Expanding the CHARMM36 United Atom Chain Model for the Inclusion of Sphingolipids.

The inclusion of accurate yet computationally inexpensive lipid force fields (FF) is pertinent for the study of lipids and lipid-containing systems using molecular dynamics (MD). Within the past decade, the implementation and further expansion of a united atom (UA) FF for lipids have been developed in the CHARMM family of FFs. The most recent version of the UA presented more accurate descriptions of lipid properties for several phospholipids with saturated and monounsaturated chains, termed C36UAr. However, the original C36UAr model lacks parameters for an important class of lipids, such as sphingolipids. The focus of this article is to broaden the scope of the C36UAr chain model to incorporate these lipids. In this study, two common sphingolipids, N-palmitoyl sphingomyelin and N-stearoyl sphingomyelin are converted to a UA-chain representation and simulated to investigate the accuracy and speed over the all-atom FF model for sphingolipids. Improvements were found among multiple parameters, for example, in the surface area per lipid (SA/lip) and hydrogen order parameters, over the all-atom simulations of these sphingomyelins in C36, while as much as halving the simulation time for simulations of the same setup otherwise. Thus, the accuracy and efficiency found in this study are consistent with those found in the C36UAr model for phospholipids and expand the application of C36UAr to a wider array of membrane models to better match that in vivo.

Chromatographic Separation and Quantitation of Sphingolipids from the Central Nervous System or Any Other Biological Tissue.

Chromatographic separation and purification of an individual lipid to homogeneity have long been introduced. Using this concept, a more precise method has been developed to identify and characterize the sphingolipid composition(s) using a small amount (30 mg) of biological sample. Sphingolipids (lipids containing sphingosine or dihydrosphingosine) are well-known regulators of the central nervous system development and play a critical role in neurodegenerative diseases. Introducing a silicic acid column chromatography, sphingolipid components have been separated to individual fractions such as ceramide, glucosyl/galactosylceramide, other neutral and acidic glycosphingolipids, including (dihydro)sphingosine and psychosine; as well as phospholipids from which individual components are quantified employing a single or combination of other advanced chromatography procedures such as thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography-mass spectrometry.

Perspective: Therapeutic Implications for Sphingolipids in Health and Disease.

Long thought to be structural components of cell membranes, sphingolipids (SLs) have emerged as bioactive molecules whose metabolism is tightly regulated. These bioactive lipids and their metabolic enzymes have been implicated in numerous disease states, including lysosomal storage disorders, multiple sclerosis, inflammation, and cancer as well as metabolic syndrome and obesity. In addition, the indications for many of these lipids to potentially serve as biomarkers for disease continue to emerge with increasing metabolomic and lipidomic studies. The implications of these studies have, in turn, led to the examination of SL enzymes and their bioactive lipids as potential therapeutic targets and as markers for therapeutic efficacy. SIGNIFICANCE STATEMENT: Many sphingolipids (SLs) and their metabolizing enzymes have been implicated in disease. This perspective highlights the potential for SLs to serve as therapeutic targets and diagnostic markers and discusses the implications for the studies and reviews highlighted in this Special Section on Therapeutic Implications for Sphingolipids in Health and Disease.

Emerging roles and therapeutic potentials of sphingolipids in pathophysiology: emphasis on fatty acyl heterogeneity.

Sphingolipids not only exert structural roles in cellular membranes, but also act as signaling molecules in various physiological and pathological processes. A myriad of studies have shown that abnormal levels of sphingolipids and their metabolic enzymes are associated with a variety of human diseases. Moreover, blood sphingolipids can also be used as biomarkers for disease diagnosis. This review summarizes the biosynthesis, metabolism, and pathological roles of sphingolipids, with emphasis on the biosynthesis of ceramide, the precursor for the biosynthesis of complex sphingolipids with different fatty acyl chains. The possibility of using sphingolipids for disease prediction, diagnosis, and treatment is also discussed. Targeting endogenous ceramides and complex sphingolipids along with their specific fatty acyl chain to promote future drug development will also be discussed.

