Dihydroceramide desaturase and dihydrosphingolipids: Debutant players in the sphingolipid arena

doi:10.1016/j.plipres.2011.12.002

Progress in Lipid Research

Volume 51, Issue 2, April 2012, Pages 82-94

https://doi.org/10.1016/j.plipres.2011.12.002 Get rights and content

Abstract

Sphingolipids are a wide family of lipids that share common sphingoid backbones, including (2S,3R)-2-amino-4-octadecane-1,3-diol (dihydrosphingosine) and (2S,3R,4E)-2-amino-4-octadecene-1,3-diol (sphingosine). The metabolism and biological functions of sphingolipids derived from sphingosine have been the subject of many reviews. In contrast, dihydrosphingolipids have received poor attention, mainly due to their supposed lack of biological activity. However, the reported biological effects of active site directed dihydroceramide desaturase inhibitors and the involvement of dihydrosphingolipids in the response of cells to known therapeutic agents support that dihydrosphingolipids are not inert but are in fact biologically active and underscore the importance of elucidating further the metabolic pathways and cell signaling networks involved in the biological activities of dihydrosphingolipids. Dihydroceramide desaturase is the enzyme involved in the conversion of dihydroceramide into ceramide and it is crucial in the regulation of the balance between sphingolipids and dihydrosphingolipids. Furthermore, given the enzyme requirement for O₂ and the NAD(P)H cofactor, the cellular redox balance and dihydroceramide desaturase activity may reciprocally influence each other. In this review both dihydroceramide desaturase and the biological functions of dihydrosphingolipids are addressed and perspectives on this field are discussed.

Section snippets

Foreword

Very early studies by Brady [1] and Stoffel [2] hypothesized that sphingosine (So) was produced by dehydrogenation of dihydrosphingosine (dhSo). Although dhSo administered in vivo produces So [3], [4] and phytosphingosine [4], [5], Ong and Brady proposed that the olefinic bond is introduced after N-acylation of the administered dhSo [6]. A later work by Merrill and Wang [7] confirmed that the double bond is inserted on the N-acylated dhSo and that this reaction occurs in the de novo

Dihydroceramide desaturase

Experimental evidence for the biochemical characterization of the dihydroceramide desaturase reaction were first reported by Geeraert et al. [10] and Michel et al. [11] in rat hepatocytes and rat liver microsomes, respectively, and then reviewed and confirmed by Schulze et al. [12]. The results obtained in these in vitro studies, which utilized dhCer analogs as substrates, showed that the desaturation reaction needed NADPH [10] or NADH [11] as electron donor and oxygen as electron acceptor,

Des1 Inhibitors

The first reported synthesized inhibitor of Des1 was compound GT11 (Fig. 7) [28]. This cyclopropene-containing sphingolipid carries out a competitive inhibition against the substrate with a K_i of 6 μM [63] and it is active both in vitro and in intact cells [64], [65]. Structure-activity relationship studies showed that the natural 2S,3R stereochemistry, the presence of a free hydroxyl function at C1 and the cyclopropene ring in place of the Cer double bond in the molecule are essential for the

Dihydroceramide and dihydrosphingolipids: innocent bystanders?

The reputation of dhCer as inactive or ineffective lipids takes origin from some studies in the early 1990’s. In these works, dhCer were shown to be unable to inhibit growth and stimulate Ceramide Activated Protein Phosphatase, a primary target enzyme for Cer, in yeast [71] and in HL-60 cells [72], [73]. These studies made use of C2-dhCer and C2-Cer, the short-chain analogs of natural long-chain Cer and dhCer. Later, it was shown that while C2-Cer was able to cause ADP-induced platelet

Des1 as a therapeutic target

The several reports mentioned above, showing that dhCer are involved in cell cycle arrest, apoptosis, autophagy, and oxidative stress underscore the interest of Des1 as a novel target for cancer therapy. Additional support to this notion is the fact that several drugs used in cancer chemotherapy decrease Des1 activity and their action appears to be related to their capacity to increase the endogenous levels of dihydroceramides and their sphingolipid metabolic products. Another evidence in

