MRI Spectrum of Brain Involvement in Sphingosine-1-Phosphate Lyase Insufficiency Syndrome.

SGPL1 encodes sphingosine-1-phosphate lyase, the final enzyme of sphingolipid metabolism. In 2017, a condition featuring steroid-resistant nephrotic syndrome and/or adrenal insufficiency associated with pathogenic SGPL1 variants was reported. In addition to the main features of the disease, patients often exhibit a range of neurologic deficits. In a handful of cases, brain imaging results were described. However, high-quality imaging results and a systematic analysis of brain MR imaging findings associated with the condition are lacking. In this study, MR images from 4 new patients and additional published case reports were reviewed by a pediatric neuroradiologist. Analysis reveals recurring patterns of features in affected patients, including isolated callosal dysgenesis and prominent involvement of the globus pallidus, thalamus, and dentate nucleus, with progressive atrophy and worsening of brain lesions. MR imaging findings of abnormal deep gray nuclei, microcephaly, or callosal dysgenesis in an infant or young child exhibiting other typical clinical features of sphingosine-1-phosphate lyase insufficiency syndrome should trigger prompt genetic testing for SGPL1 mutations.

An LC/MS/MS method for quantitation of chemopreventive sphingadienes in food products and biological samples.

Colorectal cancer (CRC) is a leading cause of cancer mortality. Diet has a significant influence on colon cancer risk. Identifying chemopreventive agents, dietary constituents, practices and/or diet supplements that promote gut health and reduce the incidence of intestinal neoplasias and CRC could significantly impact public health. Sphingadienes (SDs) are dietary sphingolipids found in plant-based food products. SDs are cytotoxic to colon cancer cells and exhibit chemopreventive properties. The aim of the present study was to develop a sensitive and robust ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantifying SDs in food products and biological samples. The assay was linear over a concentration range of 80nM to 50µM and was sensitive to a detection limit of 3.3nM. Post-extraction stability was 100% at 24h. SD content in soy oils was approximately 10nM. SDs were detected transiently in the plasma of adult mice 10min after gavage delivery of a 25mg/kg bolus and declined to baseline by 1h. SD uptake in the gut was maximal in the duodenum and peaked 1h after gavage delivery. Disappearance of SDs in the lower gastrointestinal tract suggests either rapid metabolism to yet unidentified products or potentially luminal export.

Regulation of Immune Cell Migration by Sphingosine-1-Phosphate.

Sphingosine-1-phosphate [S1P] is a potent bioactive sphingolipid molecule. In response to a stimulus, S1P is produced intracellularly by the action of two sphingosine kinases, and then it is exported to the extracellular environment or acts as an intracellular second messenger. S1P binds to its cognate G-protein coupled receptors, which are known as S1P receptors. There are five S1P receptors that have been identified in vertebrates. By activating S1P receptors, S1P controls a variety of physiological and pathological processes including cell migration, angiogenesis, vascular maturation, inflammation, and invasion, metastasis, and chemoresistance in cancer. S1P has emerged as a critical regulator of leukocyte migration and plays a central role in lymphocyte egress from the thymus and secondary lymphoid organs. In the current review article, we summarize the current understanding of the emigration of lymphocytes and other leukocytes from bone marrow, thymus and secondary lymphoid organs to the circulation, as well as the clinical implications of modulating the activity of the major S1P receptor, S1PR1.

S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism.

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.

Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation.

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.

The dihydrosphingosine-1-phosphate phosphatases of Saccharomyces cerevisiae are important regulators of cell proliferation and heat stress responses.

We have identified YSR2 and YSR3 of Saccharomyces cerevisiae as genes encoding dihydrosphingosine-1-phosphate phophatases which are involved in regulation of sphingolipid metabolism [Mao, Wadleigh, Jenkins, Hannun and Obeid (1997) J. Biol. Chem. 272, 28690-28694]. In this study, we explored the physiological roles that these enzymes may have in S. cerevisiae. Deletion of either YSR2, YSR3 or both did not affect viability or growth rate of yeast cells. However, overexpression of YSR2 significantly prolonged the doubling time of cell growth, whereas overexpression of YSR3 affected cell growth only slightly. Cell cycle analysis suggested that overexpression of either YSR2 or, to a lesser extent, YSR3 caused cell cycle arrest at the G1 phase. Disruption of YSR2, but not YSR3, conferred increased thermotolerance. On the other hand, overexpression of either YSR2 or YSR3 diminished thermotolerance. Using labelled dihydrosphingosine and dihydrosphingosine-1-P (DHS-1-P), we found that overexpression of YSR2 significantly increased ceramide formation, whereas deletion of YSR2, YSR3, or both, accumulated DHS-1-P, and deletion of YSR2 decreased ceramide formation. Together, these results show that the phenotypes of YSR2 are associated with changes in endogenous levels of the different sphingolipids. Green fluorescent protein tagging showed that in the exponentially growing cells, YSR2 and YSR3 had the same cellular localization to endoplasmic reticulum. However, YSR2 and YSR3 differ in mRNA levels: YSR2 had significantly higher mRNA levels than YSR3. This discrepancy might result in the functional differences that these proteins exhibited. In addition, this study implicates sphingolipids and their metabolism in the regulation of growth and heat stress responses of the yeast S. cerevisiae.

