The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Sarah Spiegel - One of the best experts on this subject based on the ideXlab platform.
-
sphingosine and sphingosine kinase 1 involvement in endocytic membrane trafficking
Journal of Biological Chemistry, 2017Co-Authors: Santiago Lima, Sheldon Milstien, Sarah SpiegelAbstract:The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.
-
Recycling of sphingosine is regulated by the concerted actions of sphingosine-1-phosphate phosphohydrolase 1 and sphingosine kinase 2.
The Journal of biological chemistry, 2007Co-Authors: Hervé Le Stunff, Sheldon Milstien, Michael Maceyka, Paola Giussani, Sandrine Lépine, Sarah SpiegelAbstract:In yeast, the long-chain sphingoid base phosphate phosphohydrolase Lcb3p is required for efficient ceramide synthesis from exogenous sphingoid bases. Similarly, in this study, we found that incorporation of exogenous sphingosine into ceramide in mammalian cells was regulated by the homologue of Lcb3p, sphingosine-1-phosphate phosphohydrolase 1 (SPP-1), an endoplasmic reticulum resident protein. Sphingosine incorporation into endogenous long-chain ceramides was increased by SPP-1 overexpression, whereas recycling of C(6)-ceramide into long-chain ceramides was not altered. The increase in ceramide was inhibited by fumonisin B(1), an inhibitor of ceramide synthase, but not by ISP-1, an inhibitor of serine palmitoyltransferase, the rate-limiting step in the de novo biosynthesis of ceramide. Mass spectrometry analysis revealed that SPP-1 expression increased the incorporation of sphingosine into all ceramide acyl chain species, particularly enhancing C16:0, C18:0, and C20:0 long-chain ceramides. The increased recycling of sphingosine into ceramide was accompanied by increased hexosylceramides and, to a lesser extent, sphingomyelins. Sphingosine kinase 2, but not sphingosine kinase 1, acted in concert with SPP-1 to regulate recycling of sphingosine into ceramide. Collectively, our results suggest that an evolutionarily conserved cycle of phosphorylation-dephosphorylation regulates recycling and salvage of sphingosine to ceramide and more complex sphingolipids.
-
Functional characterization of human sphingosine kinase-1.
FEBS letters, 2000Co-Authors: Victor E. Nava, Samantha Poulton, Emanuela Lacana, Hong Liu, Masako Sugiura, Keita Kono, Sheldon Milstien, Takafumi Kohama, Sarah SpiegelAbstract:Sphingosine kinase catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (SPP), a novel lipid mediator with both intra- and extracellular functions. Based on sequence identity to murine sphingosine kinase (mSPHK1a), we cloned and characterized the first human sphingosine kinase (hSPHK1). The open reading frame of hSPHK1 encodes a 384 amino acid protein with 85% identity and 92% similarity to mSPHK1a at the amino acid level. Similar to mSPHK1a, when HEK293 cells were transfected with hSPHK1, there were marked increases in sphingosine kinase activity resulting in elevated SPP levels. hSPHK1 also specifically phosphorylated D-erythro-sphingosine and to a lesser extent sphinganine, but not other lipids, such as D,L-threo-dihydrosphingosine, N,N-dimethylsphingosine, diacylglycerol, ceramide, or phosphatidylinositol. Northern analysis revealed that hSPHK1 was widely expressed with highest levels in adult liver, kidney, heart and skeletal muscle. Thus, hSPHK1 belongs to a highly conserved unique lipid kinase family that regulates diverse biological functions.
-
structure activity relationship of short chain sphingoid bases as inhibitors of sphingosine kinase
Bioorganic & Medicinal Chemistry Letters, 1999Co-Authors: Steven De Jonghe, Samantha Poulton, Chris Hendrix, Ilse Van Overmeire, Serge Van Calenbergh, Roger Busson, Piet Herdewijn, Sarah Spiegel, Denis De KeukeleireAbstract:Short-chain sphinganine analogues 8, 9, 18, and 19, as well as 3-fluoro-sphingosine analogues 25 and 26 were synthesized. Their potential as sphingosine kinase inhibitors was investigated, in combination with previously synthesized sphingosine and fluorinated sphinganine analogues.
