Ceramide Transport Research Articles

A Membrane-Assisted Mechanism for the Release of Ceramide from the CERT START Domain.

Ceramide transfer protein CERT is the mediator of nonvesicular transfer of ceramide from the ER to Golgi. In CERT, START is the domain responsible for the binding and transport of ceramide. A wealth of structural data has revealed a helix-grip fold surrounding a large hydrophobic cavity holding the ceramide. Yet, little is known about the mechanisms by which START releases the ceramide through the polar region and into the packed environment of cellular membranes. As such events do not lend themselves easily to experimental investigations, we used multiple unbiased microsecond-long molecular simulations. We propose a membrane-assisted mechanism in which the membrane acts as an allosteric effector initiating the release of ceramide and where the passage of the ceramide acyl chains is facilitated by the intercalation of a single phosphatidylcholine lipid in the cavity, practically greasing the ceramide way out. We verify using free energy calculation and experimental lipidomics data that CERT forms stable complexes with phosphatidylcholine lipids, in addition to ceramide, thus providing validation for the proposed mechanism.

Ceramide sorting into non-vesicular transport is independent of acyl chain length in budding yeast

The transport of ceramide from the endoplasmic reticulum (ER) to the Golgi is a key step in the synthesis of complex sphingolipids, the main building blocks of the plasma membrane. In yeast, ceramide is transported to the Golgi either through ATP-dependent COPII vesicles of the secretory pathway or by ATP-independent non-vesicular transport that involves tethering proteins at ER-Golgi membrane contact sites. Studies in both mammalian and yeast cells reported that vesicular transport mainly carries ceramide containing very long chain fatty acids, while the main mammalian non-vesicular ceramide transport protein CERT only transports ceramides containing short chain fatty acids. However, if non-vesicular ceramide transport in yeast similarly favors short chain ceramides remained unanswered. Here we employed a yeast GhLag1 strain in which the endogenous ceramide synthase is replaced by the cotton-derived GhLag1 gene, resulting in the production of short chain C18 rather than C26 ceramides. We show that block of vesicular transport through ATP-depletion or the use of temperature-sensitive sec mutants caused a reduction in inositolphosphorylceramide (IPC) synthesis to similar extent in WT and GhLag1 backgrounds. Since the remaining IPC synthesis is a readout for non-vesicular ceramide transport, our results indicate that non-vesicular ceramide transport is neither blocked nor facilitated when only short chain ceramides are present. Therefore, we propose that the sorting of ceramide into non-vesicular transport is independent of acyl chain length in budding yeast.

Locational and functional characterization of PI4KB in the mouse embryo.

Phosphatidylinositol 4-kinase beta (PI4KB) is a member of the PI4K family, which is mainly enriched and functions in the Golgi apparatus. The kinase domain of PI4KB catalyzes the phosphorylation of phosphatidylinositol to form phosphatidylinositol 4-phosphate, a process that regulates various sub-cellular events, such as non-vesicular cholesterol and ceramide transport, protein glycosylation, and vesicle transport, as well as cytoplasmic division. In this study, a strain of PI4KB knockout mouse, immunofluorescence, reverse transcription polymerase chain reaction and microinjection were used to characterize the cytological location and biological function of PI4KB in the mouse embryos. we found that knocking down Pi4kb in mouse embryos resulted in embryonic lethality at around embryonic day (E) 7.5. Additionally, we observed dramatic fluctuations in PI4KB expression during the development of preimplantation embryos, with high expression in the 4-cell and morula stages. PI4KB colocalized with the Golgi marker protein TGN46 in the perinuclear and cytoplasmic regions in early blastomeres. Postimplantation, PI4KB was highly expressed in the epiblast of E7.5 embryos. Treatment of embryos with PI4KB inhibitors was found to inhibit the development of the morula into a blastocyst and the normal progression of cytoplasmic division during the formation of a 4-cell embryo. These findings suggest that PI4KB plays an important role in mouse embryogenesis by regulating various intracellular vital functions of embryonic cells.

DGARM/C10orf76/ARMH3 for Ceramide Transfer Zone at the Endoplasmic Reticulum-Distal Golgi Contacts.

