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Identification of physiological modulators of nuclear hormone receptor (NHR) activity is paramount for understanding the link between metabolism and transcriptional networks that orchestrate development and cellular physiology. Using libraries of metabolic enzymes alongside their substrates and products, we identify 1-deoxysphingosines as modulators of the activity of NR2F1 and 2 (COUP-TFs), which are orphan NHRs that are critical for development of the nervous system, heart, veins, and lymphatic vessels. We show that these non-canonical alanine-based sphingolipids bind to the NR2F1/2 ligand-binding domains (LBDs) and modulate their transcriptional activity in cell-based assays at physiological concentrations. Furthermore, inhibition of sphingolipid biosynthesis phenocopies NR2F1/2 deficiency in endothelium and cardiomyocytes, and increases in 1-deoxysphingosine levels activate NR2F1/2-dependent differentiation programs. Our findings suggest that 1-deoxysphingosines are physiological regulators of NR2F1/2-mediated transcription., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

1-deoxy-sphingolipids, also known as atypical sphingolipids, are directly implicated in the development and progression of hereditary sensory and autonomic neuropathy type 1 and diabetes type 2. The mechanisms underlying their patho-physiological actions are yet to be elucidated. Accumulating evidence suggests that the biological actions of canonical sphingolipids are triggered by changes promoted on membrane organization and biophysical properties. However, little is known regarding the biophysical implications of atypical sphingolipids. In this study, we performed a comprehensive characterization of the effects of the naturally occurring 1-deoxy-dihydroceramide, 1-deoxy-ceramide Δ14Z and 1-deoxymethyl-ceramide Δ3E in the properties of a fluid membrane. In addition, to better define which structural features determine sphingolipid ability to form ordered domains, the synthetic 1-O-methyl-ceramide Δ4E and 1-deoxy-ceramide Δ4E were also studied. Our results show that natural and synthetic 1-deoxy(methyl)-sphingolipids fail to laterally segregate into ordered domains as efficiently as the canonical C16-ceramide. The impaired ability of atypical sphingolipids to form ordered domains was more dependent on the presence, position, and configuration of the sphingoid base double bond than on the structure of its C1 functional group, due to packing constraints introduced by an unsaturated backbone. Nonetheless, absence of a hydrogen bond donor and acceptor group at the C1 position strongly reduced the capacity of atypical sphingolipids to form gel domains. Altogether, the results showed that 1-deoxy(methyl)-sphingolipids induce unique changes on the biophysical properties of the membranes, suggesting that these alterations might, in part, trigger the patho-biological actions of these lipids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Sphingolipids are ubiquitous in eukaryotic plasma membranes and play major roles in human and animal physiology and disease. This class of lipids is usually defined as being derivatives of sphingosine, a long-chain 1,3-dihydroxy-2-amino alcohol. Various pathological conditions such as diabetes or neuropathy have been associated with changes in the sphingolipidome and an increased biosynthesis of structurally altered non-canonical sphingolipid derivatives. These unusual or non-canonical sphingolipids hold great promise as potential diagnostic markers. However, due to their low concentrations and the unavailability of suitable standards, the research to explore the secret of this class of 'Sphinx' lipids is ultimately hampered. Therefore, the development of efficient and facile syntheses of standard compounds is a key endeavor. Here, we present various chemical approaches for stereoselective synthesis and in-depth chemical characterization of a set of novel sphingoid bases which were recently utilized as valuable tools to explore the metabolism and biophysical properties of sphingolipids, but also to develop efficient analytical methods for their detection and quantification.

1-Deoxysphingolipids are a recently described class of sphingolipids that have been shown to be associated with several disease states including diabetic and hereditary neuropathy. The identification and characterization of 1-deoxysphingolipids and their metabolites is therefore highly important. However, exact structure determination requires a combination of sophisticated analytical techniques due to the presence of various isomers, such as ketone/alkenol isomers, carbon-carbon double-bond (C=C) isomers and hydroxylation regioisomers. Here we demonstrate that cryogenic gas-phase infrared (IR) spectroscopy of ionized 1-deoxysphingolipids enables the identification and differentiation of isomers by their unique spectroscopic fingerprints. In particular, C=C bond positions and stereochemical configurations can be distinguished by specific interactions between the charged amine and the double bond. The results demonstrate the power of gas-phase IR spectroscopy to overcome the challenge of isomer resolution in conventional mass spectrometry and pave the way for deeper analysis of the lipidome., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C 14 to C 18 , forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso -branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso -methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins., Competing Interests: The authors declare no competing interest.

