Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.     

Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism

Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.     

Inhibitors of specific ceramide synthases

Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C_14:0-Cer – C_26:0-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure–activity relationships and the potential biological impact of these compounds are discussed.     

A conserved cysteine motif essential for ceramide kinase function

Ceramide kinase (CerK) is a sphingolipid metabolizing enzyme very sensitive to oxidation; however, the determinants are unknown. We show here that the thiol-modifying agent N-ethyl-maleimide abrogates CerK activity in vitro and in a cell based assay, implying that important cysteine residues are accessible in purified as well as endogenous CerK. We replaced every 22 residues in human CerK, by an alanine, and measured activity in the resulting mutant proteins. This led to identification of a cluster of cysteines, C(347)XXXC(351)XXC(354), essential for CerK function. These findings are discussed based on homology modeling of the catalytic domain of CerK.     

In vivo metabolism of fluorescent ceramide in central nervous system myelin of adult rats

The metabolism of sphingolipids in the central nervous system (CNS) has been studied in adult rats by intraventricular administration of fluorescent ceramide (CER). Rats were sacrificed at various time points post inoculation and the fluorescence of CER, cerebrosides (CB), sulfatides (SULF) and sphingomyelin (SPM) was determined in the CNS myelin and in the pellet, containing mainly microsomes, obtained by Norton myelin preparation. The incorporation of fluorescence was more in the pellet than in the myelin at all times studied. Initially the fluorescence present in the pellet was prevalently due to untransformed CER but an increase of fluorescent products with time was observed. CB was the main product up to 2 h post inoculation, then it decreased with concomitant increase of fluorescent SULF. In the myelin we did not observe differences in incorporation and transformation of fluorescent CER with time: CB was the main fluorescent product at all times studied. At 0.5 h post inoculation the fluorescence, observed by fluorescence microscope, was located in the cell lining the ventricles while after 24 h it appeared also in the paraventricular areas.     

Regulation of neural progenitor cell motility by ceramide and potential implications for mouse brain development

We provide evidence that the sphingolipid ceramide, in addition to its pro-apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo . Ceramide ( N -palmitoyl d -erythro sphingosine and N -oleoyl d -erythro sphingosine) and the ceramide analog N -oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti-ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co-distributed with its binding partner atypical protein kinase C ζ/λ (aPKC), and Cdc42, α/β-tubulin, and β-catenin, three proteins involved in aPKC-dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide-mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo , sphingolipid depletion leads to ectopic localization of mitotic or post-mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo .     

Regulation of primary cilia formation by ceramide

The primary cilium is an important sensory organelle, the regulation of which is not fully understood. We found that in polarized Madin-Darby Canine Kidney cells, the sphingolipid ceramide is specifically distributed to a cis-Golgi compartment at the base of the primary cilium. This compartment immunostained for the centrosome marker γ-tubulin, the Rho type GTPase cell division cycle 42 (Cdc42), and atypical protein kinase Cζ/λ (aPKC), a kinase activated by ceramide and associated with a polarity protein complex consisting of partitioning defective (Par)6 and Cdc42. Inhibition of ceramide biosynthesis with Fumonisin B1 prevented codistribution of aPKC and Cdc42 in the centrosomal/pericentriolar compartment and severely impaired ciliogenesis. Cilium formation and codistribution of aPKC and Cdc42 were restored by incubation with N-acetyl or N-palmitoyl sphingosine (C2 or C16 ceramide), or the ceramide analog N-oleoyl serinol (S18). Cilium formation was also restored by the glycogen synthase kinase-3β (GSK-3β) inhibitor indirubin-3-monoxime, suggesting that regulation of ciliogenesis depends on the inhibition of GSK-3β by ceramide-activated aPKC. Consistently, inhibition of aPKC with a pseudosubstrate inhibitor prevented restoration of ciliogenesis by C2 ceramide or S18. Our data show for the first time that ceramide is required for primary cilium formation.—Wang, G., K. Krishnamurthy, and E. Bieberich. Regulation of primary cilia formation by ceramide.     