Phosphatidylinositol 4-monophosphate (PtdIns(4)P) is one of the key membrane components which mark the membrane contact sites. In the mammalian Golgi complex, PtdIns(4)P is produced at various subregions via specific mechanisms for each site. Particularly, PtdIns(4)P pools generated at the distal Golgi regions are pivotal for the determination of membrane contacts between the endoplasmic reticulum (ER) and Golgi, at which inter-organelle lipid transport takes place. In this short review, we will focus on C10orf76 (or ARMH3), which we propose to rename as DGARM after a distal Golgi armadillo repeat protein, for its function in generating a PtdIns(4)P pool crucial for ER-to-distal Golgi ceramide transport. We further discuss from the viewpoint of the evolutionary conservation of DGARM.

The ceramide transport protein CERT is involved in alkylacylglycerol transfer from the ER to the Golgi for the biosynthesis of ether phospholipid

Ether phospholipids are synthesized by a series of enzymes localized in peroxisomes, the endoplasmic reticulum (ER), and the Golgi apparatus. During this process, the lipid intermediate alkylacylglycerol (AAG) synthesized in the ER is transferred from the site of its synthesis to the Golgi apparatus. In this study, we determined whether ceramide transport protein (CERT) is a candidate for AAG transfer. A lipid transfer assay revealed that CERT can mediate AAG transfer between phospholipid liposomes. AAG transport activity was markedly inhibited by the CERT inhibitor HPA-12 and reduced when the lipid transport domain of CERT was deleted. Suppression of CERT in HEK293 cells resulted in increased levels of plasmanyl-PC, which is synthesized by the ER-residing choline/ethanolamine phosphotransferase 1 (CEPT1). The mRNA levels and enzymatic activity of plasmanyl-PC synthesizing enzymes were not increased in CERT-deficient cells, indicating that the increase in plasmanyl-PC results from AAG accumulation in the ER. Re-introduction of CERT into CERT-deficient cells caused a decrease in plasmanyl-PC. Taken together, our findings suggest for the first time that CERT is involved in the transfer of AAG from the ER to the Golgi apparatus and plays a role in the biosynthesis of ether phospholipids.

The C10orf76-PI4KB axis orchestrates CERT-mediated ceramide trafficking to the distal Golgi.

Phosphatidylinositol 4-monophosphate [PtdIns(4)P] is a precursor for various phosphoinositides but also a membrane-embedded component crucial for membrane contact sites (MCSs). Several lipid transfer proteins are recruited to MCSs by recognizing PtdIns(4)P; however, it remains poorly elucidated how the production of PtdIns(4)P for lipid transport at MCSs is regulated. Following human genome-wide screening, we discovered that the PtdIns(4)P-related genes PI4KB, ACBD3, and C10orf76 are involved in endoplasmic reticulum-to-Golgi trafficking of ceramide by the ceramide transport protein CERT. CERT preferentially utilizes PtdIns(4)P generated by PI4KB recruited to the Golgi by C10orf76 rather than by ACBD3. Super-resolution microscopy observation revealed that C10orf76 predominantly localizes at distal Golgi regions, where sphingomyelin (SM) synthesis primarily occurs, while the majority of ACBD3 localizes at more proximal regions. This study provides a proof-of-concept that distinct pools of PtdIns(4)P are generated in different subregions, even within the same organelle, to facilitate interorganelle metabolic channeling for the ceramide-to-SM conversion.

CERT1 mutations perturb human development by disrupting sphingolipid homeostasis.

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

The Small GTPase Rab14 Regulates the Trafficking of Ceramide from Endoplasmic Reticulum to Golgi Apparatus and Facilitates Classical Swine Fever Virus Assembly.

