Disruption of sphingolipid metabolism elicits apoptosis-associated reproductive defects in Drosophila

Sphingolipid signaling is thought to regulate apoptosis via mechanisms that are dependent on the concentration of ceramide relative to that of sphingosine-1-phosphate (S1P). This study reports defects in reproductive structures and function that are associated with enhanced apoptosis in Drosophila Sply05091 mutants that lack functional S1P lyase and thereby accumulate sphingolipid long chain base metabolites. Analyses of reproductive structures in these adult mutants unmasked multiple abnormalities, including supernumerary spermathecae, degenerative ovaries, and severely reduced testes. TUNEL assessment revealed increased cell death in mutant egg chambers at most oogenic stages and in affected mutant testes. These reproductive abnormalities and elevated gonadal apoptosis were also observed, to varying degrees, in other mutants affecting sphingolipid metabolism. Importantly, the reproductive defects seen in the Sply05091 mutants were ameliorated both by a second site mutation in the lace gene that restores long chain base levels towards normal and by genetic disruption of the proapoptotic genes reaper, hid and grim. These data thus provide the first evidence in Drosophila that accumulated sphingolipids trigger elevated levels of apoptosis via the modulation of known signaling pathways.     

Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis

Sphingosine-1-phosphate lyase is a widely expressed enzyme that catalyzes the essentially irreversible cleavage of the signaling molecule sphingosine 1-phosphate. To investigate whether sphingosine-1-phosphate lyase influences mammalian cell fate decisions, a recombinant human sphingosine-1-phosphate lyase fused to green fluorescent protein was expressed in HEK293 cells. The recombinant enzyme was active, localized to the endoplasmic reticulum, and reduced baseline sphingosine and sphingosine 1-phosphate levels. Stable overexpression led to diminished viability under stress, which was attributed to an increase in apoptosis and was reversible in a dose-dependent manner by exogenous sphingosine 1-phosphate. In contrast to sphingosine 1-phosphate, the products of the lyase reaction had no effect on apoptosis. Lyase enzymatic activity was required to potentiate apoptosis, because cells expressing a catalytically inactive enzyme behaved like controls. Stress increased the amounts of long- and very long-chain ceramides in HEK293 cells, and this was enhanced in cells overexpressing wild type but not catalytically inactive lyase. The ceramide increases appeared to be required for apoptosis, because inhibition of ceramide synthase with fumonisin B1 decreased apoptosis in lyase-overexpressing cells. Thus, sphingosine-1-phosphate lyase overexpression in HEK293 cells decreases sphingosine and sphingosine 1-phosphate amounts but elevates stress-induced ceramide generation and apoptosis. This identifies sphingosine-1-phosphate lyase as a dual modulator of sphingosine 1-phosphate and ceramide metabolism as well as a regulator of cell fate decisions and, hence, a potential target for diseases with an imbalance in these biomodulators, such as cancer.     

Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum

Sphingosine-1-phosphate, a product of sphingomyelin degradation, is an important element of signal transduction pathways that regulate cell proliferation and cell death. We have demonstrated additional roles for sphingosine-1-phosphate in growth and multicellular development. The specific disruption in Dictyostelium discoideum of the sphingosine-1-phosphate lyase gene, which encodes the enzyme that catalyzes sphingosine-1-phosphate degradation, results in a mutant strain with aberrant morphogenesis, as well as an increase in viability during stationary phase. The absence of sphingosine-1-phosphate lyase affects multiple stages throughout development, including the cytoskeletal architecture of aggregating cells, the ability to form migrating slugs, and the control of cell type-specific gene expression and terminal spore differentiation. This pleiotropic effect, which is due to the loss of sphingosine-1-phosphate lyase, establishes sphingolipids as pivotal regulatory molecules in a wide range of processes in multicellular development.     