A novel concatenated dimer of recombinant bovine somatotropin

A novel protein concatenated dimer structure was generated during the folding/oxidation of inclusion bodies of recombinant bovine somatotropin synthesized inEscherichia coli. The structure of this dimeric molecule was determined by peptide mapping with trypsin, and limited proteolysis by thrombin. Peptide mapping demonstrated that the two disulfide pairs in bovine somatotropin dimer were identical to those in monomer. Limited proteolysis with thrombin resulted in the cleavage of only a single peptide bond between arginine-132 and alanine-133 in bovine somatotropin dimer. This single peptide bond cleavage was sufficient to convert this dimer to a monomeric molecular weight species as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and HPLC. Since the single cleaved peptide bond is present in the large disulfide loop of bovine somatotropin, these data demonstrate that the dimeric molecule exists as a novel concatenated structure through the interlocking of the disulfide loops of this protein.     

A helix-turn motif in the C-terminal domain of histone H1

The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKTKKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone H1(o) (residues 99-121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i,i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C(alpha) protons, span from Pro100 to Val116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the alpha-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) beta-turn conformation, a sigma-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs.     

The Role of Ceramide Synthases in the Pathogenicity of Cryptococcus neoformans

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Glucosylceramides are critical for cell‐type differentiation and organogenesis,but not for cell viability in Arabidopsis

Glucosylceramides (GlcCer), glucose‐conjugated sphingolipids, are major components of the endomembrane system and plasma membrane in most eukaryotic cells. Yet the quantitative significance and cellular functions of GlcCer are not well characterized in plants and other multi‐organ eukaryotes. To address this, we examined Arabidopsis lines that were lacking or deficient in GlcCer by insertional disruption or by RNA interference (RNAi) suppression of the single gene for GlcCer synthase (GCS, At2g19880), the enzyme that catalyzes GlcCer synthesis. Null mutants for GCS (designated ‘gcs‐1’) were viable as seedlings, albeit strongly reduced in size, and failed to develop beyond the seedling stage. Heterozygous plants harboring the insertion allele exhibited reduced transmission through the male gametophyte. Undifferentiated calli generated from gcs‐1 seedlings and lacking GlcCer proliferated in a manner similar to calli from wild‐type plants. However, gcs‐1 calli, in contrast to wild‐type calli, were unable to develop organs on differentiation media. Consistent with a role for GlcCer in organ‐specific cell differentiation, calli from gcs‐1 mutants formed roots and leaves on media supplemented with the glucosylated sphingosine glucopsychosine, which was readily converted to GlcCer independent of GCS. Underlying these phenotypes, gcs‐1 cells had altered Golgi morphology and fewer cisternae per Golgi apparatus relative to wild‐type cells, indicative of protein trafficking defects. Despite seedling lethality in the null mutant, GCS RNAi suppression lines with ≤2% of wild‐type GlcCer levels were viable and fertile. Collectively, these results indicate that GlcCer are essential for cell‐type differentiation and organogenesis, and plant cells produce amounts of GlcCer in excess of that required for normal development.     

Sphingosine Forms Channels in Membranes That Differ Greatly from Those Formed by Ceramide

Ceramide channels formed in the outer membrane of mitochondria have been proposed to be the pathways by which proapoptotic proteins are released from mitochondria during the early stages of apoptosis. We report that sphingosine also forms channels in membranes, but these differ greatly from the large oligomeric barrel-stave channels formed by ceramide. Sphingosine channels have short open lifetimes and have diameters less than 2 nm, whereas ceramide channels have long open lifetimes, enlarge in size reaching diameters in excess of 10 nm. Unlike ceramide, sphingosine forms channels in erythrocyte plasma membranes that vary in size with concentration, but with a maximum possible channel diameter of 2 nm. In isolated mitochondria, a large proportion of the added sphingosine was rapidly metabolized to ceramide in the absence of externally added fatty acids or fatty-acyl-CoAs. The ceramide synthase inhibitor, fumonisin B1 failed to prevent sphingosine metabolism to ceramide and actually increased it. However, partial inhibition of conversion to ceramide was achieved in the presence of ceramidase inhibitors, indicating that reverse ceramidase activity is at least partially responsible for sphingosine metabolism to ceramide. A small amount of cytochrome c release was detected. It correlated with the level of ceramide converted from sphingosine. Thus, sphingosine channels, unlike ceramide channels, are not large enough to allow the passage of proapoptotic proteins from the intermembrane space of mitochondria to the cytoplasm.     

