Genes involved in sphingolipid metabolism

From LipidomicsWiki

Contents 1 Ceramide 2 Sphingomyelin 3 Sphingosine-1-phosphate 4 LipidomicNet Publications

Ceramide

Ceramide forms the backbone for all sphingolipids. De novo, biosynthesis of ceramide (which occurs in the endoplasmic reticulum) is initiated by the condensation of serine and palmitoyl- CoA (Fig. 31), a reaction, which is catalyzed by serine-palmitoyl CoA transferase (SPT). Mammalian SPT is a heterodimer of 35-kDa LCB1/SPT1 and 63-kDa LCB2/SPT2 subunits bound to the endoplasmic reticulum (Dickson et al. 2000, Hanada et al. 2003) and building the enzyme in a 1:1 ratio (Hanada et al. 2000). The long-chain base gene 1 (LCB1) was isolated primarily from a yeast mutant strain lacking SPT activity and requiring exogenous long-chain base for growth (Wells and Lester 1983). The protein encoded by this gene showed similarity at the amino acid level to the enzyme catalysing the serine-palmitoyl CoA transfer. Further experiments revealed that LCB1 and the homolog LCB2 are subunits of the SPT enzyme complex (Buede et al. 1991, Pinto et al. 1992).

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One of the more direct factors that affect SPT activity is the availability of both serine- and palmitoyl-CoA, and because SPT is highly selective for fatty acyl-CoA with 16 ± 1 carbon atoms, other fatty acids can be inhibitory in vivo, possibly by competing for the CoA pool (Merrill et al. 1988). SPT is the first and rate-limiting enzyme in the de novo pathway (Perry et al. 2000). The newly formed 3-ketosphinganine is subsequently reduced by NADPHdependent ketosphinganine reductase to dihydrosphingosine. The amide linkage of fatty acyl groups to dihydrosphingosine forms dihydroceramide. This reaction is catalyzed by dihydroceramide synthase, an enzyme, which acylates various long chain bases and utilizes a wide spectrum of fatty acyl-CoAs. Since there are four kinds of fatty acids in sphingolipids, there are not yet confirmed suspections of existence of more than one dihydroceramide synthase (Fig. 33). Untill now reasonable molecular and biochemical evidences are missing. However, It has been only very recently proven, that LASS5 (LAG1 longevity assurance homolog 5), is a bona fide (dihydro) ceramide synthase (Lahiri, Futerman, 2005).

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Ceramide is formed from dihydroceramide by the introduction of the trans-4, 5-double bond. The reaction is catalyzed by dihydroceramide desaturase originating from the cytosolic side of the endoplasmic reticulum. At the state of cellular homeostasis, once formed, ceramide is not accumulated but translocated to the Golgi where it serves as a starting point and metabolic precursor for all other sphingolipids such as sphingomyelin, glycosphingolipids of the lacto-, globo- and ganglioside series, and sulfatides. There are at least two pathways by which ceramide is transported to the Golgi: an ATP- and cytosol-dependent major pathway and an ATP- or cytosol-independent minor pathway (Hanada et al. 1998, Fukasawa et al. 1999, Yasuda et al. 2001). Recently, a gene called ceramide transfer protein (CERT) has been identified (Hanada et al. 2003). This gene is a splice variant of the Goodpasture antigen-binding protein (GPBPdelta26) and codes for a cytoplasmic protein with a lipid-transfer catalysing START domain, a phosphoinositde-binding pleckstrin homology (PH) domain and two phenylalanines (FF) in an acidic tract (FFAT) motif. The FFAT motif is found on diverse human lipid binding proteins (Table 8) and its combination with a PH domain which binds to the Golgi apparatus seems to predict the role of CERT (Loewen et al. 2003).