Classical swine fever virus (CSFV) is a highly pathogenic RNA virus belonging to the Flaviviridae family that can cause deadly classical swine fever (CSF) in pigs. However, the molecular details of virus replication in the host are still unclear. Our previous studies have reported that several Rab proteins mediate CSFV entry into host cells, but it is unknown whether CSFV hijacks other Rab proteins for effective viral infection. Here, we systematically studied the role of Rab14 protein in regulating lipid metabolism for promoting viral assembly. First, Rab14 knockdown and overexpression significantly affected CSFV replication, indicating the essential role of Rab14 in CSFV infection. Interestingly, Rab14 could significantly affect virus replication in the late stage of infection. Mechanistically, CSFV NS5A recruited Rab14 to the ER, followed by ceramide transportation to the Golgi apparatus, where sphingomyelin was synthesized. The experimental data of small molecule inhibitors, RNA interference, and replenishment assay showed that the phosphatidylinositol-3-kinase (PI3K)/AKT/AS160 signaling pathway regulated the function of Rab14 to affect the transport of ceramide. More importantly, sphingomyelin on the Golgi apparatus contributed to the assembly of viral particles. Blockage of the Rab14 regulatory pathway induced the reduction of the content of sphingomyelin on the Golgi apparatus, impairing the assembly of virus particles. Our study clarifies that Rab14 regulates lipid metabolism and promotes CSFV replication, which provides insight into a novel function of Rab14 in regulating vesicles to transport lipids to the viral assembly factory. IMPORTANCE The Rab protein family members participate in the viral replication of multiple viruses and play important roles in the virus infection cycle. Our previous research focused on Rab5/7/11, which regulated the trafficking of vesicles in the early stage of CSFV infection, especially in viral endocytosis. However, the role of other Rab proteins in CSFV replication is unclear and needs further clarification. Strikingly, we screened some Rabs and found the important role of Rab14 in CSFV infection. Virus infection mobilized Rab14 to regulate the vesicle to transport ceramide from the ER to the Golgi apparatus, further promoting the synthesis of sphingomyelin and facilitating virus assembly. The treatment of inhibitors showed that the lipid transport mediated by Rab14 was regulated by the PI3K/AKT/AS160 signaling pathway. Knockdown of Rab14 or the treatment with PI3K/AKT/AS160 inhibitors reduced the ceramide content in infected cells and hindered virus assembly. Our study is the first to explain that vesicular lipid transport regulated by Rab promotes CSFV assembly, which is conducive to the development of antiviral drugs.

Ligand Activation of the Aryl Hydrocarbon Receptor Upregulates Epidermal Uridine Diphosphate Glucose Ceramide Glucosyltransferase and Glucosylceramides

Ligand activation of the aryl hydrocarbon receptor (AHR) accelerates keratinocyte differentiation and the formation of the epidermal permeability barrier. Several classes of lipids, including ceramides, are critical to the epidermal permeability barrier. In normal human epidermal keratinocytes, the AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, increased RNA levels of ceramide metabolism and transport genes: uridine diphosphate glucose ceramide glucosyltransferase (UGCG), ABCA12, GBA1, and SMPD1. Levels ofabundant skin ceramides were also increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin. These included themetabolites synthesized by UGCG, glucosylceramides, and acyl glucosylceramides. Chromatin immunoprecipitation-sequence analysis and luciferase reporter assays identified UGCG as a direct AHR target. The AHR antagonist, GNF351, inhibited the 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated RNA and transcriptional increases. Tapinarof, an AHR ligand approved for the treatment of psoriasis, increased UGCG RNA, protein, and its lipid metabolites hexosylceramides as well as increased the RNA expression of ABCA12, GBA1, and SMPD1. In Ahr-null mice, Ugcg RNA and hexosylceramides were lower than those in the wild type. These results indicate that the AHR regulates the expression of UGCG, a ceramide-metabolizing enzyme required for ceramide trafficking, keratinocyte differentiation, and epidermal permeability barrier formation.

Yeast Svf1 binds ceramides and contributes to sphingolipid metabolism at the ER cis-Golgi interface.

Ceramides are essential precursors of complex sphingolipids and act as potent signaling molecules. Ceramides are synthesized in the endoplasmic reticulum (ER) and receive their head-groups in the Golgi apparatus, yielding complex sphingolipids (SPs). Transport of ceramides between the ER and the Golgi is executed by the essential ceramide transport protein (CERT) in mammalian cells. However, yeast cells lack a CERT homolog, and the mechanism of ER to Golgi ceramide transport remains largely elusive. Here, we identified a role for yeast Svf1 in ceramide transport between the ER and the Golgi. Svf1 is dynamically targeted to membranes via an N-terminal amphipathic helix (AH). Svf1 binds ceramide via a hydrophobic binding pocket that is located in between two lipocalin domains. We showed that Svf1 membrane-targeting is important to maintain flux of ceramides into complex SPs. Together, our results show that Svf1 is a ceramide binding protein that contributes to sphingolipid metabolism at Golgi compartments.

Pathogen vacuole membrane contact sites - close encounters of the fifth kind.

