scholarly journals Probing de novo sphingolipid metabolism in mammalian cells utilizing mass spectrometry

2018 ◽  
Vol 59 (6) ◽  
pp. 1046-1057 ◽  
Author(s):  
Justin M. Snider ◽  
Ashley J. Snider ◽  
Lina M. Obeid ◽  
Chiara Luberto ◽  
Yusuf A. Hannun
Keyword(s):  
Mammalian Cells ◽  
Metabolic Flux ◽  
De Novo ◽  
Bioactive Molecules ◽  
Sphingolipid Metabolism ◽  
Complete Analysis ◽  
Serine Palmitoyltransferase ◽  
One Step ◽  
Practical Tool

Sphingolipids constitute a dynamic metabolic network that interconnects several bioactive molecules, including ceramide (Cer), sphingosine (Sph), Sph 1-phosphate, and Cer 1-phosphate. The interconversion of these metabolites is controlled by a cohort of at least 40 enzymes, many of which respond to endogenous or exogenous stimuli. Typical probing of the sphingolipid pathway relies on sphingolipid mass levels or determination of the activity of individual enzymes. Either approach is unable to provide a complete analysis of flux through sphingolipid metabolism, which, given the interconnectivity of the sphingolipid pathway, is critical information to identify nodes of regulation. Here, we present a one-step in situ assay that comprehensively probes the flux through de novo sphingolipid synthesis, post serine palmitoyltransferase, by monitoring the incorporation and metabolism of the 17 carbon dihydrosphingosine precursor with LC/MS. Pulse labeling and analysis of precursor metabolism identified sequential well-defined phases of sphingolipid synthesis, corresponding to the activity of different enzymes in the pathway, further confirmed by the use of specific inhibitors and modulators of sphingolipid metabolism. This work establishes precursor pulse labeling as a practical tool for comprehensively studying metabolic flux through de novo sphingolipid synthesis and complex sphingolipid generation.

Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption

2001 ◽  
Vol 29 (6) ◽  
pp. 831-835 ◽  
Author(s):  
S. C. Linn ◽  
H. S. Kim ◽  
E. M. Keane ◽  
L. M. Andras ◽  
E. Wang ◽  
...  
Keyword(s):  
De Novo ◽  
Uv Light ◽  
Wide Spectrum ◽  
Sphingolipid Metabolism ◽  
Intrinsic Activity ◽  
Type I ◽  
Serine Palmitoyltransferase ◽  
Post Translational Modification ◽  
Sphingolipid Biosynthesis ◽  
Complex Sphingolipids

Complex sphingolipids are ‘built’ on highly bio-active backbones (sphingoid bases and ceramides) that can cause cell death when the amounts are elevated by turnover of complex sphingolipids, disruption of normal sphingolipid metabolism, or over-induction of sphingolipid biosynthesis de novo. Under normal conditions, it appears that the bioactive intermediates of this pathway (3-keto-sphinganine, sphinganine and ceramides) are kept at relatively low levels. Both the intrinsic activity of serine palmitoyltransferase (SPT) and the availability of its substrates (especially palmitoyl-CoA) can have toxic consequences for cells by increasing the production of cytotoxic intermediates. Recent work has also revealed that diverse agonists and stresses (cytokines, UV light, glucocorticoids, heat shock and toxic compounds) modulate SPT activity by induction of SPTLC2 gene transcription and/or post-translational modification. Mutation of the SPTLC1 component of SPT has also been shown to cause hereditary sensory neuropathy type I, possibly via aberrant oversynthesis of sphingolipids. Another key step of the pathway is the acylation of sphinganine (and sphingosine in the recycling pathway) by ceramide synthase, and up-regulation of this enzyme (or its inhibition to cause accumulation of sphinganine) can also be toxic for cells. Since it appears that most, if not all, tissues synthesize sphingolipids de novo, it may not be surprising that disruption of this pathway has been implicated in a wide spectrum of disease.

scholarly journals Toxoplasma gondiisalvages sphingolipids from the host Golgi through the rerouting of selected Rab vesicles to the parasitophorous vacuole

2013 ◽  
Vol 24 (12) ◽  
pp. 1974-1995 ◽  
Author(s):  
Julia D. Romano ◽  
Sabrina Sonda ◽  
Emily Bergbower ◽  
Maria Elisa Smith ◽  
Isabelle Coppens
Keyword(s):  
Toxoplasma Gondii ◽  
Mammalian Cells ◽  
De Novo ◽  
Parasitophorous Vacuole ◽  
Parasite Growth ◽  
Sphingolipid Metabolism ◽  
Obligate Intracellular ◽  
Membrane Bound ◽  
Parasite Replication

