Ceramide synthases at the centre of sphingolipid metabolism and biology

Abstract

Sphingolipid metabolism in metazoan cells consists of a complex interconnected web of numerous enzymes, metabolites and modes of regulation. At the centre of sphingolipid metabolism reside CerSs (ceramide synthases), a group of enzymes that catalyse the formation of ceramides from sphingoid base and acyl-CoA substrates. From a metabolic perspective, these enzymes occupy a unique niche in that they simultaneously regulate de novo sphingolipid synthesis and the recycling of free sphingosine produced from the degradation of pre-formed sphingolipids (salvage pathway). Six mammalian CerSs (CerS1-CerS6) have been identified. Unique characteristics have been described for each of these enzymes, but perhaps the most notable is the ability of individual CerS isoforms to produce ceramides with characteristic acyl-chain distributions. Through this control of acyl-chain length and perhaps in a compartment-specific manner, CerSs appear to regulate multiple aspects of sphingolipid-mediated cell and organismal biology. In the present review, we discuss the function of CerSs as critical regulators of sphingolipid metabolism, highlight their unique characteristics and explore the emerging roles of CerSs in regulating programmed cell death, cancer and many other aspects of biology.

Figure 1. A ‘simplified’ view of sphingolipid metabolism

De novo sphingolipid biosynthesis begins with the condensation of serine and palmitoyl-CoA catalysed by the serine palmitoyltransferase complex (SPT). Its product, 3-ketosphinganine (3-KSph) is enzymatically reduced to dihydrosphingosine (dHSph) by 3-ketosphinganine reductase (3-KR). dHSph is the substrate of CerSs, which produce a wide variety of dihydroceramides (dHCer) of various acyl-chain length (e.g. C_14:0-dHCer to C_26:0-dHCer). dHCer can be reduced to form ceramide (Cer) by dihydroceramide desaturase (DES). Ceramide can be metabolized to ceramide phosphoethanolamine (CPE) or galactosylceramide (GalCer) in the ER or transported to the Golgi via the ceramide transport protein (CERT) or through vesicular trafficking. In the Golgi, complex sphingolipids such as sphingomyelin (SM) and glycosphingolipids (GSLs) are synthesized via sphingomyelin synthases (SMS1 or SMS2), SMS-related protein (SMSr) or glycosphingolipids synthases (GCS) respectively. Ceramide may also be metabolized to ceramide 1-phosphate (C1P) via ceramide kinase (CERK) at the Golgi or plasma membrane (PM). The degradation of complex sphingolipids occurs through multiple pathways. At the outer leaflet of the plasma membrane, ceramide can be produced from sphingomyelin via secretory SMase (sSMase) and produce sphingosine via neutral CDase (nCDase). Sphingosine (Sph) can then be metabolized into S1P via SK1. Complex sphingolipids can be degraded to ceramide via the endolysosomal pathway, which contains aSMase and glycosidases (GCase). Ceramide is subsequently hydrolysed to sphingosine via acid CDase (aCDase). Free sphingosine can either be converted into S1P by SK1 or SK2 or re-synthesized into ceramide via CerS. This latter pathway is called the sphingosine salvage or recycling pathway. S1P can be hydrolysed back to sphingosine via S1P phosphatase (SPP) or degraded by S1P lyase (SPL) to ethanolamine 1-phosphate (EA1P) and hexadecenal.

Figure 2. Tissue distribution of mouse CerSs

The distribution of CerS1–CerS6 in mouse tissues based on data reported by Laviad et al. [40].

Figure 3. CerSs as regulators of de novo and salvage production of multiple ceramide species

The major contributions of each CerS isoform to de novo and salvage synthesis of different ceramide species are depicted. Cer, ceramide; dHCer, dihydroceramide; dHSph, dihydrosphingosine; Sph, sphingosine.

Figure 4. Inhibitors of CerS activity

Known inhibitors of CerS include sphingosine (A) analogues such as fumonisins (B), a toxin from Alternaria alternata f. sp. lycopersici (AAL toxin) (C) and australifungin (D) [124]. Of these compounds, the FB₁ (B) is the best characterized and most widely used to experimentally inhibit CerS. Fumonisins are produced by the fungus Fusarium moniliforme and were found to inhibit CerS activity in vitro and in vivo [125]. The structure of fumonisins consists of a hydrocarbon chain containing side chains including hydroxy and methyl groups and a single amino/amide group. In fumonisin B species, two of these hydroxy groups are esterified to tricarballylic acid (TCA). The TCA groups can be removed from the hydrocarbon backbone via intracellular esterases to form hydrolysed fumonisins (HF) or aminopolyols (AP). Hydrolysed FB₁ (HFB₁) is less potent than FB₁ at inhibiting CerS and cell growth, suggesting that both the backbone and TCA groups are required for efficient binding to these enzymes [126,127]. An interesting characteristic of HFB₁ is that it also is a substrate for the CerS reaction [127,128]. The resulting product, N-acyl-HFB₁, is considerably more potent in cells than either HFB₁ or FB₁ (more than 10-fold). However, in vitro, HFB₁ is less potent than FB₁ [127]. AAL toxin (C) is highly similar in structure to fumonisins, and similarly inhibits CerS activity and elevates sphingoid bases [129,130]. Australifungin (D), a product of the fungus Sporormiella australis, is more potent than FB₁ at inhibiting fungal CerS activity [124,131]. However, the β-ketoaldehyde moiety of australifungin can react with the free amines of sphingoid bases causing these to be sequestered, making its experimental use as an in vitro and in vivo CerS inhibitor problematic. FTY720 (fingolimod) (E), a myriocin analogue, has been shown to inhibit CerS through a complex mechanism including both uncompetitive and non-competitive modes [132,133].

Figure 5. Multiple unique and shared biological roles of CerSs

Through the generation of multiple different ceramide species, each CerS can potentially regulate a variety of biological phenomena. PCD, programmed cell death; PP1, protein phosphatase 1.