Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.     

Simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene-2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase

Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.     

Structural requirements for mitochondrial mutagenesis

Ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) and euflavine (3, 6-diamino-10-methylacridinium chloride) are superficially similar in structure and ability to intercalate into DNA. However, they exhibit qualitative differences in their ability to bring about a mitochondrial mutation (ρ⁺ → ρ^?) in Saccharomyces cerevisiae. This investigation tried to establish and compare the essential structural prerequisites in three series of planar, heterocyclic dyes: the phenanthridines (P series), the acridines (A series), and molecules with different heteroatoms related to acridines (X series). Compounds capable of bringing about the mutation in the complete absence of growth and energy sources are restricted to di-primary amines in the P series: quaternization of the ring nitrogen, and an aromatic side chain at C-6 also appear essential. Compounds in the A series are mutagenic only with growing cells; quaternization (C₁ through C₄) is essential. The 10-allyl derivative is unusual; it is highly effective even in buffer supplemented only with an energy source. The results are interpreted in terms of a model that requires interaction of the mutagen with the mitochondrial inner membrane as well as with its DNA.     

Inhibition of sodium-calcium exchange by ceramide and sphingosine

Na(+)/Ca(2+) exchange activity in Chinese hamster ovary cells expressing the bovine cardiac Na(+)/Ca(2+) exchanger was inhibited by the short chain ceramide analogs N-acetylsphingosine and N-hexanoylsphingosine (5-15 micrometer). The sphingolipids reduced exchange-mediated Ba(2+) influx by 50-70% and also inhibited the Ca(2+) efflux mode of exchange activity. The biologically inactive ceramide analog N-acetylsphinganine had only modest effects on exchange activity. Cells expressing the Delta(241-680) and Delta(680-685) deletion mutants of the Na(+)/Ca(2+) exchanger were not inhibited by ceramide; these mutants show defects in both Na(+)-dependent and Ca(2+)-dependent regulatory behavior. Another mutant, which was defective only in Na(+)-dependent regulation, was as sensitive to ceramide inhibition as the wild-type exchanger. Inhibition of exchange activity by ceramide was time-dependent and was accelerated by depletion of internal Ca(2+) stores. Sphingosine (2.5 micrometer) also inhibited the Ca(2+) influx and efflux modes of exchange activity in cells expressing the wild-type exchanger; sphingosine did not affect Ba(2+) influx in the Delta(241-680) mutant. The effects of the exogenous sphingolipids were reproduced by blocking cellular ceramide utilization pathways, suggesting that exchange activity is inhibited by increased levels of endogenous ceramide and/or sphingosine. We propose that sphingolipids impair Ca(2+)-dependent activation of the exchanger and that in cardiac myocytes, this process serves as a feedback mechanism that links exchange activity to the diastolic concentration of cytosolic Ca(2+).     

Control of inflammatory responses by ceramide,sphingosine 1-phosphate and ceramide 1-phosphate

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.     

Structural requirements of sphingosine molecules for inhibition of DNA primase: biochemical and computational analyses

Using 28 chemically well-defined compounds containing D-erythro-sphingosine and its analogues, we analyzed structure-activity relationships for DNA primase inhibition. Biochemical studies demonstrated a positively charged amino group at C2 and a long aliphatic chain to be absolutely required for inhibition. Whereas C2-amino group is intact, sphingosine 1-phosphate was totally inactive. This result could be due to cancellation of positive charge of the amino group by the interaction with negatively charged C1-phosphate, since simulations with the software INSIGHT II showed these two groups to be close enough to interact. The hydroxyl group at C3 and trans-double bond at C4-C5 were also found to be important for the inhibition. Dehydroxylation of C3, as well as saturation or cis-conversion of the trans-double bond led to decrease of inhibitory activity. Despite saturation of the double bond, introduction of a hydroxyl group into C4 of dihydrosphingosine resulted in restoration of inhibition. Conversion of the double bond into a triple bond did not abolish but rather enhanced the inhibitory activity. Among sphingosine stereoisomers, the naturally occurring D-erythro-sphingosine proved to be the strongest inhibitor. To ascertain the contribution of the total conformation to the inhibition, especially of the long aliphatic chain, we constructed a 3D-quantitative structure-activity relationship model using the computer program Catalyst/HipHop on the basis of information described above. Analysis of the hypothesis model for active compounds revealed that the orientation of aliphatic chain, represented by the dihedral angle of C2-3-4-5, correlated well with the inhibition. Modifications such as deletion of the hydroxyl group at C3 or saturation of the C4-C5 double bond caused shifts in the dihedral angle of C2-3-4-5, with concomitant decrease in inhibitory activity.     