Conclusions and perspectives

The use of mass spectrometry techniques to measure (dh)Cer and the availability of Des1 inhibitors of use as pharmacological tools have eased the advent of solid evidence arguing against the paradigm that dhCer are the innocuous counterparts of Cer. Recent reports strengthen the role of dhCer as bioactive lipids, although their actions differ from those elicited by Cer. In fact, it has been proposed that dhCer may mitigate the apoptotic effects of Cer [88]. The arising role of dhCer in the

Acknowledgements

Predoctoral fellowships from the I3P/JAE programme of the Spanish National Research Council (J.M.M.-O.), Agència de Gestió d’Ajuts Universitaris i de Recerca de la Generalitat de Catalunya (F.C.) and Doctorate School of Molecular Medicine, Università degli Studi di Milano (V.G.) are acknowledged. Financial support from Generalitat de Catalunya (Grant 2009 SGR 1072), the Spanish Ministry of Science and Innovation (Grants SAF 2008-00706 and SAF 2011-22444), the Italian Program for University

References (138)

R.O. Brady et al.
The enzymatic synthesis of sphingosine
J Biol Chem
(1958)
M.W. Crossman et al.
Biosynthesis of 4D-hydroxysphinganine by the rat. En bloc incorporation of the sphinganine carbon backbone
Biochim Biophys Acta
(1984)
M.W. Crossman et al.
Biosynthesis of phytosphingosine by the rat
J Biol Chem
(1977)
D.E. Ong et al.
In vivo studies on the introduction of the 4-t-double bond of the sphingenine moiety of rat brain ceramides
J Biol Chem
(1973)
A.H. Merrill et al.
Biosynthesis of long-chain (sphingoid) bases from serine by LM cells. Evidence for introduction of the 4-trans-double bond after de novo biosynthesis of N-acylsphinganine(s)
J Biol Chem
(1986)
Z. Mao et al.
Alkaline ceramidase 2 (ACER2) and its product dihydrosphingosine mediate the cytotoxicity of 4-HPR in tumor cells
J Biol Chem
(2010)
W. Hu et al.
Alkaline ceramidase 3 (ACER3) hydrolyzes unsaturated long-chain ceramides, and its down-regulation inhibits both cell proliferation and apoptosis
J Biol Chem
(2010)
C. Michel et al.
Characterization of ceramide synthesis. A dihydroceramide desaturase introduces the 4,5-trans-double bond of sphingosine at the level of dihydroceramide
J Biol Chem
(1997)
H. Schulze et al.
Dihydroceramide desaturase
Methods Enzymol
(2000)
C. Michel et al.
Conversion of dihydroceramide to ceramide occurs at the cytosolic face of the endoplasmic reticulum
FEBS Lett
(1997)

P. Ternes et al.

Identification and characterization of a sphingolipid delta4-desaturase family

J Biol Chem

(2002)

F. Omae et al.

Identification of an essential sequence for dihydroceramide C-4 hydroxylase activity of mouse DES2

FEBS Lett

(2004)

Y. Mizutani et al.

Identification of the human sphingolipid C4-hydroxylase, hDES2, and its up-regulation during keratinocyte differentiation

FEBS Lett

(2004)

E. Beauchamp et al.

N-Myristoylation targets dihydroceramide [Delta]4-desaturase 1 to mitochondria: partial involvement in the apoptotic effect of myristic acid

Biochimie

(2009)

T.A. Farazi et al.

The biology and enzymology of protein N-myristoylation

J Biol Chem

(2001)

M. Rahmaniyan et al.

Identification of Dihydroceramide Desaturase as a Direct in vitro target for Fenretinide

J Biol Chem

(2011)

K. Raith et al.

Ceramide analysis utilizing gas chromatography–mass spectrometry

J Chromatogr A

(2000)

J.S. Won et al.

Sphingolipid signaling and redox regulation

Free Radic Biol Med

(2006)

B. Liu et al.

Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione

J Biol Chem

(1997)

B. Liu et al.

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

J Biol Chem

(1998)

P. Signorelli et al.

Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells

Cancer Lett

(2009)

N.A. Denisova et al.