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It has been suggested that CERT acts as ceramide carrier that shuttles between ER and Golgi in a non-vesicular manner. CERT specifically extracts ceramide from phospholipid bilayer showing very little affinity to other related sphingolipids. As it has been observed, CERT is involved in transport of ceramide for sphingomyelin synthesis and not for glycosylceramide synthesis (Futerman, Riezman, 2005). Once delivered to the Golgi membrane, ceramide needs to translocate to the Gogi lumen for sphingomyelin synthesis, what occurs via “flip-flop” as a spontaneous transbilayer movement (Contreras et al 2005). Ceramide de novo synthesis is influenced by factors regulating the activity of serine palmitoyltransferase and it has been documented that LCB mRNA expression and SPT activity increase in response to various inflammatory and stress stimuli. Interleukin-1ß (Memon et al. 1998), UVB radiation (Farrell et al. 1998), fatty acids and cholesterol (Shimabukuro et al. 1998) stimulate the activity of SPT resulting in the cytotoxic accumulation of palmitate accompanied by the elevation of intracellular ceramide and induction of apoptosis. Ceramide is also generated at various subcellular locations by sphingomyelin hydrolysis (van Meer et al. 2000). This process of ceramide formation is a receptor-operated pathway (Hannun and Bell, 1989), which remained evolutionary, unchanged and is induced by different environmental and physiological stimuli. At the plasma membrane, ceramide is generated from sphingomyelin hydrolysis by acidic and/or neutral sphingomyelinases activated by diverse cytokines, death receptor ligands, differentiation factors or drugs (Levade and Jaffrezou, 1999). Stimulation of cells with 1,25-dihydroxy-vitamin D3, TNFα and CD40 ligand activates a neutral sphingomyelinase at the plasma membrane of cells, generating at the cytosolic side intracellular ceramide and phosphocholine (Geilen et al. 1996, Kolesnick and Kronke, 19984). Ceramide can also be formed through the action of an acidic sphingomyelinase that is activated by 1,2-diacylglycerol. Up till now, several sphingomyelinases have been characterized in mammalian tissues and they differ from each other by their location, Mg+2 and Zn+2 dependence and pH optimum (Levade and Jaffrezou, 1999, Hofmann et al. 2000). Ceramide catabolism starts with a ceramidase catalysing the cleavage of ceramide at the amide bond resulting in sphingosine and a free fatty acid. Three types of ceramidases have been described to date and classified according to pH optima as acid, neutral, or alkaline (Pettus et al. 2002). The deficiency of acidic ceramidase, which is located in lysosomes, provides the genetic backgroud for Farber’s disease (Sugita et al. 1972). Sphingosine released due to the action of ceramidase can be reacylated to ceramide or posphorylated by sphingosine kinase to sphingosine-1-phosphate. Ceramide can follow several metabolic pathways. It can be reutilized for the synthesis of sphingomyelin from phosphatidylcholine via choline phosphotransferase or may be phosphorylated to form ceramide-1-phosphate. The third pathway leads to glycosphingolipid synthesis. The sequential transfer of sugar residues from nucleotide sugars to ceramide results in the synthesis of glucosylceramide (GlcCer) at the cytosolic side of the Golgi, and in specialized cells, e.g. epithelial cells, ceramide serves for synthesis of galactosylceramide (GalCer) in the lumen of the ER (20). It has been proposed that GlcCer synthase in the cis- Golgi receives ceramide via the vesicular pathway whereas GlcCer synthase in the trans- Golgi receives ceramide from the ER via membrane contacts (Sadeghlar et al. 2000). The activity of galactosyltransferase 2 (GalT-2), which catalyses the synthesis of lactosylceramide, another glycosphingolipid, has been found increased in familial hypercholesterolemia and atherosclerosis (Chatterjee et al. 1982, Chatterjee et al. 1997). The increased levels of other glycosphingolipids in atherosclerotic lesions have been also reported (Mukhin et al. 1995). Ceramide-1-phosphate, an interconvertable metabolite of ceramide, is emerging as an important biological mediator. It is generated in the reaction of ceramide phosphorylation catalysed by ceramide kinase (CERK). As a bioactive lipid, ceramide-1-phosphate was demonstrated to play a role in mediation of inflammatory responses (Pettus et al, 2004). Ceramide shares some structural and functional similarities with the lipid A moiety of lipopolysaccharide (LPS), a lipid from the outer membrane of Gram-negative bacteria. The cellular responses to LPS include changes in shape, metabolism, and gene expression but also induction of a variety of biological effects that are involved in systemic inflammation and sepsis (Sweet et al. 1996). The initiation of these effects is due to the activation of monocyte/macrophages, leading to the secretion of proinflammatory cytokines such as TNFα, IL-1ß, IL-6 and IL-8. It has been suggested that LPS and ceramide recognize the same intracellular molecules (Wright and Kolesnick, 1995) and LPS was shown to mimick some ceramide properties. Ceramide-activated protein kinase (CAPK) appears to be a common target and its activation by both agonists leads to the activation of MAP kinase and the translocation of activated NF-κB. Recent data emphasize that although LPS and ceramide may share many signalling components, the signalling pathways are not identical (Wright and Kolesnick, 1995, MacKichan and DeFranco, 1999, Pfau et al. 1998)