Intracellular sphingolipid sorting drives membrane phase separation in the yeast vacuole.

The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by an increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs (CSLs). These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition-headgroup stoichiometry, longer chain lengths, and increased hydroxylations-were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.

The biological functions of sphingolipids in plant pathogenic fungi.

Sphingolipids are critically significant in a range of biological processes in animals, plants, and fungi. In mammalian cells, they serve as vital components of the plasma membrane (PM) in maintaining its structure, tension, and fluidity. They also play a key role in a wide variety of biological processes, such as intracellular signal transduction, cell polarization, differentiation, and migration. In plants, sphingolipids are important for cell development and for cell response to environmental stresses. In pathogenic fungi, sphingolipids are crucial for the initiation and the development of infection processes afflicting humans. However, our knowledge on the metabolism and function of the sphingolipid metabolic pathway of pathogenic fungi affecting plants is still very limited. In this review, we discuss recent developments on sphingolipid pathways of plant pathogenic fungi, highlighting their uniqueness and similarity with plants and animals. In addition, we discuss recent advances in the research and development of fungal-targeted inhibitors of the sphingolipid pathway, to gain insights on how we can better control the infection process occurring in plants to prevent or/and to treat fungal infections in crops.

Sphingolipids: From structural components to signaling hubs.

In late November 2019, Prof. Lina M. Obeid passed away from cancer, a disease she spent her life researching and studying its intricate molecular underpinnings. Along with her husband, Prof. Yusuf A. Hannun, Obeid laid down the foundations of sphingolipid biochemistry and oversaw its remarkable evolution over the years. Lipids are a class of macromolecules that are primarily associated with cellular architecture. In fact, lipids constitute the perimeter of the cell in such a way that without them, there cannot be cells. Hence, much of the early research on lipids identified the function of this class of biological molecules as merely structural. Nevertheless, unlike proteins, carbohydrates, and nucleic acids, lipids are elaborately diverse as they are not made up of monomers in polymeric forms. This diversity in structure is clearly mirrored by functional pleiotropy. In this chapter, we focus on a major subset of lipids, sphingolipids, and explore their historic rise from merely inert structural components of plasma membranes to lively and necessary signaling molecules that transmit various signals and control many cellular processes. We will emphasize the works of Lina Obeid since she was an integral pillar of the sphingolipid research world.

A Sphingolipidomic Profiling Approach for Comparing X-ray-Exposed and Unexposed HepG2 Cells.

An analytical method based on tandem mass spectrometry-shotgun is presently proposed to obtain sphingolipidomic profiles useful for the characterization of lipid extract from X-ray-exposed and unexposed hepatocellular carcinoma cells (HepG2). To obtain a targeted lipidic profile from a specific biological system, the best extraction method must be identified before instrumental analysis. Accordingly, four different classic lipid extraction protocols were compared in terms of efficiency, specificity, and reproducibility. The performance of each procedure was evaluated using the Fourier-transform infrared spectroscopic technique; subsequently, the quality of extracts was estimated using electrospray ionization tandem mass spectrometry. The selected procedure based on chloroform/methanol/water was successfully used in mass spectrometry-based shotgun sphingolipidomics, allowing for evaluation of the response of cells to X-ray irradiation, the most common anticancer therapy. Using a relative quantitative approach, the changes in the sphingolipid profiles of irradiated cell extracts were demonstrated, confirming that lipidomic technologies are also useful tools for studying the key sphingolipid role in regulating cancer growth during radiotherapy.

The sphingolipids ceramide and inositol phosphorylceramide protect the Leishmania major membrane from sterol-specific toxins.