Role of membrane lipids in regulation of vulnerability to oxidative stress in PC12 cells: implication for aging

Free Radic Biol Med

(2001)

R. Reinehr et al.

Involvement of NADPH oxidase isoforms and Src family kinases in CD95-dependent hepatocyte apoptosis

J Biol Chem

(2005)

P. Moskwa et al.

Glucocerebroside inhibits NADPH oxidase activation in cell-free system

Biochim Biophys Acta

(2004)

Y. Liel et al.

Monocyte dysfunction in patients with Gaucher disease: evidence for interference of glucocerebroside with superoxide generation [see comments]

Blood

(1994)

A.O. Brightman et al.

A growth factor- and hormone-stimulated NADH oxidase from rat liver plasma membrane

Biochim Biophys Acta

(1992)

C. Garcia-Ruiz et al.

Direct effect of ceramide on the mitochondrial electron transport chain leads to generation of reactive oxygen species. Role of mitochondrial glutathione

J Biol Chem

(1997)

J.M. Kraveka et al.

Involvement of the dihydroceramide desaturase in cell cycle progression in human neuroblastoma cells

J Biol Chem

(2007)

C.R. Vieira et al.

Dihydrosphingomyelin Impairs HIV-1 Infection by rigidifying liquid-ordered membrane domains

Chem Biol

(2010)

J.D. Fishbein et al.

Ceramide-mediated growth inhibition and CAPP are conserved in Saccharomyces cerevisiae

J Biol Chem

(1993)

A. Bielawska et al.

Selectivity of ceramide-mediated biology. Lack of activity of erythro- dihydroceramide

J Biol Chem

(1993)

A.H.J. Merrill

De novo sphingolipid biosynthesis: a necessary, but dangerous, pathway

J Biol Chem

(2002)

J. Chen et al.

Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat hepatocytes

J Biol Chem

(1995)

S. El Bawab et al.

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain

J Biol Chem

(1999)

F. Scarlatti et al.

Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1

J Biol Chem

(2004)

W. Zheng et al.

Ceramides and other bioactive sphingolipid backbones in health and disease: lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy

Biochim Biophys Acta

(2006)

H. Van Overloop et al.

Further characterization of mammalian ceramide kinase: substrate delivery and (stereo)specificity, tissue distribution and subcellular localization studies

J Lipid Res

(2006)

B. Ogretmen et al.

Biochemical mechanisms of the generation of endogenous long chain ceramide in response to exogenous short chain ceramide in the A549 human lung adenocarcinoma cell line: role for endogenous ceramide in mediating the action of exogenous ceramide

J Biol Chem

(2002)

C. Mao et al.

Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate

Biochim Biophys Acta

(2008)

M. Valsecchi et al.

Sphingolipidomics of A2780 human ovarian carcinoma cells treated with synthetic retinoids

J Lipid Res

(2010)

J.K. Kundu et al.

Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives

Cancer Lett

(2008)

E. Dolfini et al.

Resveratrol impairs the formation of MDA-MB-231 multicellular tumor spheroids concomitant with ceramide accumulation

Cancer Lett

(2007)

W. Stoffel et al.

Stereospecificities in the introduction of the 4t-double bond into sphinganine yielding 4t-sphingenine (sphingosine)

Hoppe Seylers Z Physiol Chem

(1971)

W. Stoffel et al.

Stereospecificities in the metabolic reactions of the four isomeric sphinganines (dihydrosphingosines) in rat liver

Hoppe Seylers Z Physiol Chem

(1973)

L. Geeraert et al.

Conversion of dihydroceramide into ceramide: involvement of a desaturase

Biochem J

(1997)

A. Enomoto et al.

Dihydroceramide:sphinganine C-4 hydroxylation requires DES2 hydroxylase and the membrane form of cytochrome b5

Biochem J

(2006)

K. Endo et al.

Degenerative spermatocyte, a novel gene encoding a transmembrane protein required for the initiation of meiosis in Drosophila spermatogenesis

Mol Gen Genet

(1996)

K. Endo et al.

Mdes, a mouse homolog of the Drosophila degenerative spermatocyte gene is expressed during mouse spermatogenesis

Dev Growth Differ

(1997)

D.L. Cadena et al.

