Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids.

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity.

LAG1 was the first longevity assurance gene discovered in Saccharomyces cerevisiae The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and in vitro assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Δlag1 cells have an increased lifespan while Δlac1 cells do not.

Eleven residues determine the acyl chain specificity of ceramide synthases.

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate-induced colitis in mice due to increased intestinal permeability.

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long-chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very-long acyl chain ceramides with concomitant increase of long chain bases and C16-ceramides, were more susceptible to dextran sodium sulphate-induced colitis, and their survival rate was markedly decreased compared with that of wild-type littermates. Using mixed bone-marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule-A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco-2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2-knockdown via CRISPR-Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long-chain bases and C16-ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC-dextran levels, indicating that altered SLs including deficiency of very-long-chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.

Oxidative stress elicited by modifying the ceramide acyl chain length reduces the rate of clathrin-mediated endocytosis.

Sphingolipids modulate clathrin-mediated endocytosis (CME) by altering the biophysical properties of membranes. We now examine CME in astrocytes cultured from ceramide synthase 2 (CerS2) null mice, which have an altered sphingolipid acyl chain composition. The rate of endocytosis of low-density lipoprotein and transferrin, which are internalized via CME, was reduced in CerS2 null astrocytes, although the rate of caveolin-mediated endocytosis was unaltered. Levels of clathrin heavy chain were increased, which was due to decreased levels of Hsc70 (also known as HSPA8), a protein involved in clathrin uncoating. Hsc70 levels were decreased because of lower levels of binding of Sp1 to position -68 in the Hsc70 promoter. Levels of Sp1 were downregulated due to oxidative stress, which was elevated fourfold in CerS2 null astrocytes. Furthermore, induction of oxidative stress in wild-type astrocytes decreased the rate of CME, whereas amelioration of oxidative stress in CerS2 null astrocytes reversed the decrease. Our data are consistent with the notion that sphingolipids not only change membrane biophysical properties but also that changes in their composition can result in downstream effects that indirectly impinge upon a number of cellular pathways, such as CME.

Regulation of very-long acyl chain ceramide synthesis by acyl-CoA-binding protein.

Ceramide and more complex sphingolipids constitute a diverse group of lipids that serve important roles as structural entities of biological membranes and as regulators of cellular growth, differentiation, and development. Thus, ceramides are vital players in numerous diseases including metabolic and cardiovascular diseases, as well as neurological disorders. Here we show that acyl-coenzyme A-binding protein (ACBP) potently facilitates very-long acyl chain ceramide synthesis. ACBP increases the activity of ceramide synthase 2 (CerS2) by more than 2-fold and CerS3 activity by 7-fold. ACBP binds very-long-chain acyl-CoA esters, which is required for its ability to stimulate CerS activity. We also show that high-speed liver cytosol from wild-type mice activates CerS3 activity, whereas cytosol from ACBP knock-out mice does not. Consistently, CerS2 and CerS3 activities are significantly reduced in the testes of ACBP-/- mice, concomitant with a significant reduction in long- and very-long-chain ceramide levels. Importantly, we show that ACBP interacts with CerS2 and CerS3. Our data uncover a novel mode of regulation of very-long acyl chain ceramide synthesis by ACBP, which we anticipate is of crucial importance in understanding the regulation of ceramide metabolism in pathogenesis.

Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.

In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD.

Effect of the sphingosine kinase 1 selective inhibitor, PF-543 on arterial and cardiac remodelling in a hypoxic model of pulmonary arterial hypertension.

Recent studies have demonstrated that the expression of sphingosine kinase 1, the enzyme that catalyses formation of the bioactive lipid, sphingosine 1-phosphate, is increased in lungs from patients with pulmonary arterial hypertension. In addition, Sk1(-/-) mice are protected from hypoxic-induced pulmonary arterial hypertension. Therefore, we assessed the effect of the sphingosine kinase 1 selective inhibitor, PF-543 and a sphingosine kinase 1/ceramide synthase inhibitor, RB-005 on pulmonary and cardiac remodelling in a mouse hypoxic model of pulmonary arterial hypertension. Administration of the potent sphingosine kinase 1 inhibitor, PF-543 in a mouse hypoxic model of pulmonary hypertension had no effect on vascular remodelling but reduced right ventricular hypertrophy. The latter was associated with a significant reduction in cardiomyocyte death. The protection involves a reduction in the expression of p53 (that promotes cardiomyocyte death) and an increase in the expression of anti-oxidant nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). In contrast, RB-005 lacked effects on right ventricular hypertrophy, suggesting that sphingosine kinase 1 inhibition might be nullified by concurrent inhibition of ceramide synthase. Therefore, our findings with PF-543 suggest an important role for sphingosine kinase 1 in the development of hypertrophy in pulmonary arterial hypertension.

Bcl2L13 is a ceramide synthase inhibitor in glioblastoma.

Therapy resistance is a major limitation to the successful treatment of cancer. Here, we identify Bcl2-like 13 (Bcl2L13), an atypical member of the Bcl-2 family, as a therapy susceptibility gene with elevated expression in solid and blood cancers, including glioblastoma (GBM). We demonstrate that mitochondria-associated Bcl2L13 inhibits apoptosis induced by a wide spectrum of chemo- and targeted therapies upstream of Bcl2-associated X protein activation and mitochondrial outer membrane permeabilization in vitro and promotes GBM tumor growth in vivo. Mechanistically, Bcl2L13 binds to proapoptotic ceramide synthases 2 (CerS2) and 6 (CerS6) via a unique C-terminal 250-aa sequence located between its Bcl-2 homology and membrane anchor domains and blocks homo- and heteromeric CerS2/6 complex formation and activity. Correspondingly, CerS2/6 activity and Bcl2L13 abundance are inversely correlated in GBM tumors. Thus, our genetic and functional studies identify Bcl2L13 as a regulator of therapy susceptibility and point to the Bcl2L13-CerS axis as a promising target to enhance responses of therapy-refractory cancers toward conventional and targeted regimens currently in clinical use.