Förster resonance energy transfer (FRET) probes are unique tools in biology, as they allow for a non-destructive monitoring of a certain state of a biomolecule or of an artificial substrate within living cells in real time. FRET substrates indicate their relative cleavage rate and thus the in situ activity of a given enzyme. In contrast to quenched probes or turn-on probes, one of the two separate signals of the FRET probes can be used as internal reference, which makes ratio-imaging and quantitation of cleavage events independent of cellular delivery possible. In this review, we describe the first examples of sphingolipid FRET probes in comparison to different alternative probes. Finally, we give an outlook on future probes and their potential application., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The generation of most sphingolipids (SPLs) starts with condensation between serine and an activated long-chain fatty acid catalyzed by serine palmitoyltransferase (SPT). SPT can also use other amino acids to generate small quantities of noncanonical SPLs. The balance between serine-derived and noncanonical SPLs is pivotal; for example, hereditary sensory and autonomic neuropathy type I results from SPT mutations that cause an abnormal accumulation of alanine-derived SPLs. The regulatory mechanism for SPT amino acid selectivity and physiological functions of noncanonical SPLs are unknown. We investigated SPT selection of amino acid substrates by measuring condensation products of serine and alanine in yeast cultures and SPT use of serine and alanine in a TSC3 knockout model. We identified the Tsc3 subunit of SPT as a regulator of amino acid substrate selectivity by demonstrating its primary function in promoting alanine utilization by SPT and confirmed its requirement for the inhibitory effect of alanine on SPT utilization of serine. Moreover, we observed downstream metabolic consequences to Tsc3 loss: serine influx into the SPL biosynthesis pathway increased through Ypk1-depenedent activation of SPT and ceramide synthases. This Ypk1-dependent activation of serine influx after Tsc3 knockout suggests a potential function for deoxy-sphingoid bases in modulating Ypk1 signaling.

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific inhibitor HPA-12 strongly diminished sphingolipid transport to inclusions of infected CERT-knockout cells, suggesting that other HPA-12-sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome., (© 2017 John Wiley & Sons Ltd.)

The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Ceramide transfer protein (CERT) mediates non‐vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate‐limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non‐homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT‐mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96‐well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis. [ABSTRACT FROM AUTHOR]

The topic of ceramidases has experienced an enormous boost during the last few years. Ceramidases catalyze the degradation of ceramide to sphingosine and fatty acids. Ceramide is not only the central hub of sphingolipid biosynthesis and degradation, it is also a key molecule in sphingolipid signaling, promoting differentiation or apoptosis. Acid ceramidase inhibition sensitizes certain types of cancer to chemo- and radio-therapy and this is suggestive of a role of acid ceramidase inhibitors as chemo-sensitizers which can act synergistically with chemo-therapeutic drugs. In this review, we summarize the development of ceramide analogues as first-generation ceramidase inhibitors together with data on their activity in cells and disease models. Furthermore, we describe the recent developments that have led to highly potent second-generation ceramidase inhibitors that act at nanomolar concentrations. In the third part, various assays of ceramidases are described and their relevance for accurately measuring ceramidase activities and for the development of novel inhibitors is highlighted. Besides potential clinical implications, the recent improvements in ceramidase inhibition and assaying may help to better understand the mechanisms of ceramide biology. [ABSTRACT FROM AUTHOR]

The equilibrium between the pro-apoptotic ceramide and pro-vital sphingosine-1-phosphate is considered to be decisive for cell death or survival. The different ceramidases thus play key roles in cell fate and might offer attractive targets for pharmacological intervention. Although until recently only moderately active inhibitors have been described, first in vivo experiments suggest activity against cancer cell survival and multi-drug resistance. Here, we provide a brief overview on the different ceramidases, and we will review the known inhibitors and current strategies for further inhibitor development. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

HPA-12 is an inhibitor of CERT-mediated non-vesicular transport of ceramide from the ER membranes to the Golgi apparatus. The inhibitor effectively blocks the synthesis of the membrane lipid sphingomyelin and may represent a novel drug prototype. Previous syntheses relied on non-commercial catalysts or specialized chemistries. Here we present a straightforward and effective method to synthesize HPA-12 from commercially available protected L-serinol in four steps. Some new analogues were synthesized and evaluated for their CERT-binding activity. [ABSTRACT FROM AUTHOR]