Vesicular trafficking and membrane fusion are well-characterized, versatile, and sophisticated means of 'long range' intracellular protein and lipid delivery. Membrane contact sites (MCS) have been studied in far less detail, but are crucial for 'short range' (10-30nm) communication between organelles, as well as between pathogen vacuoles and organelles. MCS are specialized in the non-vesicular trafficking of small molecules such as calcium and lipids. Pivotal MCS components important for lipid transfer are the VAP receptor/tether protein, oxysterol binding proteins (OSBPs), the ceramide transport protein CERT, the phosphoinositide phosphatase Sac1, and the lipid phosphatidylinositol 4-phosphate (PtdIns(4)P). In this review, we discuss how these MCS components are subverted by bacterial pathogens and their secreted effector proteins to promote intracellular survival and replication.

Mechanisms of Nonvesicular Ceramide Transport.

Ceramides, as key components of cellular membranes, play essential roles in various cellular processes, including apoptosis, cell proliferation, and cell signaling. Ceramides are the precursors of all complex sphingolipids in eukaryotic cells. They are synthesized in the endoplasmic reticulum and are further processed at the Golgi apparatus. Therefore, ceramides have to be transported between these two organelles. In mammalian cells, the ceramide transfer protein forms a contact site between the ER and the trans-Golgi region and transports ceramide utilizing its steroidogenic acute regulatory protein-related lipid transfer domain. In yeast, multiple mechanisms of nonvesicular ceramide transport have been described. This involves the nuclear-vacuolar junction protein Nvj2, the yeast tricalbin proteins, and the lipocalin-like protein Svf1. This review aims to provide a comprehensive overview of nonvesicular ceramide transport mechanisms and their relevance in cellular physiology. We will highlight the physiological and pathological consequences of perturbations in nonvesicular ceramide transport and discuss future challenges in identifying and analyzing ceramide transfer proteins.

Chlamydial Infection-Dependent Synthesis of Sphingomyelin as a Novel Anti-Chlamydial Target of Ceramide Mimetic Compounds.

The obligate intracellular bacterium Chlamydia trachomatis is the major causative agent of bacterial sexually transmitted diseases worldwide. In infected cells, the ceramide transport protein (CERT) is recruited to inclusions, where C. trachomatis replicates using host-synthesized ceramide. The ceramide is converted to sphingomyelin (SM) by a chlamydial infection-dependent SM synthesis (cidSM-synthesis) pathway, which occurs even in the absence of the SM synthases (SMS)-1 and -2 of host cells. The ceramide mimetic compound (1R,3S)-HPA-12 and the nonmimetic compound E16A, both of which are potent inhibitors of CERT, repressed the proliferation of C. trachomatis in HeLa cells. Unexpectedly, (1R,3R)-HPA-12, a ceramide mimetic compound that lacks CERT inhibitory activity, also exhibited potent anti-chlamydial activity. Using endogenous SMS-knockout mutant HeLa cells, we revealed that (1R,3R)-HPA-12 mildly inhibited cidSM-synthesis. In addition, LC-MS analysis revealed that (1R,3R)-HPA-12 is converted to a phosphocholine-conjugated metabolite in an infection-dependent manner. Imaging analysis with a fluorescent analog of ceramide suggested that cidSM-synthesis occurs in the bacterial bodies and/or inclusions. Collectively, these results suggested that (1R,3R)-HPA-12 exerts its anti-chlamydia activity not only as an inhibitor of cidSM-synthesis, but also via putative toxic effects of its phosphocholine adduct, which is most likely produced by the cidSM-synthesis route.

Herpes simplex virus 1 protein pUL21 alters ceramide metabolism by activating the interorganelle transport protein CERT

Herpes simplex virus (HSV)-1 dramatically alters the architecture and protein composition of cellular membranes during infection, but its effects upon membrane lipid composition remain unclear. HSV-1 pUL21 is a virus-encoded protein phosphatase adaptor that promotes dephosphorylation of multiple cellular and virus proteins, including the cellular ceramide (Cer) transport protein CERT. CERT mediates nonvesicular Cer transport from the endoplasmic reticulum to the trans-Golgi network, whereupon Cer is converted to sphingomyelin (SM) and other sphingolipids that play important roles in cellular proliferation, signaling, and membrane trafficking. Here, we use click chemistry to profile the kinetics of sphingolipid metabolism, showing that pUL21-mediated dephosphorylation activates CERT and accelerates Cer-to-SM conversion. Purified pUL21 and full-length CERT interact with submicromolar affinity, and we solve the solution structure of the pUL21 C-terminal domain in complex with the CERT Pleckstrin homology and steroidogenic acute regulatory-related lipid transfer domains using small-angle X-ray scattering. We identify a single amino acid mutation on the surface of pUL21 that disrupts CERT binding invitro and in cultured cells. This residue is highly conserved across the genus Simplexvirus. In addition, we identify a pUL21 residue essential for binding to HSV-1 pUL16. Sphingolipid profiling demonstrates that Cer-to-SM conversion is severely diminished in the context of HSV-1 infection, a defect that is compounded when infecting with a virus encoding the mutated form of pUL21 that lacks the ability to activate CERT. However, virus replication and spread in cultured keratinocytes or epithelial cells is not significantly altered when pUL21-mediated CERT dephosphorylation is abolished. Collectively, we demonstrate that HSV-1 modifies sphingolipid metabolism via specific protein-protein interactions.