The product of the MLD gene is a member of the membrane fatty acid desaturase family: overexpression of MLD inhibits EGF receptor biosynthesis

Biochemistry

(1997)

F. Omae et al.

DES2 protein responsible for phytoceramide biosynthesis in the mouse small intestine

Biochem J

(2004)

Cited by (83)

Targeting dihydroceramide desaturase 1 (Des1): Syntheses of ceramide analogues with a rigid scaffold, inhibitory assays, and AlphaFold2-assisted structural insights reveal cyclopropenone PR280 as a potent inhibitor
2024, Bioorganic Chemistry
Dihydroceramide desaturase 1 (Des1) catalyzes the formation of a CC double bond in dihydroceramide to furnish ceramide. Inhibition of Des1 is related to cell cycle arrest and programmed cell death. The lack of the Des1 crystalline structure, as well as that of a close homologue, hampers the detailed understanding of its inhibition mechanism and difficults the design of new inhibitors, thus making Des1 a strategic target. Based on previous structure-activity studies, different ceramides containing rigid scaffolds were designed. The synthesis and evaluation of these compounds as Des1 inhibitors allowed the identification of PR280 as a better Des 1 inhibitor in vitro (IC₅₀ = 700 nM) than GT11 and XM462, the current reference inhibitors. This cyclopropenone ceramide was obtained in a 6-step synthesis with a 24 % overall yield. The highly confident 3D structure of Des1, recently predicted by AlphaFold2, served as the basis for conducting docking studies of known Des1 inhibitors and the ceramide derivatives synthesized by us in this study. For this purpose, a complete holoprotein structure was previously constructed. This study has allowed a better knowledge of key ligand-enzyme interactions for Des1 inhibitory activity. Furthermore, it sheds some light on the inhibition mechanism of GT11.
Activation of Sphingomyelin Phosphodiesterase 3 in Liver Regeneration Impedes the Progression of Colorectal Cancer Liver Metastasis Via Exosome-Bound Intercellular Transfer of Ceramides
2023, Cellular and Molecular Gastroenterology and Hepatology
The machinery that prevents colorectal cancer liver metastasis (CRLM) in the context of liver regeneration (LR) remains elusive. Ceramide (CER) is a potent anti-cancer lipid involved in intercellular interaction. Here, we investigated the role of CER metabolism in mediating the interaction between hepatocytes and metastatic colorectal cancer (CRC) cells to regulate CRLM in the context of LR.
Mice were intrasplenically injected with CRC cells. LR was induced by 2/3 partial hepatectomy (PH) to mimic the CRLM in the context of LR. The alteration of corresponding CER-metabolizing genes was examined. The biological roles of CER metabolism in vitro and in vivo were examined by performing a series of functional experiments.
Induction of LR augmented apoptosis but promoted matrix metalloproteinase 2 (MMP2) expression and epithelial-mesenchymal transition (EMT) to increase the invasiveness of metastatic CRC cells, resulting in aggressive CRLM. Up-regulation of sphingomyelin phosphodiesterase 3 (SMPD3) was determined in the regenerating hepatocytes after LR induction and persisted in the CRLM-adjacent hepatocytes after CRLM formation. Hepatic Smpd3 knockdown was found to further promote CRLM in the context of LR by abolishing mitochondrial apoptosis and augmenting the invasiveness in metastatic CRC cells by up-regulating MMP2 and EMT through promoting the nuclear translocation of β-catenin. Mechanistically, we found that hepatic SMPD3 controlled the generation of exosomal CER in the regenerating hepatocytes and the CRLM-adjacent hepatocytes. The SMPD3-produced exosomal CER critically conducted the intercellular transfer of CER from the hepatocytes to metastatic CRC cells and impeded CRLM by inducing mitochondrial apoptosis and restricting the invasiveness in metastatic CRC cells. The administration of nanoliposomal CER was found to suppress CRLM in the context of LR substantially.
SMPD3-produced exosomal CER constitutes a critical anti-CRLM mechanism in LR to impede CRLM, offering the promise of using CER as a therapeutic agent to prevent the recurrence of CRLM after PH.
Ceramide biosynthesis is critical for establishment of the intracellular niche of Toxoplasma gondii
2022, Cell Reports
Toxoplasma gondii possesses sphingolipid synthesis capabilities and is equipped to salvage lipids from its host. The contribution of these two routes of lipid acquisition during parasite development is unclear. As part of a complete ceramide synthesis pathway, T. gondii expresses two serine palmitoyltransferases (TgSPT1 and TgSPT2) and a dihydroceramide desaturase. After deletion of these genes, we determine their role in parasite development in vitro and in vivo during acute and chronic infection. Detailed phenotyping through lipidomic approaches reveal a perturbed sphingolipidome in these mutants, characterized by a drastic reduction in ceramides and ceramide phosphoethanolamines but not sphingomyelins. Critically, parasites lacking TgSPT1 display decreased fitness, marked by reduced growth rates and a selective defect in rhoptry discharge in the form of secretory vesicles, causing an invasion defect. Disruption of de novo ceramide synthesis modestly affects acute infection in vivo but severely reduces cyst burden in the brain of chronically infected mice.