Human sphingosine-1-phosphate lyase: cDNA cloning, functional expression studies and mapping to chromosome 10q22(1)

Sphingosine-1-phosphate lyase catalyzes the last step in sphingolipid breakdown, the cleavage of phosphorylated sphingoid bases such as sphingenine-1-phosphate. The latter lipid is not only a catabolite, but can influence as an inter- and/or intracellular second messenger many cellular processes. To allow for the diagnosis of human disorders that might be linked to a deficient lyase, the human sphingosine-1-phosphate lyase cDNA was cloned. The obtained cDNA encoded a protein of 568 amino acids with a calculated molecular mass of 63492 Da. Hydropathy plots revealed the presence of one membrane span near the amino-terminal which is however not required for enzyme activity since recombinant poly-His-tagged lyase, lacking this membrane span, was functionally active. Site-directed mutagenesis disclosed the importance of the cysteine residues 218 and 317 for the cleavage reaction. Northern analysis showed the presence of rare large-sized mRNAs of 6.7, 5.8 and 4 kb and the highest expression in liver. By fluorescent in situ hybridization, the gene was mapped to chromosome 10q22.     

The immune modulator FTY720 inhibits sphingosine-1-phosphate lyase activity

FTY720 is a novel immunomodulatory agent that inhibits lymphocyte trafficking and prevents allograft rejection. FTY720 is phosphorylated in vivo, and the phosphorylated drug acts as agonist for a family of G protein-coupled receptors that recognize sphingosine 1-phosphate. Evidence suggests that FTY720-phosphate-induced activation of S1P1 is responsible for its mechanism of action. FTY720 was rationally designed by modification of myriocin, a naturally occurring sphingoid base analog that causes immunosuppression by interrupting sphingolipid metabolism. In this study, we examined interactions between FTY720, FTY720-phosphate, and sphingosine-1-phosphate lyase, the enzyme responsible for irreversible sphingosine 1-phosphate degradation. FTY720-phosphate was stable in the presence of active sphingosine-1-phosphate lyase, demonstrating that the lyase does not contribute to FTY720 catabolism. Conversely, FTY720 inhibited sphingosine-1-phosphate lyase activity in vitro. Treatment of mice with FTY720 inhibited tissue sphingosine-1-phosphate lyase activity within 12 h, whereas lyase gene and protein expression were not significantly affected. Tissue sphingosine 1-phosphate levels remained stable or increased throughout treatment. These studies raise the possibility that disruption of sphingosine 1-phosphate metabolism may account for some effects of FTY720 on immune function and that sphingosine-1-phosphate lyase may be a potential target for immunomodulatory therapy.     

Identification and characterization by electrospray mass spectrometry of endogenous Drosophila sphingadienes

Sphingolipids comprise a complex group of lipids concentrated in membrane rafts and whose metabolites function as signaling molecules. Sphingolipids are conserved in Drosophila, in which their tight regulation is required for proper development and tissue integrity. In this study, we identified a new family of Drosophila sphingolipids containing two double bonds in the long chain base (LCB). The lipids were found at low levels in wild-type flies and accumulated markedly in Drosophila Sply mutants, which do not express sphingosine-1-phosphate lyase and are defective in sphingolipid catabolism. To determine the identity of the unknown lipids, purified whole fly lipid extracts were separated on a C18-HPLC column and analyzed using electrospray mass spectrometry. The lipids contain a LCB of either 14 or 16 carbons with conjugated double bonds at C4,6. The Delta(4,6)-sphingadienes were found as free LCBs, as phosphorylated LCBs, and as the sphingoid base in ceramides. The temporal and spatial accumulation of Delta(4,6)-sphingadienes in Sply mutants suggests that these lipids may contribute to the muscle degeneration observed in these flies.     

