Serine Palmitoyltransferase Activity Research Articles

Upregulation of the serine palmitoyltransferase subunit SPTLC2 by endoplasmic reticulum stress inhibits the hepatic insulin response

Endoplasmic reticulum (ER) stress is induced by various conditions, such as inflammation and the presence of excess nutrients. Abnormal accumulation of unfolded proteins leads to the activation of a collective signaling cascade, termed the unfolded protein response (UPR). ER stress is reported to perturb hepatic insulin response metabolism while promoting insulin resistance. Here, we report that ER stress regulates the de novo biosynthesis of sphingolipids via the activation of serine palmitoyltransferase (SPT), a rate-limiting enzyme involved in the de novo biosynthesis of ceramides. We found that the expression levels of Sptlc1 and Sptlc2, the major SPT subunits, were upregulated and that the cellular concentrations of ceramide and dihydroceramide were elevated by acute ER stress inducers in primary hepatocytes and HepG2 cells. Sptlc2 was upregulated and ceramide levels were elevated by tunicamycin in the livers of C57BL/6J wild-type mice. Analysis of the Sptlc2 promoter demonstrated that the transcriptional activation of Sptlc2 was mediated by the spliced form of X-box binding protein 1 (sXBP1). Liver-specific Sptlc2 transgenic mice exhibited increased ceramide levels in the liver and elevated fasting glucose levels. The insulin response was reduced by the inhibition of the phosphorylation of insulin receptor β (IRβ). Collectively, these results demonstrate that ER stress induces activation of the de novo biosynthesis of ceramide and contributes to the progression of hepatic insulin resistance via the reduced phosphorylation of IRβ in hepatocytes.

Human Rhinovirus Infection of the Respiratory Tract Affects Sphingolipid Synthesis.

The 17q21 asthma susceptibility locus includes asthma risk alleles associated with decreased sphingolipid synthesis, likely resulting from increased expression of ORMDL3. ORMDL3 inhibits serine-palmitoyl transferase (SPT), the rate-limiting enzyme of de novo sphingolipid synthesis. There is evidence that decreased sphingolipid synthesis is critical to asthma pathogenesis. Children with asthma and 17q21 asthma risk alleles display decreased sphingolipid synthesis in blood cells. Reduced SPT activity results in airway hyperreactivity, a hallmark feature of asthma. 17q21 asthma risk alleles are also linked to childhood infections with human rhinovirus (RV). This study evaluates the interaction of RV with the de novo sphingolipid synthesis pathway, and the alterative effects of concurrent SPT inhibition in SPT-deficient mice and human airway epithelial cells. In mice, RV infection shifted lung sphingolipid synthesis gene expression to a pattern that resembles genetic SPT deficiency, including decreased expression of Sptssa, a small SPT subunit. This pattern was pronounced in lung epithelial cellular adhesion molecule (EpCAM+) cells and reproduced in human bronchial epithelial cells. RV did not affect Sptssa expression in lung CD45+ immune cells. RV increased sphingolipids unique to the de novo synthesis pathway in mouse lung and human airway epithelial cells. Interestingly, these de novo sphingolipid species were reduced in the blood of RV-infected wild-type mice. RV exacerbated SPT deficiency-associated airway hyperreactivity. Airway inflammation was similar in RV-infected wild-type and SPT-deficient mice. This study reveals the effects of RV infection on the de novo sphingolipid synthesis pathway, elucidating a potential mechanistic link between 17q21 asthma risk alleles and rhinoviral infection.

A Dansyl-Modified Sphingosine Kinase Inhibitor DPF-543 Enhanced De Novo Ceramide Generation.