-
Molecular Cloning and Functional Characterization of Murine Sphingosine Kinase
The Journal of biological chemistry, 1998Co-Authors: Takafumi Kohama, Lisa C Edsall, M. Marek Nagiec, Robert B. Dickson, Sarah SpiegelAbstract:Abstract Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison withSaccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylatedd-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide,d,l-threo-dihydrosphingosine orN,N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.
Edward H. Schuchman - One of the best experts on this subject based on the ideXlab platform.
-
sphingoid long chain bases prevent lung infection by pseudomonas aeruginosa
Embo Molecular Medicine, 2014Co-Authors: Yael Pewznerjung, Shaghayegh Tavakoli Tabazavareh, Lukasz Japtok, Tammar Joseph, Burkhard Tuemmler, Heike Grassmé, Katrin Anne Becker, Jörg Steinmann, S. Lang, Edward H. SchuchmanAbstract:Cystic fibrosis patients and patients with chronic obstructive pulmonary disease, trauma, burn wound, or patients requiring ventilation are susceptible to severe pulmonary infection by Pseudomonas aeruginosa. Physiological innate defense mechanisms against this pathogen, and their alterations in lung diseases, are for the most part unknown. We now demonstrate a role for the sphingoid long chain base, sphingosine, in determining susceptibility to lung infection by P. aeruginosa. Tracheal and bronchial sphingosine levels were significantly reduced in tissues from cystic fibrosis patients and from cystic fibrosis mouse models due to reduced activity of acid ceramidase, which generates sphingosine from ceramide. Inhalation of mice with sphingosine, with a sphingosine analog, FTY720, or with acid ceramidase rescued susceptible mice from infection. Our data suggest that luminal sphingosine in tracheal and bronchial epithelial cells prevents pulmonary P. aeruginosa infection in normal individuals, paving the way for novel therapeutic paradigms based on inhalation of acid ceramidase or of sphingoid long chain bases in lung infection.
-
simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene 2 3 dicarboxyaldehyde derivatization after hydrolysis with ceramidase
Analytical Biochemistry, 2005Co-Authors: Arie Dagan, Shimon Gatt, Edward H. SchuchmanAbstract:Abstract Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine–NDA and ceramidase-derived sphingosine–NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.
Douglas L Mann - One of the best experts on this subject based on the ideXlab platform.
-
sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor alpha in the adult mammalian cardiac myocyte
Journal of Biological Chemistry, 1997Co-Authors: Hakan Oral, Gerald W Dorn, Douglas L MannAbstract:Abstract To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-α (TNF-α), we examined sphingosine levels in diluent and TNF-α-stimulated cardiac myocytes. TNF-α stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-α-stimulated cardiac myocytes showed that TNF-α stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-α on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-α in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-α in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-α. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-α in isolated cardiac myocytes.
-
sphingosine mediates the immediate negative inotropic effects of tumor necrosis factor α in the adult mammalian cardiac myocyte
Journal of Biological Chemistry, 1997Co-Authors: Hakan Oral, Gerald W Dorn, Douglas L MannAbstract:Abstract To determine whether activation of the neutral sphingomyelinase pathway was responsible for the immediate (<30 min) negative inotropic effects of tumor necrosis factor-α (TNF-α), we examined sphingosine levels in diluent and TNF-α-stimulated cardiac myocytes. TNF-α stimulation of adult feline cardiac myocytes provoked a rapid (<15 min) increase in the hydrolysis of [14C]sphingomyelin in cell-free extracts, as well as an increase in ceramide mass, consistent with cytokine-induced activation of the neutral sphingomyelinase pathway. High performance liquid chromatographic analysis of lipid extracts from TNF-α-stimulated cardiac myocytes showed that TNF-α stimulation produced a rapid (<30 min) increase in free sphingosine levels. Moreover, exogenous D-sphingosine mimicked the effects of TNF-α on intracellular calcium homeostasis, as well as the negative inotropic effects of TNF-α in isolated contracting myocytes; time course studies showed that exogenous D-sphingosine produced abnormalities in cell shortening that were maximal at 5 min. Finally, blocking sphingosine production using an inhibitor of ceramidase, n-oleoylethanolamine, completely abrogated the negative inotropic effects of TNF-α in isolated contracting cardiac myocytes. Additional studies employing biologically active ceramide analogs and sphingosine 1-phosphate suggested that neither the immediate precursor of sphingosine nor the immediate metabolite of sphingosine, respectively, were likely to be responsible for the immediate negative inotropic effects of TNF-α. Thus, these studies suggest that sphingosine mediates the immediate negative inotropic effects of TNF-α in isolated cardiac myocytes.
