Yeast sphingolipids: metabolism and biology

Sphingolipids have recently emerged as important bioactive molecules in addition to being critical structural components of cellular membranes. These molecules have been implicated in regulating cell growth, differentiation, angiogenesis, apoptosis, and senescene. To study sphingolipid mediated biology, it is necessary to investigate sphingolipid metabolism and its regulation. The yeast Saccharomyces cerevisiae has allowed such studies to take place as the sphingolipid metabolic and regulatory pathways appear conserved across species. Using yeast genetic approaches most enzymes of sphingolipid metabolism have been identified and cloned which has led to identification of their mammalian homologues. Many of the yeast enzymes are targets of fungal toxins thus underscoring the importance of this pathway in yeast cell regulation. This review focuses on the yeast sphingolipid metabolic pathway and its role in regulation of yeast biology. Implication of the insights gained from yeast to mammalian cell regulation are discussed.     

Autophagy paradox and ceramide

Sphingolipid molecules act as bioactive lipid messengers and exert their actions on the regulation of various cellular signaling pathways. Sphingolipids play essential roles in numerous cellular functions, including controlling cell inflammation, proliferation, death, migration, senescence, tumor metastasis and/or autophagy. Dysregulated sphingolipid metabolism has been also implicated in many human cancers. Macroautophagy (referred to here as autophagy) “self-eating” is characterized by nonselective sequestering of cytosolic materials by an isolation membrane, which can be either protective or lethal for cells. Ceramide (Cer), a central molecule of sphingolipid metabolism, has been extensively implicated in the control of autophagy. The increasing evidence suggests that Cer is highly involved in mediating two opposing autophagic pathways, which regulate either cell survival or death, which is referred here as autophagy paradox. However, the underlying mechanism that regulates the autophagy paradox remains unclear. Therefore, this review focuses on recent studies with regard to the regulation of autophagy by Cer and elucidates the roles and mechanisms of action of Cer in controlling autophagy paradox. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Sphingosine kinase,sphingosine-1-phosphate,and apoptosis

The sphingolipid metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) play an important role in the regulation of cell proliferation, survival, and cell death. Cer and Sph usually inhibit proliferation and promote apoptosis, while the further metabolite S1P stimulates growth and suppresses apoptosis. Because these metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines cell fate. The relevance of this "sphingolipid rheostat" and its role in regulating cell fate has been borne out by work in many labs using many different cell types and experimental manipulations. A central finding of these studies is that Sph kinase (SphK), the enzyme that phosphorylates Sph to form S1P, is a critical regulator of the sphingolipid rheostat, as it not only produces the pro-growth, anti-apoptotic messenger S1P, but also decreases levels of pro-apoptotic Cer and Sph. Given the role of the sphingolipid rheostat in regulating growth and apoptosis, it is not surprising that sphingolipid metabolism is often found to be disregulated in cancer, a disease characterized by enhanced cell growth, diminished cell death, or both. Anticancer therapeutics targeting SphK are potentially clinically relevant. Indeed, inhibition of SphK has been shown to suppress gastric tumor growth [Cancer Res. 51 (1991) 1613] and conversely, overexpression of SphK increases tumorigenicity [Curr. Biol. 10 (2000) 1527]. Moreover, S1P has also been shown to regulate angiogenesis, or new blood vessel formation [Cell 99 (1999) 301], which is critical for tumor progression. Furthermore, there is intriguing new evidence that S1P can act in an autocrine and/or paracrine fashion [Science 291 (2001) 1800] to regulate blood vessel formation [J. Clin. Invest. 106 (2000) 951]. Thus, SphK may not only protect tumors from apoptosis, it may also increase their vascularization, further enhancing growth. The cytoprotective effects of SphK/S1P may also be important for clinical benefit, as S1P has been shown to protect oocytes from radiation-induced cell death in vivo [Nat. Med. 6 (2000) 1109]. Here we review the growing literature on the regulation of SphK and the role of SphK and its product, S1P, in apoptosis.     