Identification and characterization of 3-ketosphinganine reductase activity encoded at the BT_0972 locus in Bacteroides thetaiotaomicron
2022, Journal of Lipid Research
Citation Excerpt :
As a positive control for 3-KDSR activity, we then cotransformed S. cerevisiae 3-KDSR (TSC10) and confirmed the conversion of 3-KDS to SA (Fig. 2A–C). As SA can also be produced by ceramide hydrolysis (36–38), a potential contaminant from complex bacterial media, we employed the use of PAalk, a labeled analog of PA, and successfully generated SAalk in a BtSPT-TSC10-dependent manner, validating our screening platform. We then constructed expression plasmids for our B. thetaiotaomicron KDSR candidates and individually transformed them into our BtSPT E. coli background.
Bacterial sphingolipid synthesis is important for the fitness of gut commensal bacteria with an implied potential for regulating mammalian host physiology. Multiple steps in bacterial sphingolipid synthesis pathways have been characterized previously, with the first step of de novo sphingolipid synthesis being well conserved between bacteria and eukaryotes. In mammals, the subsequent step of de novo sphingolipid synthesis is catalyzed by 3-ketosphinganine reductase, but the protein responsible for this activity in bacteria has remained elusive. In this study, we analyzed the 3-ketosphinganine reductase activity of several candidate proteins in Bacteroides thetaiotaomicron chosen based on sequence similarity to the yeast 3-ketosphinganine reductase gene. We further developed a metabolomics-based 3-ketosphinganine reductase activity assay, which revealed that a gene at the locus BT_0972 encodes a protein capable of converting 3-ketosphinganine to sphinganine. Taken together, these results provide greater insight into pathways for bacterial sphingolipid synthesis that can aid in future efforts to understand how microbial sphingolipid synthesis modulates host-microbe interactions.
Cholesterol and ceramide: An unlikely pair
2022, Cholesterol: From Chemistry and Biophysics to the Clinic
Cholesterol (Chol) has been commonly considered as a structural component in lipid membranes, with the capacity to induce formation of liquid-ordered platforms that might in turn have a decisive effect on membrane trafficking and protein clustering. Meanwhile, sphingolipids have appeared as important pieces of the “membrane puzzle,” with many of them playing bioactive roles in a variety of cellular processes. Among sphingolipids, some of them (e.g., sphingomyelins, SM) appear to exhibit a preferential interaction with Chol, while others (such as ceramides, Cer) have been described to compete with Chol for the same interaction sites, mostly in the SM amide group, due to the high hydrophobicity of both molecules. However, publications in the last decade have demonstrated that Chol and Cer may not always displace each other; rather, they can interact in a mutually stabilizing fashion, particularly in the presence of SM. More importantly, experiments have shown that this interaction may also occur in predominantly fluid (but heterogeneous) membranes. Furthermore, while the early experiments to probe this interaction explored the effects of C16:0 sphingolipids, newer reports have shown that Chol-Cer interactions are also possible in the presence of C24:0 and/or C24:1 sphingolipids, which are (together with C16:0) among the most abundant sphingolipid species occurring in natural membranes. In this manuscript, we explore the latest reports on this issue, which are mainly centered on different scenarios where Chol-Cer interactions may appear, and the putative relevance of such interactions on membrane functionality.
On the nature of ceramide-mitochondria interactions – Dissection using comprehensive mitochondrial phenotyping
2021, Cellular Signalling
Sphingolipids are a unique class of lipids owing to their non-glycerol-containing backbone, ceramide, that is constructed from a long-chain aliphatic amino alcohol, sphinganine, to which a fatty acid is attached via an amide bond. Ceramide plays a star role in the initiation of apoptosis by virtue of its interactions with mitochondria, a control point for a downstream array of signaling cascades culminating in apoptosis. Many pathways converge on mitochondria to elicit mitochondrial outer membrane permeabilization (MOMP), a step that corrupts bioenergetic service. Although much is known regarding ceramides interaction with mitochondria and the ensuing cell signal transduction cascades, how ceramide impacts the elements of mitochondrial bioenergetic function is poorly understood. The objective of this review is to introduce the reader to sphingolipid metabolism, present a snapshot of mitochondrial respiration, elaborate on ceramides convergence on mitochondria and the upstream players that collaborate to elicit MOMP, and introduce a mitochondrial phenotyping platform that can be of utility in dissecting the fine-points of ceramide impact on cellular bioenergetics.