Sphingosine-1-phosphate (S1P) synthesized by sphingosine kinase (SPHK) is a signaling molecule, involved in cell proliferation, growth, differentiation, and survival. Indeed, a sharp increase of S1P is linked to a pathological outcome with inflammation, cancer metastasis, or angiogenesis, etc. In this regard, SPHK/S1P axis regulation has been a specific issue in the anticancer strategy to turn accumulated sphingosine (SPN) into cytotoxic ceramides (Cers). For these purposes, there have been numerous chemicals synthesized for SPHK inhibition. In this study, we investigated the comparative efficiency of dansylated PF-543 (DPF-543) on the Cers synthesis along with PF-543. DPF-543 deserved attention in strong cytotoxicity, due to the cytotoxic Cers accumulation by ceramide synthase (CerSs). DPF-543 exhibited dual actions on Cers synthesis by enhancing serine palmitoyltransferase (SPT) activity, and by inhibiting SPHKs, which eventually induced an unusual environment with a high amount of 3-ketosphinganine and sphinganine (SPA). SPA in turn was consumed to synthesize Cers via de novo pathway. Interestingly, PF-543 increased only the SPN level, but not for SPA. In addition, DPF-543 mildly activates acid sphingomyelinase (aSMase), which contributes a partial increase in Cers. Collectively, a dansyl-modified DPF-543 relatively enhanced Cers accumulation via de novo pathway which was not observed in PF-543. Our results demonstrated that the structural modification on SPHK inhibitors is still an attractive anticancer strategy by regulating sphingolipid metabolism.

Childhood amyotrophic lateral sclerosis caused by excess sphingolipid synthesis.

Amyotrophic lateral sclerosis (ALS) is a progressive, neurodegenerative disease of the lower and upper motor neurons with sporadic or hereditary occurrence. Age of onset, pattern of motor neuron degeneration and disease progression vary widely among individuals with ALS. Various cellular processes may drive ALS pathomechanisms, but a monogenic direct metabolic disturbance has not been causally linked to ALS. Here we show SPTLC1 variants that result in unrestrained sphingoid base synthesis cause a monogenic form of ALS. We identified four specific, dominantly acting SPTLC1 variants in seven families manifesting as childhood-onset ALS. These variants disrupt the normal homeostatic regulation of serine palmitoyltransferase (SPT) by ORMDL proteins, resulting in unregulated SPT activity and elevated levels of canonical SPT products. Notably, this is in contrast with SPTLC1 variants that shift SPT amino acid usage from serine to alanine, result in elevated levels of deoxysphingolipids and manifest with the alternate phenotype of hereditary sensory and autonomic neuropathy. We custom designed small interfering RNAs that selectively target the SPTLC1 ALS allele for degradation, leave the normal allele intact and normalize sphingolipid levels in vitro. The role of primary metabolic disturbances in ALS has been elusive; this study defines excess sphingolipid biosynthesis as a fundamental metabolic mechanism for motor neuron disease.

Detailed Mechanism of ORMDLs Function

Sphingolipids (SLs) are one of the major lipid types present in eukaryotic cells. SLs not only serve as an important constituent of the plasma membrane but also as bioactive lipids which regulate several signal transduction processes such as cell growth and differentiation. The de novo biosynthesis begins in the endoplasmic reticulum (ER) with the condensation of serine and palmitoyl Co-A by the rate-limiting enzyme, serine palmitoyltransferase (SPT). SPT is homeostatically regulated by small ER proteins: ORMDLs in mammalian cells and Orms in yeast. The ORMDLs stereo-specifically senses D-erythro ceramide to regulate SPT activity in response to the cellular SL levels. Mammalian cells have three ORMDL isoforms that show high amino acid sequence similarity, but why cells need three ORMDLs is still unknown. Here we are investigating the role of different ORMDL isoforms and the amino-acid regions of ORMDL involved in SPT regulation. We are also looking at regulation of individual ORMDL isoform protein turnover, as an additional mechanism by which SLs are maintained in the cells. The successful generation of ORMDL2/3, ORMDL1/2, and ORMDL1/3 double-knockout cell lines by CRISPR-Cas9 reveals that cells are compensating for the missing ORMDLs by increasing the protein levels of the remaining ORMDL. Surprisingly, we do not see any changes in the mRNA levels of the endogenous ORMDL in double-knockout cell lines, suggesting that ORMDL protein levels are post-transcriptionally regulated. We find an important clue to regulation by finding that ORMDLs are short-lived proteins and that their degradation is regulated by p62 receptor-mediated selective autophagy. Characterization of the ORMDL isoforms shows that individual ORMDL isoforms are alone sufficient to regulate SPT activity. Lipidomic results show that there is no significant change in total ceramide levels in the ORMDL double-knockout cell line, confirming that each individual ORMDL isoform is sufficient to control ceramide or SL levels in cells. Based on our findings, we still do not know why cells need three different ORMDL isoforms. We hypothesize that each ORMDL isoforms respond to different ceramide levels in cell. To test this hypothesis, we used a cell-free system to measure the response of each isoform to different levels of ceramide. We found that ORMDL isoforms response to different ceramide levels, to downregulate the activity of SPT. To investigate which region of ORMDL is important for its function, we have generated three deletion mutant constructs on cytosolic N- terminus & C-terminus and two polyA substitution mutant constructs on the cytosolic loop of mORMDL1 by using site-directed mutagenesis approach. We have confirmed the co-localization of mutant constructs in the ER by immunofluorescence staining. Further, we have established a platform to test our mutant constructs and we are in the process of testing the mutant constructs. These data strongly suggest that cells require an optimal amount of ORMDL protein and sense the different ceramide levels to control overall SL levels in cells by regulating SPT enzyme activity.