The accessibility of sterols in mammalian cells to exogenous sterol-binding agents has been well-described previously, but sterol accessibility in distantly related protozoa is unclear. The human pathogen Leishmania major uses sterols and sphingolipids distinct from those used in mammals. Sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids, but the surface exposure of ergosterol in Leishmania remains unknown. Here, we used flow cytometry to test the ability of the L. major sphingolipids inositol phosphorylceramide (IPC) and ceramide to shelter ergosterol by preventing binding of the sterol-specific toxins streptolysin O and perfringolysin O and subsequent cytotoxicity. In contrast to mammalian systems, we found that Leishmania sphingolipids did not preclude toxin binding to sterols in the membrane. However, we show that IPC reduced cytotoxicity and that ceramide reduced perfringolysin O- but not streptolysin O-mediated cytotoxicity in cells. Furthermore, we demonstrate ceramide sensing was controlled by the toxin L3 loop, and that ceramide was sufficient to protect L. major promastigotes from the anti-leishmaniasis drug amphotericin B. Based on these results, we propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus, L. major could serve as a genetically tractable protozoan model organism for understanding toxin-membrane interactions.

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains.

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains to develop a set of photoswitchable sphingolipids with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-ß-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable liquid-ordered domains.

Multiplex Quantification of Plasma Biomarkers for Patients with Gaucher Disease Type 1.

Gaucher disease (GD) is a lysosomal storage disorder caused by a deficiency of the enzyme beta-glucocerebrosidase. This leads to the accumulation of glycolipids in macrophages and ultimately results in tissue damage. Recent metabolomic studies highlighted several potential biomarkers in plasma specimens. In hopes of better understanding the distribution, importance, and clinical significance of these potential markers, a UPLC-MS/MS method was developed and validated to quantify lyso-Gb1 and six related analogs (with the following modifications on the sphingosine moiety: -C2 H4 (-28 Da), -C2 H4 +O (-12 Da), -H2 (-2 Da), -H2 +O (+14 Da), +O (+16 Da), and +H2 O (+18 Da)), sphingosylphosphorylcholine, and N-palmitoyl-O-phosphocholineserine in plasma specimens of treated and untreated patients. This 12-min UPLC-MS/MS method involves a purification step via solid-phase extraction followed by evaporation under nitrogen flow and resuspension in an organic mix compatible with HILIC chromatography. This method is currently used for research purposes and might be used for monitoring, prognostics, and follow-up. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Fluorescently Labeled Ceramides and 1-Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies.

Ceramides (Cer) are bioactive sphingolipids that have been proposed as potential disease biomarkers since they are involved in several cellular stress responses, including apoptosis and senescence. 1-Deoxyceramides (1-deoxyCer), a particular subtype of noncanonical sphingolipids, have been linked to the pathogenesis of type II diabetes. To investigate the metabolism of these bioactive lipids, as well as to have a better understanding of the signaling processes where they participate, it is essential to expand the toolbox of fluorescent sphingolipid probes exhibiting complementary subcellular localization. Herein, we describe a series of new sphingolipid probes tagged with two different organic fluorophores, a far-red/NIR-emitting coumarin derivative (COUPY) and a green-emitting BODIPY. The assembly of the probes involved a combination of olefin cross metathesis and click chemistry reactions as key steps, and these fluorescent ceramide analogues exhibited excellent emission quantum yields, being the Stokes' shifts of the COUPY derivatives much higher than those of the BODIPY counterparts. Confocal microscopy studies in HeLa cells confirmed an excellent cellular permeability for these sphingolipid probes and revealed that most of the vesicles stained by COUPY probes were either lysosomes or endosomes, whereas BODIPY probes accumulated either in Golgi apparatus or in nonlysosomal intracellular vesicles. The fact that the two sets of fluorescent Cer probes have such different staining patterns indicates that their subcellular distribution is not entirely defined by the sphingolipid moiety but rather influenced by the fluorophore.

Plasma Lysosphingolipid Biomarker Measurement by Liquid Chromatography Tandem Mass Spectrometry.