Sphingosine phosphate lyase expression is essential for normal development in Caenorhabditis elegans

Sphingolipids are ubiquitous membrane constituents whose metabolites function as signaling molecules in eukaryotic cells. Sphingosine 1-phosphate, a key sphingolipid second messenger, regulates proliferation, motility, invasiveness, and programmed cell death. These effects of sphingosine 1-phosphate and similar phosphorylated sphingoid bases have been observed in organisms as diverse as yeast and humans. Intracellular levels of sphingosine 1-phosphate are tightly regulated by the actions of sphingosine kinase, which is responsible for its synthesis and sphingosine-1-phosphate phosphatase and sphingosine phosphate lyase, the two enzymes responsible for its catabolism. In this study, we describe the cloning of the Caenorhabditis elegans sphingosine phosphate lyase gene along with its functional expression in Saccharomyces cerevisiae. Promoter analysis indicates tissue-specific and developmental regulation of sphingosine phosphate lyase gene expression. Inhibition of C. elegans sphingosine phosphate lyase expression by RNA interference causes accumulation of phosphorylated and unphosphorylated long-chain bases and leads to poor feeding, delayed growth, reproductive abnormalities, and intestinal damage similar to the effects seen with exposure to Bacillus thuringiensis toxin. Our results show that sphingosine phosphate lyase is an essential gene in C. elegans and suggest that the sphingolipid degradative pathway plays a conserved role in regulating animal development.     

TheDPL1Gene Is Involved in Mediating the Response to Nutrient Deprivation inSaccharomyces cerevisiae

Sphingosine-1-phosphate is a sphingolipid metabolite involved in the regulation of cell proliferation in mammalian cells. The major route of sphingosine-1-phosphate degradation is through cleavage at the C_2–3bond by sphingosine phosphate lyase. The recent identification of the first dihydrosphingosine/sphingosine phosphate lyase gene inSaccharomyces cerevisiaeestablishes that phosphorylated sphingoid base metabolism is conserved throughout evolution. Thedpl1Δ deletion mutant, which accumulates endogenous phosphorylated sphingoid bases, exhibits unregulated proliferation upon approach to stationary phase. The increased proliferation rate during respiratory growth was associated with failure to appropriately recruit cells into the G₁phase of the cell cycle. Several genes were found to be overexpressed or prematurely expressed during nutrient deprivation in thedpl1Δ strain, including glucose-repressible genes and G₁cyclins. These studies implicate a role forDPL1and phosphorylated sphingoid bases in the regulation of global responses to nutrient deprivation in yeast.     

Sphingosine-1-phosphate lyase SPL is an endoplasmic reticulum-resident, integral membrane protein with the pyridoxal 5'-phosphate binding domain exposed to the cytosol

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that functions as a bioactive lipid molecule. S1P is degraded either by S1P lyase or by S1P phosphohydrolase. The gene encoding mammalian S1P lyase, SPL, has been identified. Here, we characterize the SPL protein in its expression, localization, and topology. The expression levels of the SPL protein correlated well with the dihydrosphingosine-1-phosphate (DHS1P) lyase activity in most tissues. However, liver and heart exhibited high DHS1P lyase activities compared to their SPL protein levels. The SPL mRNA expression was temporally regulated during mouse embryonal development. Immunofluorescence microscopy demonstrated that SPL is localized at the endoplasmic reticulum. Proteinase K digestion studies revealed that the large hydrophilic domain, containing the active site, faces the cytosol. This active site orientation is opposite to that of S1P phosphohydrolase, indicating that the degradation of S1P by two S1P-degrading enzymes occurs in spatially separated sides of the endoplasmic reticulum.     

