Upstream of Growth and Differentiation Factor 1 (uog1), a Mammalian Homolog of the Yeast Longevity Assurance Gene 1 (LAG1), RegulatesN-Stearoyl-sphinganine (C18-(Dihydro)ceramide) Synthesis in a Fumonisin B1-independent Manner in Mammalian Cells

Abstract

The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B 1 , an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1, upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3 H]sphinganine or L-3-[ 3 H]serine demonstrated that uog1 conferred fumonisin B 1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids of uog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB 1 , and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B 1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays of uog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that the N-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.Interest in determining the regulatory mechanisms of ceramide metabolism has been stimulated over the past decade by the realization that ceramides formed by turnover of complex sphingolipids, and by de novo synthesis, influence key aspects of cell growth, regulation, differentiation, and death (1-6). Ceramides are formed de novo by N-acylation of sphinganine to dihydroceramide, which is subsequently desaturated by dihydroceramide desaturase (7-9). The N-acyltransferase(s), which are referred to herein as (dihydro)ceramide synthase(s), acylate various long chain bases, including sphinganine, sphingosine, and 4-hydroxysphinganine, utilize a wide spectrum of fatty acyl-CoAs, and are inhibited by the mycotoxin, fumonisin B 1 (FB 1 ) 1 (10 -12). Kinetic evidence has been obtained for multiple (dihydro)ceramide synthases, but no biochemical or molecular evidence has been obtained to prove their existence. One reason for suggesting that multiple (dihydro)ceramide synthases exist is that FB ...

The sphingolipid salvage pathway in ceramide metabolism and signaling

“…For example, LASS1 shows significant preference for C 18:0 fatty acid CoA, whereas LASS5 and LASS6 preferentially catalyze the acylation of (dihydro)sphingosine with myristoyl-, palmitoyl-and stearoyl-CoA rather than very long-chain fatty acid CoAs [31,34]. Initial studies regarding the subcellular localization of LASS family members demonstrate that, in over-expression models of genes, tagged-LASS 1/4/5/6 members were commonly observed to reside at the endoplasmic reticulum [31,32,35]. Dihydroceramides with distinct fatty acyl moieties are then desaturated by dihydroceramide desaturase to form the corresponding ceramides [36].…”

“…Ceramide synthase activity [71] has been observed in mitochondria-associated membranes where free sphingoid bases are converted to (dihydro)ceramide by (dihydro)ceramide synthases [23]. Moreover, ceramideenriched mitochondria upon treatment with phorbol esters were seen to localize around the nuclear envelope where LASS5 as well as other LASS family members are present [31,32,35]. These spatial changes in subcellular compartments (mitochondria, endoplasmic reticulum, and mitochondria-associated membranes) may play a key role in allowing the accumulation of C 16:0 -ceramide in mitochondria and/or mitochondria-associated membranes.…”

A novel mechanism of dasatinib-induced apoptosis in chronic myeloid leukemia; ceramide synthase and ceramide clearance genes

“…Different length of ceramides were synthesized by different CerS genes [20]. While CerS1 is responsible for synthesis of C18-ceramide [21], CerS2 and CerS4 increase the levels of C24-ceramides and C22-ceramides, respectively [22,23]. C14-ceramides and C16-ceramides were sythesised by CerS5 and CerS6 genes [24,25].…”

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

“…One of the key enzymes of the de novo pathway is (dihydro)ceramide synthase, and it has been shown that in Saccharomyces cerevisiae, longevity assurance gene (LAG) members (LAG1p and Lac1p) play a role in the regulation of life span (14) and are also required for ceramide synthase activity (15,16). Recent studies also revealed that one of the mouse homologues of these proteins, known as upstream of growth and differentiation factor 1 (mUOG1 or mouse LASS1) specifically regulates the synthesis of stearoyl (C 18 )-containing sphingolipids, including C 18 -ceramide (17,18). The human homologue of the LAG1 was also identified (named as LASS1; ref.…”