Plasma lysosphingolipids are highly elevated in patients with Gaucher, Krabbe, Fabry, and Niemann-Pick diseases and tend to accumulate to a greater extent than their respective primary sphingolipids in the plasma of affected patients. In this chapter, we describe two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods to measure plasma concentrations of four lysosphingolipids species. The first method described measures glucosylsphingosine (lyso-GL1) and galactosylsphingosine (psychosine), biomarkers that accumulate in Gaucher and Krabbe diseases, respectively. The second method measures globotriaosylsphingosine (lyso-Gb3) and sphingosylphosphorylcholine (lyso-SPM), biomarkers for Fabry and Niemann-Pick diseases, respectively. Each method utilizes isotope-labeled internal standards and multipoint calibration curves to quantify the analytes of interest. Briefly, plasma samples are mixed with five volumes of LC-MS grade methanol containing internal standard, and protein is removed via centrifugation. Supernatant is dried and resuspended in initial mobile phase. Samples are separated by liquid chromatography using either a BEH amide column (lyso-GL1 + psychosine) or a C18 column (lyso-Gb3 + lyso-SPM). Protonated analytes are measured by selected reaction monitoring (SRM) in positive electrospray ionization mode. Using these methods, we have observed elevations of these lyso- species in Gaucher, Fabry, and Niemann-Pick and successfully distinguished different subtypes reflecting the disease severity.

Vacuole-Specific Lipid Release for Tracking Intracellular Lipid Metabolism and Transport in Saccharomyces cerevisiae.

Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the endoplasmic reticulum-vacuole-tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.

Diversity in sphingolipid metabolism across land plants.

Sphingolipids are essential metabolites found in all plant species. They are required for plasma membrane integrity, tolerance of and responses to biotic and abiotic stresses, and intracellular signalling. There is extensive diversity in the sphingolipid content of different plant species, and in the identities and roles of enzymes required for their processing. In this review, we survey results obtained from investigations of the classical genetic model Arabidopsis thaliana, from assorted dicots with less extensive genetic toolkits, from the model monocot Oryza sativa, and finally from the model bryophyte Physcomitrium patens. For each species or group, we first broadly summarize what is known about sphingolipid content. We then discuss the most insightful and puzzling features of modifications to the hydrophobic ceramides, and to the polar headgroups of complex sphingolipids. Altogether, these data can serve as a framework for our knowledge of sphingolipid metabolism across the plant kingdom. This chemical and metabolic heterogeneity underpins equally diverse functions. With greater availability of different tools for analytical measurements and genetic manipulation, our field is entering an exciting phase of expanding our knowledge of the biological functions of this persistently cryptic class of lipids.

Very-Long-Chain Unsaturated Sphingolipids Mediate Oleate-Induced Rat ß-Cell Proliferation.

Fatty acid (FA) signaling contributes to ß-cell mass expansion in response to nutrient excess, but the underlying mechanisms are poorly understood. In the presence of elevated glucose, FA metabolism is shifted toward synthesis of complex lipids, including sphingolipids. Here, we tested the hypothesis that sphingolipids are involved in the ß-cell proliferative response to FA. Isolated rat islets were exposed to FA and 16.7 mmol/L glucose for 48-72 h, and the contribution of the de novo sphingolipid synthesis pathway was tested using the serine palmitoyltransferase inhibitor myriocin, the sphingosine kinase (SphK) inhibitor SKI II, or knockdown of SphK, fatty acid elongase 1 (ELOVL1) and acyl-CoA-binding protein (ACBP). Rats were infused with glucose and the lipid emulsion ClinOleic and received SKI II by gavage. ß-Cell proliferation was assessed by immunochemistry or flow cytometry. Sphingolipids were analyzed by liquid chromatography-tandem mass spectrometry. Among the FAs tested, only oleate increased ß-cell proliferation. Myriocin, SKI II, and SphK knockdown all decreased oleate-induced ß-cell proliferation. Oleate exposure did not increase the total amount of sphingolipids but led to a specific rise in 24:1 species. Knockdown of ACBP or ELOVL1 inhibited oleate-induced ß-cell proliferation. We conclude that unsaturated very-long-chain sphingolipids produced from the available C24:1 acyl-CoA pool mediate oleate-induced ß-cell proliferation in rats.

Penta-deuterium-labeled 4E, 8Z-sphingadienine for rapid analysis in sphingolipidomics study.