Sphingomyelin

Sphingomyelin (SM) in mammalian cells has been found to colocalize with cholesterol mainly in the plasma membrane, in lysosomal and Golgi membranes, as well as in the polar surface of circulating lipoproteins (Merrill and Jones, 1990, Koval and Pagano, 1991). In plasma lipoproteins sphingomyelin is the second most abundant polar lipid after phosphatidylcholine. It interacts strongly with cholesterol. Due to its unique physicochemical properties, SM is enriched in specialized lipid microdomains such as rafts and caveolae. Turnover of sphingomyelin is a critical process and influences the maintenance of membrane integrity and synthesis of new membranes (van Blitterswijk et al. 2001). Sphingomyelin synthesis de novo is mediated by sphingomyelin synthase, which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide forming sphingomyelin and diacylglycerol (DAG) (Fig. 30) (Ullman, Radin, 1974). This enzyme is able also to catalyse the reverse reaction and generate phosphatidylcholine from sphingomyelin and DAG (Marggraf et al. 1981). Recently, a novel family of membrane proteins responsible for SM synthesis has been identified (Huitema et al. 2004). Two members of this family SMS1 and SMS2 fulfill the criteria for functional sphingomyelin synthase. SMS1 seems to represent the well-known Golgi-associated SM synthase while SMS2 is located to the plasma membrane. This fact needs further explanations since the presence of sphingomyelin synthase in the plasma membrane may impair ceramide signalling by converting ceramide back to sphingomyelin and generating diacylglycerol. Sphingomyelin synthesized in the Golgi is transported to the plasma membrane via vesicular transport (Diringer et al. 1972). The degradation of sphingomyelin leads to the release of ceramide and free sphingosine, known signalling molecules. However, aside from generation of second messengers, stimulation of sphingomyelin hydrolysis has been shown to induce cholesterol movement from the cell surface to intracellular membranes (Koval and Pagano 1991). This suggests that sphingomyelin metabolism plays an essential role not only in signalling pathways but also in the alteration of membrane physical properties. Sphingomyelinases, enzymes catalysing hydrolysis of sphingomyelin, have been classified into five categories based upon their pH optima, cellular localization, and cation dependence and are summerized in the table below

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The neutral membrane-bound Mg2+-independent sphingomyelinase (N-SMase) and the lysosomal acid sphingomyelinase (A-SMase) have been the best studied for their roles in ceramide generation. An increase in N-SMase activity, a corresponding decrease in SM, and an increase in ceramide have been demonstrated in response to TNFα, Fas ligand, 1α,25- dihydroxyvitamin D3, γ-interferon, chemotherapeutic agents, heat stress, ischemia/ reperfusion, and interleukin-1 (Liu et al. 1998, Tepper et al. 1995). In addition, both arachidonic acid and glutathione depletion have been shown to activate this enzyme (Liu et al. 1998, Jayadev et al. 1997, Levade et al.1994/1999). The acid sphingomyelinase gene codes for both lysosomal (cation-independent) and secretory sphingomyelinase (fully or partially dependent on Zn+² for enzymatic activity) (Schissel et al., 1998b). Abnormalities in sphingomyelin metabolism have been associated with atherosclerosis, cancer and genetic disorders (e.g. Niemann-Pick disease).