The obligate intracellular protozoan Toxoplasma gondii actively invades mammalian cells and, upon entry, forms its own membrane-bound compartment, named the parasitophorous vacuole (PV). Within the PV, the parasite replicates and scavenges nutrients, including lipids, from host organelles. Although T. gondii can synthesize sphingolipids de novo, it also scavenges these lipids from the host Golgi. How the parasite obtains sphingolipids from the Golgi remains unclear, as the PV avoids fusion with host organelles. In this study, we explore the host Golgi–PV interaction and evaluate the importance of host-derived sphingolipids for parasite growth. We demonstrate that the PV preferentially localizes near the host Golgi early during infection and remains closely associated with this organelle throughout infection. The parasite subverts the structure of the host Golgi, resulting in its fragmentation into numerous ministacks, which surround the PV, and hijacks host Golgi–derived vesicles within the PV. These vesicles, marked with Rab14, Rab30, or Rab43, colocalize with host-derived sphingolipids in the vacuolar space. Scavenged sphingolipids contribute to parasite replication since alterations in host sphingolipid metabolism are detrimental for the parasite's growth. Thus our results reveal that T. gondii relies on host-derived sphingolipids for its development and scavenges these lipids via Golgi-derived vesicles.

scholarly journals In Vivo Kinetics of mRNA Splicing and Transport in Mammalian Cells

2002 ◽  
Vol 22 (19) ◽  
pp. 6706-6718 ◽  
Author(s):  
A. Audibert ◽  
D. Weil ◽  
F. Dautry
Keyword(s):  
Mammalian Cells ◽  
Rna Degradation ◽  
Complete Analysis ◽  
First Order ◽  
Cellular Mrnas ◽  
Kinetics Of ◽  
Progressive Accumulation ◽  
Transcriptional Induction

ABSTRACT The kinetics of pre-mRNA processing in living cells is poorly known, preventing a detailed analysis of the regulation of these reactions. Using tetracycline-regulated promoters we performed, during a transcriptional induction, a complete analysis of the maturation of two cellular mRNAs, those for LT-α and β-globin. In both cases, splicing was appropriately described by first-order reactions with corresponding half-lives ranging between 0.4 and 7.5 min, depending on the intron. Transport also behaved as a first-order reaction during the early phase of β-globin expression, with a nuclear dwelling time of 4 min. At a later time, analysis was prevented by the progressive accumulation within the nucleus of mature mRNA not directly involved in export. Our results further establish for these genes that (i) splicing components are never limiting, even when expression is induced in naive cells, (ii) there is no significant RNA degradation during splicing and transport, and (iii) precursor-to-product ratios at steady state can be used for the determination of splicing rates. Finally, the comparison between the kinetics of splicing during transcriptional induction and during transcriptional shutoff reveals a novel coupling between transcription and splicing.

scholarly journals The concept of sphingolipid rheostat in skin: a driving force for new active ingredients in cosmetic applications

OCL ◽  
2018 ◽  
Vol 25 (5) ◽  
pp. D507 ◽  
Author(s):  
Iuliana Popa
Keyword(s):  
Fatty Acids ◽  
Polyunsaturated Fatty Acids ◽  
Stratum Corneum ◽  
Driving Force ◽  
Mammalian Cells ◽  
De Novo ◽  
Oral Treatment ◽  
Sphingolipid Metabolism ◽  
Intercellular Signaling ◽  
Sphingosine 1 Phosphate

Skin is a representative model of the complex metabolism that lipids may trigger. It is known that the biosynthesis of these lipids in mammalian cells generally ensures the cell membranes stability and participates to the signaling function. In the inner layers of the skin, the “de-novo” synthesis is the driving force ensuring proliferation, development and intercellular signaling. To promote stratum corneum formation, lipid catabolism leads to the renewal of ceramides, fatty acids and cholesterol that are responsible for the cohesion of the stratum corneum, its permeability, hydration, moisturization and signalling with the outer skin layers, appendages and inner layers secretion (cytokines, neuropeptides). Some actives applied in local treatments (i.e., peptides, n-3 polyunsaturated fatty acids (PUFA), ceramides, urea or an aqueous extract of Gromwell) and in oral treatment (i.e., sphingomyelin, n-3 polyunsaturated fatty acids (PUFA)) promote sphingosine 1-phosphate (S1P) production by the sphingolipid rheostat via triggering the salvage process along with autophagy and detoxification in aged skin. This review gives some basis for using the concept of sphingolipid metabolism rheostat in skin as the driving force for the development of new cosmetic actives ingredients or for repositioning the benefits of other actives for the skin.