SPTLC1 Mutations Associated with Early Onset Amyotrophic Lateral Sclerosis Impair ORMDL Regulation

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration of lower and upper motor neurons leading to progressive muscle weakness, swallowing difficulties, and respiratory insufficiency. The majority of ALS cases occur sporadically, but a significant number (>10%) are hereditary. Although a variety of cellular processes have been implicated in ALS, a specific monogenic metabolic disruption has not been reported. Thus, the recent discovery of four dominantly inherited SPTLC1 mutations that resulting in childhood-onset ALS in seven families is significant. SPTLC1 encodes a subunit of serine palmitoyltransferase (SPT), the committed and rate-limiting enzyme of sphingolipid synthesis. The ALS mutations flank the membrane spanning domain of SPTLC1, which is not required for ER targeting, association with the SPTLC2 and ssSPT subunits, or enzymatic activity. Rather, this domain is critical for binding of the ORMDL proteins that regulate SPT. This suggested that the SPTLC1 mutations likely result in unrestrained SPT activity. Our studies reveal that these highly penetrant SPTLC1 mutations do indeed abrogate negative regulation of SPT by the ORMDL proteins and result in elevated levels of sphingolipids. Using cultured cells, patient fibroblasts, iPSC-induced MNs, and mouse models, we are investigating the importance of ORMDL regulation in the maintenance of sphingolipid homeostasis and potential mechanisms of sphingolipid mediated motor neuron death. Significantly, the ALS SPTLC1 mutations confer disease pathology by a distinctly different mechanism than SPTLC1 and SPTLC2 mutations associated with hereditary sensory and autonomic neuropathy, type 1 (HSAN1). Whereas the ALS mutations result in elevated levels of canonical sphingolipids, the HSAN1 mutations, which compromise amino acid substrate selectivity of SPT, cause accumulation of atypical deoxy-sphingoid bases that are implicated in neuronal cell death.

Antifungal activity of myriocin against Fusarium graminearum and its inhibitory effect on deoxynivalenol production in wheat grains

Fusarium graminearum, a devastating pathogenic fungus of wheat (Triticum aestivum L.), causes fusarium head blight (FHB) and produces mycotoxins such as deoxynivalenol (DON). This study aimed to explore the potency of myriocin to control postharvest infection of F. graminearum in wheat grains. Myriocin showed excellent antifungal activity against F. graminearum, as evidenced through inhibition on spore germination and mycelial growth. The mechanism of action was investigated by analyzing changes of cell membrane permeability and serine palmitoyl transferase (SPT) activity. Results indicated that myriocin inhibited F. graminearum by disrupting permeability of membrane and result in release of cellular contents. Myriocin suppressed the SPT activity and molecular docking also proved the sC interaction of myriocin-SPT. Significant morphological alterations were observed in SEM imaging, including holes on the hyphae, contraction and disruption of mycelia. In addition, myriocin also showed inhibitory effect on toxigenic activity of F. graminearum in wheat grains by reducing more than 99.0% of deoxynivalenol (DON) and its derivative 15-acetyldeoxynivalenol (15-ADON) synthesis. Quantitative real-time PCR analysis indicated that TRI5 gene expression was downregulated by myriocin and results in the reduction of toxicity. Overall, myriocin exerted effective fungicidal activity to control F. graminearum contamination in wheat grains and provided a lead compound to develop more alternatives for pathogen control.