Alfred H. Merrill - One of the best experts on this subject based on the ideXlab platform.
-
thematic review series sphingolipids biodiversity of sphingoid bases Sphingosines and related amino alcohols
Journal of Lipid Research, 2008Co-Authors: Sarah T Pruett, Anatoliy S Bushnev, Kerri Hagedorn, Madhura Adiga, Cameron M Sullards, Christopher A. Haynes, Dennis C Liotta, Alfred H. MerrillAbstract:Sphingolipids are composed of a structurally related family of backbones termed sphingoid bases, which are sometimes referred to as “long-chain bases” or “Sphingosines” after the original designation of the first isolated compound from brain as “sphingosin” by J. L. W. Thudichum in 1884 (1). Today, the term “sphingosine” is usually reserved for (2S,3R,4E)-2-aminooctadec-4-ene-1,3-diol (compound 6 in Fig. 1), which has important biological functions in cell signaling per se (2, 3) as well as after derivatization to the 1-phosphate (compound 9 in Fig. 1) (2, 4, 5), N-acylated metabolites (ceramides; compound 4 in Fig. 1) (2, 6, 7), and more complex phosphosphingolipids and glycosphingolipids with head groups attached to the hydroxyl on carbon 1. The structural diversity of the latter compounds is widely appreciated, with hundreds of head group variants for mammals alone, as was reviewed recently (8, 9) and addressed at a number of “omics” web sites, such as SphinGOMAP (www.sphingomap.org), the Japanese Lipid Bank (http://www.lipidbank.jp) and Glycoforum (http://www.glycoforum.gr.jp/), the Lipid Maps Consortium (www.lipidmaps.org), the Consortium for Functional Glycomics (http://www.functionalglycomics.org/fg/), and the Complex Carbohydrate Research Center at the University of Georgia (http://www.ccrc.uga.edu/~moremen/glycomics/). Fig. 1. Biosynthesis and turnover of the three major categories of sphingoid bases in mammalian cells. DHR, dihydroceramide; SPT, serine palmitoyltransferase. Somewhat less well appreciated is that sphingoid bases also display considerable structural diversity, as was elegantly reviewed by K. A. Karlsson almost 40 years ago (10, 11). In remembrance of Herbert E. Carter, who first elucidated the structure of sphingosine 6 and dihydrosphingosine 2 (12) and “proposed to designate those lipides derived from sphingosine as sphingolipides” (13), this thematic review summarizes and updates points made previously regarding the structural diversity of sphingoid bases (10, 11) and expands the topic to include sphingoid bases and sphingoid base-like compounds that have been discovered in intervening years. In addition to being fascinating for their biodiversity, some of these naturally occurring compounds (and synthetic analogs) are promising drug leads, while others cause disease, as exemplified by the fumonisins (14).
-
sphingolipid metabolism and cell growth regulation
The FASEB Journal, 1996Co-Authors: Sarah Spiegel, Alfred H. MerrillAbstract:Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. The current paradigm for their action is that complex sphingolipids such as gangliosides interact with growth factor receptors, the extracellular matrix, and neighboring cells, whereas the backbones--sphingosine and other long-chain or "sphingoid" bases, ceramides, and sphingosine 1-phosphate--activate or inhibit protein kinases and phosphatases, ion transporters, and other regulatory machinery. Tumor necrosis factor-alpha, interleukin 1beta, and nerve growth factor, for example, induce sphingomyelin hydrolysis to ceramide. Other agonists, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine and activate sphingosine kinase to form sphingosine 1-phosphate. These metabolites either stimulate or inhibit growth and may be cytotoxic (in some cases via induction of apoptosis), depending on which products are formed (or added exogenously), the cellular levels (...