A novel cross-linked RNase A dimer with enhanced enzymatic properties

A new cross-linked ribonuclease A (RNase A) dimer composed of monomeric units covalently linked by a single amide bond between the side-chains of Lys(66) and Glu(9) is described. The dimer was prepared in the absence of water by incubating a lyophilized preparation of RNase, sealed under vacuum, in an oven at 85 degrees C. It was determined that the in vacuo procedure does not induce any significant conformational changes to the overall structure of RNase A, yet the amide cross-link has an increased acid lability, indicating that it is exposed and conformationally strained. Examination of X-ray crystallographic structures indicates that Lys(66) and Glu(9) are not close enough for the in vacuo dimer to adopt any of the known domain-swapped conformations. Therefore, the in vacuo RNase A dimer appears to be a novel dimeric structure. The in vacuo RNase A dimer also exhibits a twofold increase in activity over monomeric RNase A on a per monomer basis. This doubling of enzymatic activity was shown using dsRNA and ssRNA as substrates. In addition to this enhanced ability to degrade RNA, the dimer is not inhibited by the cellular ribonuclease inhibitor protein (cRI).     

Rapid transmembrane diffusion of ceramide and dihydroceramide spin-labelled analogues in the liquid ordered phase

In order to study the basic physical phenomena underlying complex lipid transbilayer movement in biological membranes, we have measured the transmembrane diffusion of spin-labelled analogues of sphingolipids in phosphatidylcholine (PC) large unilamellar vesicles in the absence or presence of cholesterol, going from a fluid ( liquid disordered) l_d, phase to a more viscous, liquid ordered (l_o), phase. We have found cholesterol to reduce the transverse diffusion of glucosylceramide (GlcCer) and galactosylceramide (GalCer) in a concentration-dependent manner. However, surprisingly, we could neither detect any influence of cholesterol on the rapid flip-flop of ceramide nor on the flip-flop of dihydroceramide, for which the τ_1/2 of flip-flop remains in the order of 1 minute at 20°C in the presence of cholesterol. As a consequence of rapid flip-flop of ceramide in both the l_o and the l_d phase, ceramide is likely to distribute between the two monolayers of a membrane, and could in principle partition into segregated domains in each side of the plasma membrane of eukaryotic cells.     

A novel family of phospholipase D homologues that includes phospholipid synthases and putative endonucleases: identification of duplicated repeats and potential active site residues.

Phosphatidylcholine-specific phospholipase D (PLD) enzymes catalyze hydrolysis of phospholipid phosphodiester bonds, and also transphosphatidylation of phospholipids to acceptor alcohols. Bacterial and plant PLD enzymes have not been shown previously to be homologues or to be homologous to any other protein. Here we show, using sequence analysis methods, that bacterial and plant PLDs show significant sequence similarities both to each other, and to two other classes of phospholipid-specific enzymes, bacterial cardiolipin synthases, and eukaryotic and bacterial phosphatidylserine synthases, indicating that these enzymes form an homologous family. This family is suggested also to include two Poxviridae proteins of unknown function (p37K and protein K4), a bacterial endonuclease (nuc), an Escherichia coli putative protein (o338) containing an N-terminal domain showing similarities with helicase motifs V and VI, and a Synechocystis sp. putative protein with a C-terminal domain likely to possess a DNA-binding function. Surprisingly, four regions of sequence similarity that occur once in nuc and o338, appear twice in all other homologues, indicating that the latter molecules are bi-lobed, having evolved from an ancestor or ancestors that underwent a gene duplication and fusion event. It is suggested that, for each of these enzymes, conserved histidine, lysine, aspartic acid, and/or asparagine residues may be involved in a two-step ping pong mechanism involving an enzyme-substrate intermediate.     

Functional characterization of enzymes catalyzing ceramide phosphoethanolamine biosynthesis in mice

Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.