Structural requirements of lysophospholipid-regulated mitochondrial Ca2+ transport

Analogues of lysophosphatidylcholine, including PAF (platelet-activating-factor) and HePC (an experimental anticancer drug), were studied for their influence on mitochondrial Ca2+ transport and membrane potential. Lysophospholipids released Ca2+ from mitochondria and reduced the maximal Ca2+ uptake. The structure-activity relations indicate that deprotonated head groups like phosphocholines yield active compounds while partially protonated head groups like phosphoethanolamines are essentially inactive. Structural requirements for the apolar part of the molecules were acyl or alkyl chain lengths of less than 18 carbon atoms at the C1-position of the glycerol backbone and residues of small size and/or low polarity at the C2-position. Choline lysophospholipids, but not ethanolamine lysophospholipids, may therefore induce mitochondrial Ca2+ efflux and become mediators of ischaemic tissue damage where dysregulated phospholipase A2 activity and an impairment of mitochondrial function are supposed to play a crucial role.     

N-hexyl-O-glucosyl sphingosine, an inhibitor of glucosyl ceramide -glucosidase

A synthetic analog of glucocerebroside, N-hexyl-O-glucosyl sphingosine, was found to inhibit the glucosidase in rat spleen that hydrolyzes glucocerebroside. At a concentration of 1 micro m, the analog inhibited the enzyme by 48%. The mode of action appeared to be competitive, probably aided by tight binding of the amine group to a carboxyl group near the enzyme's active site. Increasing or decreasing the chain length of the n-alkyl group attached to the nitrogen atom led to decreased effectiveness. The inhibitory effect was maximal at pH 7.0, but it was still considerable at the enzyme's optimal pH, 5.0. It is suggested that the compound may be useful for inducing an animal model of Gaucher's disease.     

Molecular cloning and characterization of a human mitochondrial ceramidase

We have recently purified a rat brain membrane-bound nonlysosomal ceramidase (El Bawab, S., Bielawska, A., and Y. A. Hannun (1999) J. Biol. Chem. 274, 27948-27955). Using peptide sequences obtained from the purified rat brain enzyme, we report here the cloning of the human isoform. The deduced amino acid sequence of the protein did not show any similarity with proteins of known function but was homologous to three putative proteins from Arabidospis thaliana, Mycobacterium tuberculosis, and Dictyostelium discoideum. Several blocks of amino acids were highly conserved in all of these proteins. Analysis of the protein sequence revealed the presence at the N terminus of a signal peptide followed by a putative myristoylation site and a putative mitochondrial targeting sequence. The predicted molecular mass was 84 kDa, and the isoelectric point was 6.69, in agreement with rat brain purified enzyme. Northern blot analysis of multiple human tissues showed the presence of a major band corresponding to a size of 3.5 kilobase. Analysis of this major band on the blot indicated that the enzyme is ubiquitously expressed with higher levels in kidney, skeletal muscle, and heart. The enzyme was then overexpressed in HEK 293 and MCF7 cells using the pcDNA3. 1/His-ceramidase construct, and ceramidase activity (at pH 9.5) increased by 50- and 12-fold, respectively. Next, the enzyme was characterized using lysate of overexpressing cells. The results confirmed that the enzyme catalyzes the hydrolysis of ceramide in the neutral alkaline range and is independent of cations. Finally, a green fluorescent protein-ceramidase fusion protein was constructed to investigate the localization of this enzyme. The results showed that the green fluorescent protein-ceramidase fusion protein presented a mitochondrial localization pattern and colocalized with mitochondrial specific probes. These results demonstrate that this novel ceramidase is a mitochondrial enzyme, and they suggest the existence of a topologically restricted pathways of sphingolipid metabolism.     

Structural requirements of mitochondrial D-beta-hydroxybutyrate dehydrogenase with respect to its coenzyme

The purpose of this work was to test structural analogs of NAD+ in order to know enzyme requirements of chemical structure of coenzyme to get catalytic activity and, in a other hand to see which chemical parts of the coenzyme were involved in the coenzyme binding to the active site. The binding of the coenzyme analog at the catalytic site requires an adenosine diphosphoribose structure without any additional phosphate group on the ribose linked to adenine.     