The PLB2 gene of Saccharomyces cerevisiae confers resistance to lysophosphatidylcholine and encodes a phospholipase B/lysophospholipase.

The PLB1 gene of Saccharomyces cerevisiae encodes a protein that demonstrates phospholipase B, lysophospholipase, and transacylase activities. Several genes with significant homology to PLB1 exist in the S. cerevisiae genome, raising the possibility that other proteins may contribute to the total phospholipase B/lysophospholipase/transacylase activities of the cell. We report the isolation of a previously uncharacterized gene that is highly homologous to PLB1 and that, when overexpressed, confers resistance to 1-palmitoyllysophosphatidylcholine. This gene, which is located adjacent to the PLB1 gene on the left arm of chromosome XIII and which we refer to as PLB2, encodes a phospholipase B/lysophospholipase. Unlike PLB1, this gene product does not contain significant transacylase activity. The PLB2 gene product shows lysophospholipase activity toward lysophosphatidylcholine, lysophosphatidylserine, and lysophosphatidylethanolamine. Whereas deletion of either PLB1 or PLB2 resulted in the loss of 80% of cellular lysophospholipase activity, a plb1/plb2 double deletion mutant is completely devoid of lysophospholipase activity toward the preferred substrate lysophosphatidylcholine. Overexpression of PLB2 was associated with an increase in total cellular phospholipase B/lysophospholipase activity, as well as the appearance of significant lysophospholipase activity in the medium. Moreover, overexpression of PLB2 was associated with saturation at a higher cell density, and an increase in total cellular phospholipid content, but no change in phospholipid composition or fatty acid incorporation into cellular lipids. Deletion of PLB2 was not lethal and did not result in alteration of membrane phospholipid composition or content. PLB2 gene expression was found to be maximal during exponential growth conditions and was decreased in late phase, in a manner similar to other genes involved in phospholipid metabolism.

YAP1 confers resistance to the fatty acid synthase inhibitor cerulenin through the transporter Flr1p in Saccharomyces cerevisiae.

In this study, we utilized a genetic approach to identify genes which render yeast cells resistant to cerulenin (Cer), a potent and noncompetitive inhibitor of fatty acid synthase (FAS). Overexpression of the yeast transcription factor Yap1p was found to confer Cer resistance (CerR). This resistance was shown to be less pronounced in a strain deleted for YCF1, a multidrug resistance ABC transporter, supporting previous observations that implicated YCF1 in mediating CerR. However, isolation of YAP1 as a high-copy CerR gene in a ycf1delta strain suggested that YAP1-induced CerR was mediated by additional downstream effectors. Overexpression of neither glutathione reductase nor a predicted aryl alcohol dehydrogenase (the products of two YAP1-regulated genes involved in detoxification) conferred CerR. Overexpression of ATR1, another YAP1-regulated gene previously implicated in conferring resistance to a number of cytotoxic drugs, was also incapable of making cells resistant to Cer. In contrast, overexpression of Flr1p, a yeast transporter of the major facilitator superfamily which is also under the control of YAP1, was sufficient to confer CerR in an otherwise wild-type background. Moreover, CerR was markedly diminished in a strain deleted for FLR1. These findings implicate members of both of the transporter superfamilies involved in multiple drug resistance (MDR) in the acquisition of CerR in yeast. Furthermore, our studies indicate that yeast may be a useful model system in which to investigate the role of FAS in cancer biology and the effects of Cer on eukaryotic cell growth.