The use of deuterium-incorporated bioactive compounds is an efficient method for tracing their metabolic fate and for quantitative analysis by mass spectrometry without complicated HPLC separation even if their amounts are extremely small. Plant sphingolipids and their metabolites, which have C4, 8-olefins on a common backbone as a sphingoid base, show unique and fascinating bioactivities compared to those of sphingolipids in mammals. However, the functional and metabolic mechanisms of exogenous plant sphingolipids have not been elucidated due to the difficulty in distinguishing exogenous sphingolipids from endogenous sphingolipids having the same polarity and same molecular weight by mass spectrometric analysis. Their roles might be elucidated by the use of deuterated probes with original biological and physicochemical properties. In this study, we designed (2S,3R,4E,8Z)-2-aminooctadeca-4,8-diene-17,17,18,18,18-d5-1,3-diol (penta-deuterium-labeled 4E, 8Z-sphingadienine) as a tracer for exogenous metabolic studies. In addition, the sphingadienine was confirmed to be metabolized in HEK293 cells and showed distinct peaks in mass spectrometric analysis.

Compartmentation and functions of sphingolipids.

Sphingolipids (SLs) are one of the three major lipid classes in all eukaryotic cells. They function as structural molecules of membranes and can also act as highly active signaling molecules. SL biosynthesis is mainly occurring at the endoplasmic reticulum and the Golgi apparatus. However, SL intermediates are also generated at other organelles such as the plasma membrane and the lysosome. SL biosynthesis is therefore highly compartmentalized. Maintaining SL levels is necessary for the function of multiple trafficking pathways. One major challenge is to decipher the complex regulatory networks controlling SL biosynthesis, the coordination of vesicular and non-vesicular SL transport as well as their role in trafficking. Recent investigations have shed new light on the regulation of SL biosynthesis. Here, we review how SL biosynthesis is coordinated, how SLs are transported and how their levels affect trafficking pathways. Finally, we discuss recently developed methods to study SL metabolism with spatio-temporal resolution.

Unique symbiont-derived sphingolipids: Dietary amino acids source branch formation.

Metabolites derived from symbionts have the potential to regulate host pathophysiological conditions, especially in the gut. In a recent issue of Nature, Oh et al. clarify unique molecular structures of α-galactosylceramides derived from B. fragilis and their immune-modulatory functions against host natural killer T (NKT) cells.

Lipidomics Analysis of Outer Membrane Vesicles and Elucidation of the Inositol Phosphoceramide Biosynthetic Pathway in Bacteroides thetaiotaomicron.

Approximately one-third of the human colonic microbiome is formed by bacteria from the genus Bacteroides. These bacteria produce a large amount of uniformly sized outer membrane vesicles (OMVs), which are equipped with hydrolytic enzymes that play a role in the degradation of diet- and host-derived glycans. In this work, we characterize the lipid composition of membranes and OMVs from Bacteroides thetaiotaomicron VPI-5482. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated that OMVs carry sphingolipids, glycerophospholipids, and serine-dipeptide lipids. Sphingolipid species represent more than 50% of the total lipid content of OMVs. The most abundant sphingolipids in OMVs are ethanolamine phosphoceramide (EPC) and inositol phosphoceramide (IPC). Bioinformatics analysis allowed the identification of the BT1522-1526 operon putatively involved in IPC synthesis. Mutagenesis studies revealed that BT1522-1526 is essential for the synthesis of phosphatidylinositol (PI) and IPC, confirming the role of this operon in the biosynthesis of IPC. BT1522-1526 mutant strains lacking IPC produced OMVs that were indistinguishable from the wild-type strain, indicating that IPC sphingolipid species are not involved in OMV biogenesis. Given the known role of sphingolipids in immunomodulation, we suggest that OMVs may act as long-distance vehicles for the delivery of sphingolipids in the human gut. IMPORTANCE Sphingolipids are essential membrane lipid components found in eukaryotes that are also involved in cell signaling processes. Although rare in bacteria, sphingolipids are produced by members of the phylum Bacteroidetes, human gut commensals. Here, we determined that OMVs carry sphingolipids and other lipids of known signaling function. Our results demonstrate that the BT1522-1526 operon is required for IPC biosynthesis in B. thetaiotaomicron.