Sphingosine-1-Phosphate Phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg(+)-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Sphingosine-1-phosphate phosphatases

Sphingosine-1-phosphate is a potent proliferative, survival, and morphogenetic factor, acting as an extracellular ligand for the EDG family of G-protein-coupled receptors and possibly intracellularly through as yet, unidentified targets. It is produced within most, if not all cells by phosphorylation of sphingosine, and is an abundant serum lipid that is released from activated platelets. Sphingosine and sphingosine-1-phosphate are in dynamic equilibrium with each other due to the activities of sphingosine kinase and sphingosine-1-phosphate phosphatase (SPPase). Several SPPase genes have now been cloned, first from yeast and more recently from mammalian cells. By sequence homology, these enzymes can be classified as a subset of membrane bound, Type 2 lipid phosphohydrolases that contain conserved residues within three domains predicted to be at the active site of the enzyme. Outside of the consensus motif, there is very little homology between SPPases and the other type 2 lipid phosphohydrolases in the LPP/PAP family. Type 2 phosphatase activity is Mg+-independent and insensitive to N-ethylmaleimide, and substrate specificity is broad for LPP enzymes, whereas SPPases are highly selective for sphingolipid substrates. SPPase activity in yeast and mammalian cells regulates intracellular sphingosine-1-phosphate levels, and also alters the levels of sphingosine and ceramide, two other signaling molecules that often oppose the actions of sphingosine-1-phosphate. Thus, loss of SPPase in yeast results in high sphingosine-1-phosphate levels and cells are more resistant to stress, and in mammalian cells, overexpression of SPPase elevates ceramide levels and provokes apoptosis.     

Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.     

Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism

Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the host's sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival.     

Sphingolipid-mediated Signalling in Plants

A plethora of biological effects, ranging from cellular survivalto apoptosis, has been assigned to sphingolipids and, in particular,to the sphingolipid metabolites ceramide, sphingosine and sphingosine-1-phosphate.One aspect of sphingolipid biology that is currently attractinga great deal of interest in animals and yeast is their rolein cell signalling. In contrast, much less is known about sphingolipidsin plants, although available information suggests that thesecompounds may also fulfil important signalling roles. Thereare suggestions that sphingolipid metabolites may be involvedin diverse processes including pathogenesis, membrane stabilityand the response to drought. Here, we review current informationon the role of sphingolipid metabolites and highlight theiremerging roles in plant signalling. Copyright 2001 Annals ofBotany Company Sphingolipid, cerebrosides, glucosylceramides, sphingosine-1-phosphate, pathogenesis, stomata, guard cells, calcium, signal transduction, cell signalling     

Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.     

Integration of lipidomics and transcriptomics data towards a systems biology model of sphingolipid metabolism

Background  

Sphingolipids play important roles in cell structure and function as well as in the pathophysiology of many diseases. Many of the intermediates of sphingolipid biosynthesis are highly bioactive and sometimes have antagonistic activities, for example, ceramide promotes apoptosis whereas sphingosine-1-phosphate can inhibit apoptosis and induce cell growth; therefore, quantification of the metabolites and modeling of the sphingolipid network is imperative for an understanding of sphingolipid biology.     

Death and taxis: what non-mammalian models tell us about sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a signaling molecule that regulates critical events including mammalian cell proliferation, survival, migration and cell-cell interactions. Most of these signals are triggered by engagement of sphingosine-1-phosphate receptors of the Edg family. However, accumulating evidence derived from investigation of non-mammalian models that lack Edg receptors suggests that sphingosine-1-phosphate-like molecules can act through alternative mechanisms and thereby contribute to morphogenesis, development, reproduction and survival. This review provides an overview of sphingosine-1-phosphate metabolism, the isolation of genes in this pathway employing yeast genetics, the evidence for its influence on non-mammalian development, and the pertinence of these findings to human disease.     

Sphingosine-1-phosphate and lipid phosphohydrolases

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-1 (EDG-1) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S1P are low and tightly regulated in a spatial-temporal manner not only by sphingosine kinase (SPHK) but also by degradation catalyzed by S1P lyase, specific S1P phosphohydrolases, and by general lipid phosphate phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S1P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S1P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S1P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S1P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing sphingosine and ceramide, bioactive sphingolipids that often have opposing biological actions to S1P. By regulating the cellular ratio between ceramide/sphingosine and S1P, S1P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S1P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S1P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S1P and how turnover of S1P can regulate sphingolipid metabolites signaling.