Olivier Cuvillier - One of the best experts on this subject based on the ideXlab platform.
-
Biochemical methods for quantifying sphingolipids: ceramide, sphingosine, sphingosine kinase-1 activity, and sphingosine-1-phosphate.
Methods in molecular biology (Clifton N.J.), 2012Co-Authors: Leyre Brizuela, Olivier CuvillierAbstract:Sphingolipids (ceramide, sphingosine, and sphingosine-1-phosphate) are bioactive lipids with important biological functions in proliferation, apoptosis, angiogenesis, and inflammation. Herein, we describe easy and rapid biochemical methods with the use of radiolabeled molecules ((3)H, (32)P) for their mass determination. Quantitation of sphingosine kinase-1 activity, the most studied isoform, is also included.
-
Overcoming MDR-associated chemoresistance in HL-60 acute myeloid leukemia cells by targeting sphingosine kinase-1.
Leukemia, 2005Co-Authors: Elisabeth Bonhoure, Olivier Cuvillier, Dimitri Pchejetski, Nassera Aouali, Hamid Morjani, Thierry Levade, T KohamaAbstract:We examined the involvement of sphingosine kinase-1, a critical regulator of the sphingolipid balance, in susceptibility to antineoplastic agents of either sensitive or multidrug-resistant acute myeloid leukemia cells. Contrary to parental HL-60 cells, doxorubicin and etoposide failed to trigger apoptosis in chemoresistant HL-60/Doxo and HL-60NP16 cells overexpressing MRP1 and MDR1, respectively. Chemosensitive HL-60 cells displayed sphingosine kinase-1 inhibition coupled with ceramide generation. In contrast, chemoresistant HL-60/ Doxo and HL-60/VP16 had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment. Enforced expression of sphingosine kinase-1 in chemosensitive HL-60 cells resulted in marked inhibition of apoptosis that was mediated by blockade of mitochondrial cytochrome c efflux hence suggesting a control of apoptosis at the pre-mitochondrial level. Incubation with cell-permeable ceramide of chemoresistant cells led to a sphingosine kinase-1 inhibition and apoptosis both prevented by sphingosine kinase-1 over-expression. Furthermore, F-12509a, a new sphingosine kinase inhibitor, led to ceramide accumulation, decrease in sphingosine 1-phosphate content and caused apoptosis equally in chemosensitive and chemoresistant cell lines that is inhibited by adding sphingosine 1-phosphate or overexpressing sphingosine kinase-1. F-12509a induced classical apoptosis hallmarks namely nuclear fragmentation, caspase-3 cleavage as well as downregulation of antiapoptotic XIAP, and release of cytochrome c and SMAC/Diablo.
-
Suppression of ceramide-mediated programmed cell death by sphingosine-1-phosphate.
Nature, 1996Co-Authors: Olivier Cuvillier, Philip G. Vanek, Grisha Pirianov, Omar A Coso, J. Silvio Gutkind, Burkhard Kleuser, Sarah SpiegelAbstract:Ceramide is an important regulatory participant of programmed cell death (apoptosis) induced by tumour-necrosis factor (TNF)-alpha and Fas ligand, members of the TNF superfamily. Conversely, sphingosine and sphingosine-1-phosphate, which are metabolites of ceramide, induce mitogenesis and have been implicated as second messengers in cellular proliferation induced by platelet-derived growth factor and serum. Here we report that sphingosine-1-phosphate prevents the appearance of the key features of apoptosis, namely intranucleosomal DNA fragmentation and morphological changes, which result from increased concentrations of ceramide. Furthermore, inhibition of ceramide-mediated apoptosis by activation of protein kinase C results from stimulation of sphingosine kinase and the concomitant increase in intracellular sphingosine-1-phosphate. Finally sphingosine-1-phosphate not only stimulates the extracellular signal-regulated kinase (ERK) pathway, it counteracts the ceramide-induced activation of stress-activated protein kinase (SAPK/JNK). Thus, the balance between the intracellular levels of ceramide and sphingosine-1-phosphate and their regulatory effects on different family members of mitogen-activated protein kinases determines the fate of the cell.
