Publications

• Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties.

  Russo S.B., Tidhar R., Futerman A.H., Cowart L.A.

  <h4>Background</h4>Myristate is a novel potential substrate for sphingoid base synthesis.<h4>Results</h4>Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases.<h4>Conclusion</h4>d16:0 and d16:1 sphingolipids consti ... >> More

  <h4>Background</h4>Myristate is a novel potential substrate for sphingoid base synthesis.<h4>Results</h4>Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases.<h4>Conclusion</h4>d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles.<h4>Significance</h4>This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection. The enzyme serine palmitoyltransferase (SPT) catalyzes the formation of the sphingoid base "backbone" from which all sphingolipids are derived. Previous studies have shown that inhibition of SPT ameliorates pathological cardiac outcomes in models of lipid overload, but the metabolites responsible for these phenotypes remain unidentified. Recent in vitro studies have shown that incorporation of the novel subunit SPTLC3 broadens the substrate specificity of SPT, allowing utilization of myristoyl-coenzyme A (CoA) in addition to its canonical substrate palmitoyl-CoA. However, the relevance of these findings in vivo has yet to be determined. The present study sought to determine whether myristate-derived d16 sphingolipids are represented among myocardial sphingolipids and, if so, whether their function and metabolic routes were distinct from those of palmitate-derived d18 sphingolipids. Data showed that d16:0 sphingoid bases occurred in more than one-third of total dihydrosphingosine and dihydroceramides in myocardium, and a diet high in saturated fat promoted their de novo production. Intriguingly, d16-ceramides demonstrated highly limited N-acyl chain diversity, and in vitro enzyme activity assays showed that these bases were utilized preferentially to canonical bases by CerS1. Functional differences between myristate- and palmitate-derived sphingolipids were observed in that, unlike d18 sphingolipids and SPTLC2, d16 sphingolipids and SPTLC3 did not appear to contribute to myristate-induced autophagy, whereas only d16 sphingolipids promoted cell death and cleavage of poly(ADP-ribose) polymerase in cardiomyocytes. Thus, these results reveal a previously unappreciated component of cardiac sphingolipids with functional differences from canonical sphingolipids. << Less

  J. Biol. Chem. 288:13397-13409(2013) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain.

  Tidhar R., Ben-Dor S., Wang E., Kelly S., Merrill A.H. Jr., Futerman A.H.

  In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine ac ... >> More

  In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity. << Less

  J. Biol. Chem. 287:3197-3206(2012) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Biosynthesis of the anti-lipid-microdomain sphingoid base 4,14-sphingadiene by the ceramide desaturase FADS3.

  Jojima K., Edagawa M., Sawai M., Ohno Y., Kihara A.

  Sphingolipids are multifunctional lipids. Among the sphingolipid-component sphingoid bases, 4,14-sphingadiene (SPD) is unique such that it has a cis double bond with a bent structure. Although SPD was discovered half a century ago, its tissue distribution, biosynthesis, and degradation remain poor ... >> More

  Sphingolipids are multifunctional lipids. Among the sphingolipid-component sphingoid bases, 4,14-sphingadiene (SPD) is unique such that it has a cis double bond with a bent structure. Although SPD was discovered half a century ago, its tissue distribution, biosynthesis, and degradation remain poorly understood. Here, we established a specific and quantitative method for SPD measurement and found that SPD exists in a wide range of mammalian tissues. SPD was especially abundant in kidney, where the amount of SPD was ~2/3 of sphingosine, the most abundant sphingoid base in mammals. Although SPD is metabolized to ceramides and SPD 1-phosphate with almost the same efficiency as sphingosine, it is less susceptible to degradation by a cleavage reaction, at least in vitro. We identified the fatty acid desaturase family protein FADS3 as a ceramide desaturase that produces SPD ceramides by desaturating ceramides containing sphingosine. SPD sphingolipids were preferentially localized outside lipid microdomains, suggesting that SPD has different functions compared to other sphingoid bases in the formation of lipid microdomains. In summary, we revealed the biosynthesis and degradation pathways of SPD and its characteristic membrane localization. Our findings contribute to the elucidation of the molecular mechanism underlying the generation of sphingolipid diversity. << Less

  FASEB J. 34:3318-3335(2020) [PubMed] [EuropePMC]

  This publication is cited by 24 other entries.

• Human homologues of LAG1 reconstitute Acyl-CoA-dependent ceramide synthesis in yeast.

  Guillas I., Jiang J.C., Vionnet C., Roubaty C., Uldry D., Chuard R., Wang J., Jazwinski S.M., Conzelmann A.

  Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are member ... >> More

  Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum. lag1delta lac1delta double mutants in Saccharomyces cerevisiae lack an acyl-CoA-dependent ceramide synthase and are either very sick or nonviable, depending on the genetic background. LAG1 and LAC1 are members of a large eukaryotic gene family that shares the Lag1 motif, and some members of this family additionally contain a DNA-binding HOX homeodomain. Here we show that several human LAG1 homologues can rescue the viability of lag1delta lac1delta yeast cells and restore acyl-CoA-dependent ceramide and sphingolipid biosynthesis. When tested in a microsomal assay, Lac1p and Lag1p had a strong preference for C26:0-CoA over C24:0-CoA, C20-CoA, and C16-CoA, whereas some human homologues preferred C24:0-CoA and CoA derivatives with shorter fatty acids. This suggests that LAG1 proteins are related to substrate recognition and to the catalytic activity of ceramide synthase enzymes. CLN8, another human LAG1 homologue implicated in ceroid lipofuscinosis, could not restore viability to lag1delta lac1delta yeast mutants. << Less

  J. Biol. Chem. 278:37083-37091(2003) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

  Luttgeharm K.D., Cahoon E.B., Markham J.E.

  Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG o ... >> More

  Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism. << Less

  Biochem. J. 473:593-603(2016) [PubMed] [EuropePMC]

  This publication is cited by 16 other entries.

• A fluorescent assay for ceramide synthase activity.

  Kim H.J., Qiao Q., Toop H.D., Morris J.C., Don A.S.

  The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, g ... >> More

  The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compound's lipid tail and amine headgroup. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays. << Less

  J. Lipid Res. 53:1701-1707(2012) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.