Structural insights into the assembly and substrate selectivity of human SPT-ORMDL3 complex.

Human serine palmitoyltransferase (SPT) complex catalyzes the initial and rate-limiting step in the de novo biosynthesis of all sphingolipids. ORMDLs regulate SPT function, with human ORMDL3 being related to asthma. Here we report three high-resolution cryo-EM structures: the human SPT complex, composed of SPTLC1, SPTLC2 and SPTssa; the SPT-ORMDL3 complex; and the SPT-ORMDL3 complex bound to two substrates, PLP-L-serine (PLS) and a non-reactive palmitoyl-CoA analogue. SPTLC1 and SPTLC2 form a dimer of heterodimers as the catalytic core. SPTssa participates in acyl-CoA coordination, thereby stimulating the SPT activity and regulating the substrate selectivity. ORMDL3 is located in the center of the complex, serving to stabilize the SPT assembly. Our structural and biochemical analyses provide a molecular basis for the assembly and substrate selectivity of the SPT and SPT-ORMDL3 complexes, and lay a foundation for mechanistic understanding of sphingolipid homeostasis and for related therapeutic drug development.

CRISPR/Cas9 deletion of ORMDLs reveals complexity in sphingolipid metabolism

The serine palmitoyltransferase (SPT) complex catalyzes the rate-limiting step in the de novo biosynthesis of ceramides, the precursors of sphingolipids. The mammalian ORMDL isoforms (ORMDL1-3) are negative regulators of SPT. However, the roles of individual ORMDL isoforms are unclear. Using siRNA against individual ORMDLs, only single siORMDL3 had modest effects on dihydroceramide and ceramide levels, whereas downregulation of all three ORMDLs induced more pronounced increases. With the CRISPR/Cas9-based genome-editing strategy, we established stable single ORMDL3 KO (ORMDL3-KO) and ORMDL1/2/3 triple-KO (ORMDL-TKO) cell lines to further understand the roles of ORMDL proteins in sphingolipid biosynthesis. While ORMDL3-KO modestly increased dihydroceramide and ceramide levels, ORMDL-TKO cells had dramatic increases in the accumulation of these sphingolipid precursors. SPT activity was increased only in ORMDL-TKO cells. In addition, ORMDL-TKO but not ORMDL3-KO dramatically increased levels of galactosylceramides, glucosylceramides, and lactosylceramides, the elevated N-acyl chain distributions of which broadly correlated with the increases in ceramide species. Surprisingly, although C16:0 is the major sphingomyelin species, it was only increased in ORMDL3-KO, whereas all other N-acyl chain sphingomyelin species were significantly increased in ORMDL-TKO cells. Analysis of sphingoid bases revealed that although sphingosine was only increased 2-fold in ORMDL-TKO cells, levels of dihydrosphingosine, dihydrosphingosine-1-phosphate, and sphingosine-1-phosphate were hugely increased in ORMDL-TKO cells and not in ORMDL3-KO cells. Thus, ORMDL proteins may have a complex, multifaceted role in the biosynthesis and regulation of cellular sphingolipids.

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis. However, the role of ORMDL3 in the synthesis of LTs is not known. In this study, we used peritoneal-derived mast cells isolated from ORMDL3 KO or control mice and examined their calcium mobilization, degranulation, NF-κB inhibitor-α phosphorylation, and TNF-α production. We found that peritoneal-derived mast cells with ORMDL3 KO exhibited increased responsiveness to antigen. Detailed lipid analysis showed that compared with WT cells, ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids but also of LT signaling mediators LTB4, 6t-LTB4, LTC4, LTB5, and 6t-LTB5. The crosstalk between ORMDL3 and 5-LO metabolic pathways was supported by the finding that endogenous ORMDL3 and 5-LO are localized in similar endoplasmic reticulum domains in human mast cells and that ORMDL3 physically interacts with 5-LO. Further experiments showed that 5-LO also interacts with the long-chain 1 and long-chain 2 subunits of SPT. In agreement with these findings, 5-LO knockdown increased ceramide levels, and silencing of SPTLC1 decreased arachidonic acid metabolism to LTs to levels observed upon 5-LO knockdown. These results demonstrate functional crosstalk between the LT and sphingolipid metabolic pathways, leading to the production of lipid signaling mediators.