Involvement of a Cluster of Basic Amino Acids in Phosphorylation-Dependent Functional Repression of the Ceramide Transport Protein CERT.

Ceramide transport protein (CERT) mediates ceramide transfer from the endoplasmic reticulum to the Golgi for sphingomyelin (SM) biosynthesis. CERT is inactivated by multiple phosphorylation at the serine-repeat motif (SRM), and mutations that impair the SRM phosphorylation are associated with a group of inherited intellectual disorders in humans. It has been suggested that the N-terminal phosphatidylinositol 4-monophosphate [PtdIns(4)P] binding domain and the C-terminal ceramide-transfer domain of CERT physically interfere with each other in the SRM phosphorylated state, thereby repressing the function of CERT; however, it remains unclear which regions in CERT are involved in the SRM phosphorylation-dependent repression of CERT. Here, we identified a previously uncharacterized cluster of lysine/arginine residues that were predicted to be located on the outer surface of a probable coiled-coil fold in CERT. Substitutions of the basic amino acids in the cluster with alanine released the SRM-dependent repression of CERT activities, i.e., the synthesis of SM, PtdIns(4)P-binding, vesicle-associated membrane protein-associated protein (VAP) binding, ceramide-transfer activity, and localization to the Golgi, although the effect on SM synthesis activity was only partially compromised by the alanine substitutions, which moderately destabilized the trimeric status of CERT. These results suggest that the basic amino acid cluster in the coiled-coil region is involved in the regulation of CERT function.

Compartmentalization of casein kinase 1 γ CSNK1G controls the intracellular trafficking of ceramide

SummaryCasein kinase 1 γ (CK1G) is involved in the regulation of various cellular functions. For instance, the ceramide transport protein (CERT), which delivers ceramide to the Golgi apparatus for the synthesis of sphingomyelin (SM), is inactivated when it receives multiple phosphorylation by CK1G. Using human genome-wide gene disruption screening with an SM-binding cytolysin, we found that loss of the C-terminal region of CK1G3 rendered the kinase hyperactive in cells. Deletion of the C-terminal 20 amino acids or mutation of cysteine residues expected to be palmitoylated sites redistributed CK1G3 from cytoplasmic punctate compartments to the nucleocytoplasm. Wild-type CK1G3 exhibited a similar redistribution in the presence of 2-bromopalmitate, a protein palmitoylation inhibitor. Expression of C-terminal mutated CK1G1/2/3 similarly induced the multiple phosphorylation of the CERT SRM, thereby down-regulating de novo SM synthesis. These findings revealed that CK1Gs are regulated by a compartmentalization-based mechanism to access substrates present in specific intracellular organelles.

Function of ceramide transfer protein for biogenesis and sphingolipid composition of extracellular vesicles.

The formation of extracellular vesicles (EVs) is induced by the sphingolipid ceramide. How this pathway is regulated is not entirely understood. Here, we report that the ceramide transport protein (CERT) mediates a non‐vesicular transport of ceramide between the endoplasmic reticulum (ER) and the multivesicular endosome at contact sites. The process depends on the interaction of CERT's PH domain with PI4P generated by PI4KIIα at endosomes. Furthermore, a complex is formed between the START domain of CERT, which carries ceramide, and the Tsg101 protein, which is part of the endosomal sorting complex required for transport (ESCRT‐I). Inhibition of ceramide biosynthesis reduces CERT‐Tsg101 complex formation. Overexpression of CERT increases EV secretion while its inhibition reduces EV formation and the concentration of ceramides and sphingomyelins in EVs. In conclusion, we discovered a function of CERT in regulating the sphingolipid composition and biogenesis of EVs, which links ceramide to the ESCRT‐dependent pathway.

