Fumonisin-induced blockade of ceramide synthase in sphingolipid biosynthetic pathway alters aortic input impedance spectrum of pigs.
The sphingolipid signaling pathway appears to play an important role in regulating vascular tone. We examined the effect of fumonisin B(1), a fungal toxin in corn that blocks ceramide synthase in the sphingolipid signaling pathway, on the ascending aortic impedance spectrum of pigs. Sixteen pigs were fed culture material containing fumonisin B(1) (20 mg/kg body wt) (n = 7) or a control diet (n = 9) daily for 3 days and then instrumented under alpha-chloralose anesthesia for measurement of ascending aortic pressure and flow. Fumonisin ingestion increased serum sphinganine and sphingosine concentrations. Fumonisin ingestion also decreased cardiac output and characteristic impedance and increased the frequency of the first minimum impedance modulus, systemic vascular resistance, and the terminal, first, and second harmonic reflection coefficients, without changing mean arterial pressure. Thus blockade of ceramide synthase is accompanied by decreased vascular tone in systemic conduit arteries and increased vascular tone in systemic resistance vessels. The results indicate that the sphingolipid signaling pathway influences vascular tone in alpha-chloralose-anesthetized pigs. (+info)
Two mammalian longevity assurance gene (LAG1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-CoA donors.
Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [3H](dihydro)ceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (FB1). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids. (+info)
Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.
Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%. (+info)
Defects in cell growth regulation by C18:0-ceramide and longevity assurance gene 1 in human head and neck squamous cell carcinomas.
In this study, endogenous long chain ceramides were measured in 32 human head and neck squamous cell carcinoma (HNSCC) and 10 nonsquamous head and neck carcinoma tumor tissues, as compared with adjacent noncancerous tissues, by liquid chromatography/mass spectroscopy. Interestingly, only one specific ceramide, C(18:0)-ceramide, was selectively down-regulated in the majority of HNSCC tumor tissues. On the other hand, in nonsquamous tumor tissues, this selectivity for C18-ceramide was not detected. These data suggested the hypotheses that decreased levels of C18-ceramide might impart a growth advantage to HNSCC cells and that increased generation of C18-ceramide may be involved in the inhibition of growth. These roles were examined by reconstitution of C18-ceramide at physiologically relevant concentrations in UM-SCC-22A cells (squamous cell carcinoma of hypopharynx) via overexpression of mammalian upstream regulator of growth and differentiation factor 1 (mUOG1), a mouse homologue of longevity assurance gene 1 (mLAG1), which has been shown to specifically induce the generation of C18-ceramide. Liquid chromatography/mass spectroscopy analysis showed that overexpression of the mLAG1/mUOG1 resulted in increased levels of only C(18:0)-ceramide by approximately 2-fold, i.e. concentrations similar to those of normal head and neck tissues. Importantly, increased generation of C18-ceramide by mLAG1/mUOG1 inhibited cell growth (approximately 70-80%), which mechanistically involved the modulation of telomerase activity and induction of apoptotic cell death by mitochondrial dysfunction. In conclusion, this study demonstrates, for the first time, a biological role for LAG1 and C18-ceramide in the regulation of growth of HNSCC. (+info)
LASS5 is a bona fide dihydroceramide synthase that selectively utilizes palmitoyl-CoA as acyl donor.
We demonstrated recently (Riebeling, C., Allegood, J.C., Wang, E., Merrill, A. H. Jr., and Futerman, A. H. (2003) J. Biol. Chem. 278, 43452-43459) that upon over-expression in human embryonic kidney cells, longevity assurance gene homolog 5 (LASS5, previously named TRH4) elevates the synthesis of (dihydro)ceramides selectively enriched in palmitic acid. To determine whether LASS5 is a bona fide dihydroceramide synthase or, alternatively, whether it modifies an endogenous dihydroceramide synthase, we over-expressed LASS5 with a hemagglutinin (HA) tag at the C terminus, solubilized it using digitonin, and purified it by immunoprecipitation. Solubilized LASS5-HA displays the same fatty acid selectivity as the membrane-bound enzyme. After elution from agarose beads, only one band could be detected by SDS-PAGE, and its identity was confirmed to be LASS5 by mass spectrometry. Dihydroceramide synthase activity of the eluted LASS5-HA protein was totally dependent on exogenously added phospholipids. Moreover, eluted LASS5-HA was highly selective toward palmitoyl-CoA as acyl donor and was inhibited by the (dihydro)ceramide synthase inhibitor, fumonisin B1. This study identifies LASS5 as a genuine dihydroceramide synthase and demonstrates that mammalian dihydroceramide synthases do not require additional subunits for their activity. (+info)
The CLN9 protein, a regulator of dihydroceramide synthase.
A new variant of a group of pediatric neurodegenerative diseases known as neuronal ceroid lipofuscinosis (NCL) or Batten disease has been identified. It is termed CLN9-deficient. CLN9-deficient fibroblasts have a distinctive phenotype of rapid growth and increased apoptosis and diminished levels of ceramide, dihydroceramide, and sphingomyelin. Transfection with CLN8 but not other NCL genes corrected growth and apoptosis in CLN9-deficient cells, although the entire CLN8 sequence was normal. CLN8 is one of the TRAM-Lag1-CLN8 proteins containing a Lag1 motif. The latter imparts (dihydro)ceramide synthase activity to yeast cells. Transfection with the yeast gene Lag1 Sc and the human homolog LASS1 increased ceramide levels and partially corrected growth and apoptosis in CLN9-deficient cells. LASS2,-4,,-5, and -6 also corrected growth and apoptosis. Dihydroceramide levels and dihydroceramide synthase activity were markedly diminished in CLN9-deficient cells. Sequencing of LASS1, LASS2, LASS4, LASS5, and LASS6 genes was normal, and expression levels were increased or normal in CLN9-deficient cells by reverse transcription-PCR. N-(4-Hydroxyphenyl)retinamide (4-HPR), a dihydroceramide synthase activator, corrected growth and apoptosis and increased dihydroceramide synthase activity. Ceramide levels dropped further, and there was no increase in de novo ceramide synthesis, probably due to the effects of 4-HPR as activator of dihydroceramide synthase and inhibitor of dihydroceramide desaturase. Fumonisin B1, a dihydroceramide synthase inhibitor, exaggerated the CLN9-deficient phenotype of accelerated growth, decreased ceramide and increased apoptosis. This was neutralized by 4-HPR. We conclude that the CLN9 protein may be a regulator of dihydroceramide synthase and that 4-HPR could be developed as a treatment for CLN9-deficient patients. (+info)
Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.
Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific. (+info)
LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity.
The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland. (+info)