The DPL1 gene is involved in mediating the response to nutrient deprivation in Saccharomyces 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, bond by sphingosine phosphate lyase. The recent identification of the first dihydrosphingosine/sphingosine phosphate lyase gene in Saccharomyces cerevisiae establishes that phosphorylated sphingoid base metabolism is conserved throughout evolution. The dpl1delta 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 G1 phase of the cell cycle. Several genes were found to be overexpressed or prematurely expressed during nutrient deprivation in the dpl1delta strain, including glucose-repressible genes and G1 cyclins. These studies implicate a role for DPL1 and phosphorylated sphingoid bases in the regulation of global responses to nutrient deprivation in yeast.

Characterization of sphingosine kinase (SK) activity in Saccharomyces cerevisiae and isolation of SK-deficient mutants.

Sphingosine kinase (SK) catalyses the phosphorylation of sphingosine to generate sphingosine 1-phosphate, which is a second messenger involved in the proliferative responses of mammalian cells. Although the yeast Saccharomyces cerevisiae has similar phosphorylated sphingoid bases which appear to be involved in growth regulation and the response to stress, SK activity had not been previously demonstrated in yeast. In this study, an in vitro system was set up to characterize yeast SK activity. Activity was detected in the cytosol at neutral pH and 37 degreesC. Yeast SK phosphorylated the sphingoid bases sphingosine, dihydrosphingosine and phytosphingosine. (d,l)-threo-dihydrosphingosine, an inhibitor of mammalian SK, did not inhibit the yeast enzyme. Unique properties of yeast SK were an optimal temperature of 43 degreesC, and in vivo activation during nutrient deprivation. Spontaneous mutants with diminished SK activity were isolated utilizing a screen for resistance to sphingosine in a sphingosine-phosphate-lyase deletion background. Abnormal growth and heat sensitivity were observed in these mutants. These findings suggest that SK may function as a stress-response protein in yeast.

Identification of the first mammalian sphingosine phosphate lyase gene and its functional expression in yeast.

Sphingosine-1-phosphate (S-1-P) has been shown to participate in the proliferative signal transduction pathways of mammalian cells. Sphingosine-1-phosphate lyase (SPL) catalyzes the breakdown of S-1-P. Using the C. elegans SPL nucleotide sequence, we identified a mouse EST as a putative candidate for the homologous gene encoding this enzyme. Sequencing of the mouse EST revealed an open reading frame of 1707 nucleotides. This putative mouse SPL gene is 62% similar and 39% identical to the C. elegans SPL gene and 59% homologous and 39.6% identical to the yeast SPL gene. Expression of the mouse SPL gene in a yeast strain-delta bst1, which carries a deletion of the SPL gene and is hypersensitive to sphingosine, restored a sphingosine-resistant phenotype, suggesting this mouse gene can functionally complement the yeast defect when expressed. In vitro enzyme assay using extracts from these sphingosine-resistant transformants confirmed the SPL activities encoded by this mouse cDNA clone. Northern analysis indicated the mouse SPL gene is expressed at various levels in different tissues. Chromosomal localization mapped this SPL gene to Chromosome 10 at 32 cM. Here, we report the identification of the first mammalian sphingosine phosphate lyase gene.

The BST1 gene of Saccharomyces cerevisiae is the sphingosine-1-phosphate lyase.

Sphingolipids elicit a wide variety of eukaryotic cellular responses, most involving regulation of cell growth, differentiation, and apoptosis. Sphingosine 1-phosphate, a sphingolipid catabolite, is mitogenic in fibroblasts and inhibits the chemotactic mobility and invasiveness of human tumor cells. Sphingosine 1-phosphate degradation requires cleavage at the C2-3 carbon bond by sphingosine phosphate lyase. A yeast genetic approach was used to clone the first sphingosine phosphate lyase gene, BST1. BST1 overexpression conferred resistance to sphingosine in yeast. BST1 deletion produced sensitivity to exogenous D-erythro-sphingosine and phytosphingosine and intracellular accumulation of sphingosine 1-phosphate upon exposure to exogenous sphingosine. This study confirms that sphingoid base metabolism is similar in all eukaryotes and suggests that yeast genetics may be useful in the isolation and identification of other genes involved in sphingolipid signaling and metabolism.

Ceramide: an intracellular mediator of apoptosis and growth suppression.

Ceramide is an endogenous lipid molecule generated by hydrolysis of membrane sphingomyelin, in response to cellular stimulation by hormones and cytokines. Ceramide appears to have a role in mediating biological responses in a wide variety of cell types. These responses are generally considered anti-proliferative, but endpoints are varied and include differentiation, growth inhibition, senescence and apoptotic cell death. Mechanisms for ceramide action involve regulation of protein phosphorylation via stimulation of a serine/threonine protein phosphatase, a proline-directed kinase and possibly other direct and/or indirect targets.