingolipids using amine-reactive tags (iTRAQ).

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.

Novel lipogenic enzyme ELOVL7 is involved in prostate cancer growth through saturated long-chain fatty acid metabolism.

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.

Imaging lipid membrane domains with lipid-specific probes.

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.