The control of the balance between ceramide and sphingosine-1-phosphate by sphingosine kinase: oxidative stress and the seesaw of cell survival and death

Sphingolipids are components of all eukaryotic cells that play important roles in a wide variety of biological processes. Ceramides and sphingosine-1-phosphate (S1P) are signaling molecules that regulate cell fate decisions in a wide array of species including yeast, plants, vertebrates, and invertebrates. Ceramides favor anti-proliferative and cell death pathways such as senescence and apoptosis, whereas S1P stimulates cell proliferation and survival pathways. The control of cell fate by these two interconvertible lipids has been called the sphingolipid rheostat or sphingolipid biostat. Sphingosine kinase, the enzyme that synthesizes S1P, is a crucial enzyme in regulation of the balance of these sphingolipids. Sphingosine kinase has been shown to play dynamic roles in the responses of cells to stress, leading to modulation of cell fate through a variety of signaling pathways impinging on the processes of cell proliferation, apoptosis, autophagy and senescence. This review summarizes the roles of sphingosine kinase signaling in these processes and the mechanisms mediating these responses. In addition, we discuss the evidence tying sphingosine kinase-mediated stress responses to the process of aging.     

Sphingosine kinase/sphingosine 1-phosphate signalling in central nervous system

Sphingolipids were once regarded as inert structural components of cell membranes. Now these metabolites are generally believed to be important bioactive molecules that control a wide repertoire of cellular processes such as proliferation and survival of cells. Along with these ubiquitous cell functions observed in many peripheral tissues sphingolipid metabolites, especially sphingosine 1-phosphate, exert important neuron-specific functions such as regulation of neurotransmitter release. This review summarizes physiological and pathological roles of sphingolipid metabolites emphasizing the role of sphingosine 1-phosphate in the central nervous system.     

Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death （PCD）. However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 （/br11-1）, which fails to generate reactive oxygen intermediates （ROIs）, is incapable of initiating PCD when the mutant is challenged by fumonisin B l （FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 （LCB 1） subunit of serine palmitoyltransferase （SPT）, which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Membranes in balance: mechanisms of sphingolipid homeostasis

Sphingolipids and their metabolites play key cellular roles both as structural components of membranes and as signaling molecules that mediate responses to physiologic cues and stresses. Despite progress during the last two decades in defining the enzymatic machinery responsible for synthesizing and degrading sphingolipids, comparatively little is known about how these enzymes are regulated to ensure sphingolipid homeostasis. Here, we review new insights into how cells sense and control sphingolipid biosynthesis and transport. We also discuss emerging evidence that sphingolipid metabolism is closely coordinated with that of sterols and glycerolipids and with other processes that occur in the secretory pathway. An improved understanding of sphingolipid homeostasis promises to shed light on basic processes in cell biology and disease, including how cells establish and maintain the complex membrane composition and architecture that is a defining feature of eukaryotic cell biology.     

Jasmonates modulate sphingolipid metabolism and accelerate cell death in the ceramide kinase mutant acd5

Sphingolipids are structural components of the lipid bilayer that acts as signaling molecules in many cellular processes, including cell death. Ceramides, key intermediates in sphingolipid metabolism, are phosphorylated by the ceramide kinase ACCELERATED CELL DEATH5 (ACD5). The loss of ACD5 function leads to ceramide accumulation and spontaneous cell death. Here, we report that the jasmonate (JA) pathway is activated in the Arabidopsis (Arabidopsis thaliana) acd5 mutant and that methyl JA treatment accelerates ceramide accumulation and cell death in acd5. Moreover, the double mutants of acd5 with jasmonate resistant1-1 and coronatine insensitive1-2 exhibited delayed cell death, suggesting that the JA pathway is involved in acd5-mediated cell death. Quantitative sphingolipid profiling of plants treated with methyl JA indicated that JAs influence sphingolipid metabolism by increasing the levels of ceramides and hydroxyceramides, but this pathway is dramatically attenuated by mutations affecting JA pathway proteins. Furthermore, we showed that JAs regulate the expression of genes encoding enzymes in ceramide metabolism. Together, our findings show that JAs accelerate cell death in acd5 mutants, possibly by modulating sphingolipid metabolism and increasing ceramide levels.     