Sphingosine-1-phosphate

The sphingosine metabolite sphingosine-1-phosphate (S1P) is produced upon phosphorylation of sphingosine by sphingosine kinase during the breakdown of sphingomyelin (Fig. 30). Sphingosine kinase is a highly conserved enzyme, which is activated by several stimuli. S1P can be further degraded to phosphoethanolamine and hexadecanal by sphingosine-1-phosphate lyase. It can be also converted back to sphingosine via the action of sphingosine-1-phosphate phosphatase thus increasing the level of sphingosine in the cytosol and cell membranes. Elevated concentration of sphingosine is potentially toxic for the cells as sphingosine modulates critical molecules such as protein kinase C and ion-channels and disturbs cell membranes (Hannun et al. 2001). Sphingosine is also a substrate for sphingosine kinase to produce sphingosine-1-phosphate (Fig. 31). Extracellular S1P can be generated through two pathways: phosphorylation of extracellular sphingosine by sphingosine kinase released from endothelial cells or release of intracellular S1P from various cell types. S1P is present both in plasma and serum. Its concentration is regulated by the balance between synthesis through sphingosine kinase and degradation by phosphohydrolase or lyase. However, the concentration of S1P is relatively low, possibly reflecting the low expression of sphingosine kinase. Blood platelets are an exception as they release substantial amounts of S1P from α-granules where it is stored by platelet activation and have a robust sphingosine kinase activity and lack of S1P-lyse (Olivera and Spiegel, 2001). Sphingosine kinase is the enzyme that catalyses the phosphorylation of sphingosine and its two isoforms SPHK1 and SPHK2 have been identified as separate genes (Kohama et al.1998, Liu et al. 2000). These kinases have homology to the diacylglycerol kinase catalytic site and to calmodulin-binding motifs (Kohama et al 1998). SPHK1 is localized in the cytosol, however some growth factors can induce its translocation to the plasma membrane (Spiegel and Milstien, 2003 and lipid-protein interactions may play a major role in the regulation of membrane translocation and activation of SPHK1 (Stahelin at al, 2005). Growth and survival factors regulate the activity of SPHK1 and S1P synthesised in the presence of this isoform has been found to exert mitogenic and anti-apoptotic effects (Olivera et al, 1999). In contrast, little is known about SPHK2. Whereas SPHK1 is stimulated by several simuli, SPHK2 is activated specifically in the response to EGF (Olivera et al, 2005). Although, these both isoforms have similar amino acid sequence, they differ in their kinetic properties, developmental and tissue expression suggesting that they may have distinct physiological properties. Interestingly, SPHK2 contains a 9-amino acid motif that is similar to that present in BH3-only proteins, what would partially explain why SPKH2 supresses growth and induces apoptosis (Liu at al, 2003). Similarly to other BH3-only proteins, SPHK2 has been localized to the endoplasmic reticulum. Additionally, recently, it has been documented that this isoform is present in nuclei and due to its localization, is able to inhibit DNA synthesis (Igarashi et al. 2003). Interestingly, S1P activity is dependent on the isoform of the sphingosine kinase, which takes part in S1P biosynthesis. S1P is now recognized as a potent bioactive lipid with multiple functions and it is suggested to be involved in the regulation of cell shape changes, platelet aggregation, neurite retraction and smooth muscle cell chemotaxis (Spiegel, 1999). Various stimuli increase the intracellular concentration of S1P by activating sphingosine kinase. The list includes growth and survival factors, such as PDGF, serum, NGF as well as muscarinic acetylcholine agonists and TNFα. Free plasma levels of S1P are tightly regulated by protein binding to albumin and HDL. S1P export from mammalian cells is not yet well documented. It has been shown that mammalian cells of haematopoietic origin actively export/secrete S1P (Yatomi et al. 2001), the precise mechanism of this transport, however, is not known. S1P is considered as a unique lipid mediator acting both internally and externally. As a second messenger is S1P implicated in the regulation of Ca+2 mobilization, in cellular growth and proliferation and survival induced by platelet-derived growth factor, nerve growth factor and serum. Through its high affinity G protein-coupled receptors, S1P acts as an extracellular physiological mediator regulating heart rate (Sugiyama et al. 2000), coronary artery blood flow (Ohmori et al. 2003), blood pressure (Karliner 2002), endothelial integrity in the lung (English et al. 2002, Garcia et al. 2001) and most recently it has been shown to regulate the recirculation of lymphocytes (Rosen et al. 2003, Xie et al. 2003).
The structure of S1P is similar to that of the glycerophospholipid, lysophosphatidic acid (LPA), a platelet-derived mediator that acts through its G protein-coupled receptor to influence target cells. Lysophosphatidic acid is one of the simplest natural phospholipids (Moolenaar 1995). Its important role as a precursor of phospholipid synthesis in both eukaryotic and prokaryotic cells has been known since a long time, and recently it has emerged as an intracellular signalling molecule. The appearance and functional properties of both LPA and S1P suggest similar roles in development, wound healing and tissue regeneration. LPA and S1P also evoke cellular effector functions, which are dependent on cytoskeletal responses such as contraction, secretion, adhesion and chemotaxis. The list of biological responses to both molecules (Fig. 34) includes also cell aggregation and proliferation, hypertrophy, differentiation, migration, in neuroblastoma cells: cell rounding, neurite retraction and inhibition of melanoma and breast cancer cell motility (An et al. 1998).

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LipidomicNet Publications

• Brodesser S, et al., (2011) | Dihydroceramide desaturase inhibition by a cyclopropanated dihydroceramide analog in cultured keratinocytes. J Lipids Article ID 724015 (Partner 15A)