Regulation of Sphingomyelin Synthesis by Cholesterol

The most abundant sphingolipid of the plasma membrane (PM), sphingomyelin (SM), binds to cholesterol in a manner dependent on the structure of fatty acids in the SM, and it is a major determinant of the cholesterol content of the PM. Synthesis of SM in the Golgi apparatus produces an mol‐equivalent of diacylglycerol (DAG), a signaling lipid implicated in the fission of secretory vesicles from the Golgi. We speculate that coupled synthesis of SM and DAG in the terminal compartments of the Golgi results in fission of secretory vesicles enriched in SM and integral membrane proteins that favor sphingolipid and cholesterol‐rich membrane. Two classes of SMs are synthesized in the Golgi apparatus that are distinguished by the length of the fatty acyl chain on the ceramide backbone: long chain (LC; ≤C20), and very long chain (VLC; >C20) SMs. Whereas the precursor to LC‐SMs is supplied to the Golgi via non‐vesicular transport of LC‐ceramide from the ER to the Golgi by ceramide transport protein (CERT), it is speculated that VLC‐ceramide is supplied to the Golgi via bulk membrane trafficking pathways. From the Golgi, LC‐SM and VLC‐SM are trafficked to the PM in SM‐rich secretory vesicles that also contain a select cohort of secreted proteins (Deng et al., 2018).Cholesterol extraction increases sphingomyelin synthesis (Perry and Ridgway, 2006), however, the two classes of SMs are differentially regulated; we discovered that only synthesis of very long chain (>C20) SMs was increased. Furthermore, cholesterol loading reduced the synthesis of VLC‐SM, only. We then asked, what cellular components regulate VLC‐SM synthesis and how do they influence the cholesterol content of the PM and other organelles. Synthesis of VLC‐SM was inhibited by interfering with COPII‐mediated export from the ER and by depletion of ER‐localized ceramide synthase 2, which produces VLC‐ceramide. Reduced VLC‐SM synthesis resulted in the depletion of cholesterol from the PM inner leaflet and a concomitant accumulation in the cytoplasmic leaflets of the membranes of lysosomes and other intracellular organelles. Increased synthesis of VLC‐SMs by cholesterol depletion resulted in increased the level of diacylglycerol (DAG) in the Golgi and the appearance in cytoplasmic vesicles containing TGN46 which is secreted in SM‐rich secretory vesicles. The results suggest that VLC‐SM synthesis is regulated by cholesterol content on the PM and cholesterol depletion‐induced synthesis of VLC‐SM enhances SM‐specific secretory pathway from the Golgi to the PM.

Hyperosmotic Stress Induces Phosphorylation of CERT and Enhances Its Tethering throughout the Endoplasmic Reticulum

The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER–Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER–Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.

Natural Ligand-Mimetic and Nonmimetic Inhibitors of the Ceramide Transport Protein CERT.

Lipid transfer proteins (LTPs) are recognized as key players in the inter-organelle trafficking of lipids and are rapidly gaining attention as a novel molecular target for medicinal products. In mammalian cells, ceramide is newly synthesized in the endoplasmic reticulum (ER) and converted to sphingomyelin in the trans-Golgi regions. The ceramide transport protein CERT, a typical LTP, mediates the ER-to-Golgi transport of ceramide at an ER-distal Golgi membrane contact zone. About 20 years ago, a potent inhibitor of CERT, named (1R,3S)-HPA-12, was found by coincidence among ceramide analogs. Since then, various ceramide-resembling compounds have been found to act as CERT inhibitors. Nevertheless, the inevitable issue remains that natural ligand-mimetic compounds might directly bind both to the desired target and to various undesired targets that share the same natural ligand. To resolve this issue, a ceramide-unrelated compound named E16A, or (1S,2R)-HPCB-5, that potently inhibits the function of CERT has recently been developed, employing a series of in silico docking simulations, efficient chemical synthesis, quantitative affinity analysis, protein–ligand co-crystallography, and various in vivo assays. (1R,3S)-HPA-12 and E16A together provide a robust tool to discriminate on-target effects on CERT from off-target effects. This short review article will describe the history of the development of (1R,3S)-HPA-12 and E16A, summarize other CERT inhibitors, and discuss their possible applications.