Role of lysosomal acid ceramidase in the metabolism of ceramide in human skin fibroblasts

A 2.8-fold accumulation of ceramide was demonstrated in cultured skin ftbroblasts from a patient with Farber's disease, an inborn error of metabolism in which acid ceramidase activity is deficient. To investigate the role of acid ceramidase in the metabolism of ceramide in fibroblasts, we have investigated the lysosomal degradation of ceramide that was taken up by fibroblasts from an exogenous lipid suspension. Fluorescent 4-nitrobenz-2-oxa-1,3-diazole-7-aminododecanoyl-sphingosine (NBD-ceramide) from an exogenous ceramide suspension was incorporated into the intracellular structures of fibroblasts at 37 °C. Study of the cellular uptake of exogenous [³H]oleylsphingosine showed that the rate of ceramide accumulation was nearly identical in Farber's disease and normal fibroblasts. The deficiency of acid ceramidase in Farber's fibroblasts resulted in the decrease of cellular degradation and uptake of ceramide and the increase of retention time of ceramide in these diseased cells. Studies of subcellular fractionation of these fibroblasts showed that the accumulated ceramide was located in the lysosomal fraction. As a result, the density of the lysosomal fraction of Farber's fibroblasts was found to be less than that of controls. These results suggest the defect of cellular metabolism in this inherited disease is located within the lysosome.     

Involvement of neutral ceramidase in ceramide metabolism at the plasma membrane and in extracellular milieu

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.     

Cycloeucalenol-obtusifoliol isomerase. Structural requirements for transformation or binding of substrates and inhibitors

The molecular features of 19 synthetic substrates and ground-state analogues of cycloeucalenol, the natural substrate of cycloeucalenol - obtusifoliol isomerase, a membrane-bound enzyme specific to higher plants, and of 9 synthetic carbocationic analogues of the high-energy intermediate occurring during the reaction catalyzed by the isomerase, were related to their ability to be transformed by this enzyme (catalytical competence) and their potency as inhibitors of this enzyme. With substrates and ground-state analogues it has been possible to determine at least two critical domains: significant binding requires the presence of the 3 beta-hydroxyl group on the ring A with the correct stereochemistry together with absence of a 4 beta-methyl group. Moreover initial enzyme-substrate interaction appears to be dependent upon the accessibility of the 3 beta-oxygen. Substitutions on the ring B do not preclude binding whereas they are of great influence on substrate transformation. Modifications of the ring A and other modifications suggest that ground-state and high-energy intermediate analogues bind two different conformations of the isomerase active site.     

Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: Review of sphingosine kinase inhibitors

Sphingosine 1-phosphate (S1P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S1P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell–cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [ and ]. Given the wide variety of cellular and physiological processes in which S1P is involved, it is immediately obvious why the mechanisms governing S1P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the sphingosine kinase (SK)/S1P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S1P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Ceramidase and ceramide synthesis in human kidney and cerebellum. Description of a new alkaline ceramidase.

It has been shown that tissues of patients with Farber's disease characteristically lack acid (pH 4.0) ceramidase. In normal cerebellum, however, ceramide cleavage and the reverse reaction, free fatty acid-dependent ceramide synthesis, both occur not only at pH 4.0 but also at pH 9.0, although normal kidney exhibits these activities only at pH 4.0. Both tissues are capable of snythesizing ceramide via an acyl-COA-dependent pathway at neutral pH. The synthetic analog of ceramide, N-oleoyl-ethanolamine, is a potent inhibitor of ceramidase.     

Discovery of novel sphingosine kinase 1 inhibitors

Sphingosine kinase 1 (SK1) is an important enzyme that regulates the balance between ceramide and sphingosine-1-phosphate (S1P). Potent and novel SK1 inhibitors (6ag, 9ab and 12aa) have been discovered through a series of modifications of sphingosine (1), the substrate of this enzyme.     

Structural requirements for biologically active jasmonates: Induction of protease inhibitors and cotyledon senescence

Methyl jasmonate and jasmonate isomers were resolved for studies in stereoselectivity of biological action. Chemical modifications included fluorination at the chiral C7 carbon, trifluorination at the C12 of the pentenyl sidechain, shortening of this pentenyl to a butenyl moiety, and synthesis of the (+) and (–)-epi-methyl jasmonates. The effects of these modifications and stereospecificity were monitored using three assays: proteinase inhibitor II promoter driving the reporter -glucuronidase; endogenous trypsin proteinase inhibitor induction; and sunflower cotyledon senescence induction. The methyl esters were more active than the corresponding acids, the R absolute configuration at C3 was most effective in all assays, fluorination at C7 abolished activity in the proteinase inhibitor assays, and the trifluorination at C12 or shortening to a butenyl did not alter activity relative to the active (–)- methyl jasmonate, but both of these latter modifications revealed a delayed response in the senescence conductivity measures.