Functions and metabolism of sphingolipids in Saccharomyces cerevisiae

We describe recent advances in understanding sphingolipid functions and metabolism in the baker’s yeast Saccharomyces cerevisiae. One milestone has been reached in yeast sphingolipid research with the complete or nearly complete identification of genes involved in sphingolipid synthesis and breakdown. Other advances include roles for sphingolipid long-chain bases as signaling molecules that regulate growth, responses to heat stress, cell wall synthesis and repair, endocytosis and dynamics of the actin cytoskeleton. We touch briefly on other sphingolipid functions so that readers unfamiliar with the field will gain a broader view of sphingolipid research. These functions include roles in protein trafficking/exocytosis, lipid rafts or microdomains, calcium homeostasis, longevity and cellular aging, nutrient uptake, cross-talk with other lipids and the interaction of sphingolipids and antifungal drugs.     

Molecular facets of sphingolipids: Mediators of diseases

Sphingolipids constitute a biologically active lipid class that is significantly important from both structural and regulatory aspects. The manipulation of sphingolipid metabolism is currently being studied as a novel strategy for cancer therapy. The basics of this therapeutic approach lie in the regulation property of sphingolipids on cellular processes, which are important in a cell's fate, such as cell proliferation, apoptosis, cell cycle arrest, senescence, and inflammation. Furthermore, the mutations in the enzymes catalyzing some specific reactions in the sphingolipid metabolism cause mortal lysosomal storage diseases like Fabry, Gaucher, Niemann-Pick, Farber, Krabbe, and Metachromatic Leukodystrophy. Therefore, the alteration of the sphingolipid metabolic pathway determines the choice between life and death. Understanding the sphingolipid metabolism and regulation is significant for the development of new therapeutic approaches for all sphingolipid-related diseases, as well as for cancer. An important feature of the sphingolipid metabolic pathway is the compartmentalization into endoplasmic reticulum, the Golgi apparatus, lysosome and plasma membrane, and this compartmentalization makes the transport of sphingolipids critical for proper functioning. This paper focuses on the structures, metabolic pathways, localization, transport mechanisms, and diseases of sphingolipids in Saccharomyces cerevisiae and humans, and provides the latest comprehensive information on sphingolipid research.     

High-content analysis of proapoptotic EphA4 dependence receptor functions using small-molecule libraries

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.     

Synthesis of 7 alpha- and 7 beta-spermidinylcholesterol, squalamine analogues

Stereoselective synthesis of squalamine dessulfates analogues, 7 alpha and 7 beta-N-[3N-(4-aminobutyl) aminopropyl]aminocholesterol are reported, using 7 alpha and 7 beta-aminocholesterol as a key intermediate. It's the first example in which the position of spermidine is modified at the steroid ring. These molecules showed a comparable antibacteria and fungi activities to squalamine. Then, they have a cytotoxic activity on a human non-small cell bronchopulmonary carcinoma line (NSCLC-N6).     

Sphingolipids as modulators of cancer cell death: Potential therapeutic targets

Through modifications in the fine membrane structure, cell-cell or cell-matrix interactions, and/or modulation of intracellular signaling pathways, sphingolipids can affect the tumorigenic potential of numerous cell types. Whereas ceramide and its metabolites have been described as regulators of cell growth and apoptosis, these lipids as well as other sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer treatments, and their susceptibility to non-apoptotic cell deaths. This review summarizes our current knowledge on the properties of sphingolipids in the regulation of cancer cell death and tumor development. It also provides an update on the potential perspectives of manipulating sphingolipid metabolism and using sphingolipid analogues in anticancer therapy.     

Sphingolipids as modulators of cancer cell death: potential therapeutic targets

Through modifications in the fine membrane structure, cell-cell or cell-matrix interactions, and/or modulation of intracellular signaling pathways, sphingolipids can affect the tumorigenic potential of numerous cell types. Whereas ceramide and its metabolites have been described as regulators of cell growth and apoptosis, these lipids as well as other sphingolipid molecules can modulate the ability of malignant cells to grow and resist anticancer treatments, and their susceptibility to non-apoptotic cell deaths. This review summarizes our current knowledge on the properties of sphingolipids in the regulation of cancer cell death and tumor development. It also provides an update on the potential perspectives of manipulating sphingolipid metabolism and using sphingolipid analogues in anticancer therapy.     

A house divided: ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

Ceramide synthase-dependent ceramide generation and programmed cell death: involvement of salvage pathway in regulating postmitochondrial events

The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.