View all citing articles on Scopus

¹: These authors contributed equally to this review.

View full text

ReviewDihydroceramide desaturase and dihydrosphingolipids: Debutant players in the sphingolipid arena

Abstract

Section snippets

Foreword

Dihydroceramide desaturase

Des1 Inhibitors

Dihydroceramide and dihydrosphingolipids: innocent bystanders?

Des1 as a therapeutic target

Conclusions and perspectives

Acknowledgements

J Biol Chem

Biochim Biophys Acta

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Methods Enzymol

FEBS Lett

J Biol Chem

FEBS Lett

FEBS Lett

Biochimie

J Biol Chem

J Biol Chem

J Chromatogr A

Free Radic Biol Med

J Biol Chem

J Biol Chem

Cancer Lett

Free Radic Biol Med

J Biol Chem

Biochim Biophys Acta

Blood

Biochim Biophys Acta

J Biol Chem

J Biol Chem

Chem Biol

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Biochim Biophys Acta

J Lipid Res

J Biol Chem

Biochim Biophys Acta

J Lipid Res

Cancer Lett

Cancer Lett

Stereospecificities in the introduction of the 4t-double bond into sphinganine yielding 4t-sphingenine (sphingosine)

Hoppe Seylers Z Physiol Chem

Stereospecificities in the metabolic reactions of the four isomeric sphinganines (dihydrosphingosines) in rat liver

Hoppe Seylers Z Physiol Chem

Conversion of dihydroceramide into ceramide: involvement of a desaturase

Biochem J

Dihydroceramide:sphinganine C-4 hydroxylation requires DES2 hydroxylase and the membrane form of cytochrome b5

Biochem J

Degenerative spermatocyte, a novel gene encoding a transmembrane protein required for the initiation of meiosis in Drosophila spermatogenesis

Mol Gen Genet

Mdes, a mouse homolog of the Drosophila degenerative spermatocyte gene is expressed during mouse spermatogenesis

Dev Growth Differ

The product of the MLD gene is a member of the membrane fatty acid desaturase family: overexpression of MLD inhibits EGF receptor biosynthesis

Biochemistry

DES2 protein responsible for phytoceramide biosynthesis in the mouse small intestine

Biochem J

Review
Dihydroceramide desaturase and dihydrosphingolipids: Debutant players in the sphingolipid arena