scholarly journals Sphingosine-1-phosphate phosphohydrolase in regulation of sphingolipid metabolism and apoptosis

2002 ◽  
Vol 158 (6) ◽  
pp. 1039-1049 ◽  
Author(s):  
Hervé Le Stunff ◽  
Ismael Galve-Roperh ◽  
Courtney Peterson ◽  
Sheldon Milstien ◽  
Sarah Spiegel
Keyword(s):  
Cell Fate ◽  
De Novo ◽  
Fumonisin B1 ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Serine Palmitoyltransferase ◽  
Intracellular Membranes ◽  
Sphingosine 1 Phosphate ◽  
Ceramide Accumulation ◽  
Membrane Treatment

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein–coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.

scholarly journals A Dansyl-Modified Sphingosine Kinase Inhibitor Dpf-543 Enhanced de Novo Ceramide Generation

2021 ◽  
Author(s):  
Maftuna Shamshiddinova ◽  
Shokhid Gulyamov ◽  
Hee Jung Kim ◽  
Seo Hyeon Jung ◽  
Dong Jae Baek ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Sphingosine Kinase ◽  
Acid Sphingomyelinase ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Serine Palmitoyltransferase ◽  
Comparative Efficiency ◽  
Sphingosine 1 Phosphate

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P was linked to the pathological outcome with inflammation, cancer metastasis or angiogenesis etc. In this regard, the SPHK/S1P axis regulation has been a specific issue in anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhance the serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs which eventually induced an unusual environment of the high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase) that contributes a partial increase on Cers. Collectively, a dansyl-modified DPF-543 relatively en-hanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

scholarly journals A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation

2021 ◽  
Vol 22 (17) ◽  
pp. 9190
Author(s):  
Maftuna Shamshiddinova ◽  
Shokhid Gulyamov ◽  
Hee-Jung Kim ◽  
Seo-Hyeon Jung ◽  
Dong-Jae Baek ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Sphingosine Kinase ◽  
Acid Sphingomyelinase ◽  
Sphingolipid Metabolism ◽  
Ceramide Synthase ◽  
Serine Palmitoyltransferase ◽  
Comparative Efficiency ◽  
Sphingosine 1 Phosphate

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

scholarly journals Perinatal origins of chronic lung disease: mechanisms–prevention–therapy—sphingolipid metabolism and the genetic and perinatal origins of childhood asthma

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Emily Wasserman ◽  
Stefan Worgall
Keyword(s):  
Childhood Asthma ◽  
Early Life ◽  
De Novo ◽  
Association Studies ◽  
Critical Time ◽  
Bioactive Molecules ◽  
Sphingolipid Metabolism ◽  
Genome Wide Association Studies ◽  
Risk Alleles ◽  
Asthma Risk

AbstractChildhood asthma derives from complex host-environment interactions occurring in the perinatal and infant period, a critical time for lung development. Sphingolipids are bioactive molecules consistently implicated in the pathogenesis of childhood asthma. Genome wide association studies (GWAS) initially identified a link between alleles within the 17q21 asthma-susceptibility locus, childhood asthma, and overexpression of the ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3), an inhibitor of de novo sphingolipid synthesis. Subsequent studies of pediatric asthma offer strong evidence that these asthma-risk alleles correlate with early-life aberrancies of sphingolipid homeostasis and asthma. Relationships between sphingolipid metabolism and asthma-related risk factors, including maternal obesity and respiratory viral infections, are currently under investigation. This review will summarize how these perinatal and early life exposures can synergize with 17q21 asthma risk alleles to exacerbate disruptions of sphingolipid homeostasis and drive asthma pathogenesis.

scholarly journals Neutrophil elastase increases airway ceramide levels via upregulation of serine palmitoyltransferase

2018 ◽  
Vol 314 (1) ◽  
pp. L206-L214 ◽  
Author(s):  
Sophia Karandashova ◽  
Apparao B. Kummarapurugu ◽  
Shuo Zheng ◽  
Charles E. Chalfant ◽  
Judith A. Voynow
Keyword(s):  
Neutrophil Elastase ◽  
De Novo ◽  
Mass Spectrometry Analysis ◽  
Sphingolipid Metabolism ◽  
Cytokine Signaling ◽  
Serine Palmitoyltransferase ◽  
Protein Levels ◽  
Cell Counts ◽  
The Impact ◽  
Long Chain

Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high-mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long-chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long-chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by qRT-PCR and Western analyses, respectively. NE increased lung SPT long-chain subunit 2 (SPTLC2) protein levels but not SPTLC1 and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2 h before NE administration. Myriocin decreased BAL d18:1/22:0 and d18:1/24:1 ceramide, KC, and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.

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