ORMDL3 and Asthma: Linking Sphingolipid Regulation to Altered T Cell Function.

Orosomucoid like 3 (ORMDL3) encodes an ER-resident transmembrane protein that regulates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme for sphingolipid biosynthesis in cells. A decade ago, several genome wide association studies revealed single nucleotide polymorphisms associated with increased ORMDL3 protein expression and susceptibility to allergic asthma. Since that time, numerous studies have investigated how altered ORMDL3 expression might predispose to asthma and other autoimmune/inflammatory diseases. In this brief review, we focus on growing evidence suggesting that heightened ORMDL3 expression specifically in CD4+ T lymphocytes, the central orchestrators of adaptive immunity, constitutes a major underlying mechanism of asthma pathogenesis by skewing their differentiation and function. Furthermore, we explore how sphingolipid modulation in T cells might be responsible for these effects, and how further studies may interrogate this intriguing hypothesis.

Membrane disruption of Fusarium oxysporum f. sp. niveum induced by myriocin from Bacillus amyloliquefaciens LZN01.

Myriocin, which is produced by Bacillus amyloliquefaciens LZN01, can inhibit the growth of Fusarium oxysporum f. sp. niveum (Fon). In the present study, the antifungal mechanism of myriocin against Fon was investigated with a focus on the effects of myriocin on the cell membrane. Myriocin decreased the membrane fluidity and destroyed the membrane integrity of Fon. Significant microscopic morphological changes, including conidial shrinkage, the appearance of larger vacuoles and inhomogeneity of electron density, were observed in myriocin-treated cells. A membrane-targeted mechanism of action was also supported by transcriptomic and proteomic analyses; a total of 560 common differentially expressed genes (DEGs) and 285 common differentially expressed proteins (DEPs) were identified. The DEGs were further verified by using RT-qPCR. The combined analysis between the transcriptome and proteome revealed that the expression of some membrane-related genes and proteins, mainly those related to sphingolipid metabolism, glycerophospholipid metabolism, steroid biosynthesis, ABC transporters and protein processing in the endoplasmic reticulum, was disordered. Myriocin affected the serine palmitoyl transferase (SPT) activity as evidenced through molecular docking. Our results indicate that myriocin has significant antifungal activity owing to its ability to induce membrane damage in Fon.

Wnt-controlled sphingolipids modulate Anthrax Toxin Receptor palmitoylation to regulate oriented mitosis in zebrafish

Oriented cell division is a fundamental mechanism to control asymmetric stem cell division, neural tube elongation and body axis extension, among other processes. During zebrafish gastrulation, when the body axis extends, dorsal epiblast cells display divisions that are robustly oriented along the animal-vegetal embryonic axis. Here, we use a combination of lipidomics, metabolic tracer analysis and quantitative image analysis to show that sphingolipids mediate spindle positioning during oriented division of epiblast cells. We identify the Wnt signaling as a regulator of sphingolipid synthesis that mediates the activity of serine palmitoyltransferase (SPT), the first and rate-limiting enzyme in sphingolipid production. Sphingolipids determine the palmitoylation state of the Anthrax receptor, which then positions the mitotic spindle of dividing epiblast cells. Our data show how Wnt signaling mediates sphingolipid-dependent oriented division and how sphingolipids determine Anthrax receptor palmitoylation, which ultimately controls the activation of Diaphanous to mediate spindle rotation and oriented mitosis.

Production of leukotriene signaling mediators is limited by ORMDL3/serine palmitoyltransferase/5-lipoxygenase crosstalk

Abstract Leukotrienes and sphingolipids are critical lipid mediators participating in cellular signal transduction and development of various diseases. Metabolic pathways initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to leukotrienes and serine palmitoyltransferase (SPT) de novo synthesis of sphingolipids. Previous studies showed that endoplasmic reticulum membrane protein ORMDL3 inhibits the activity of SPT and sphingolipid synthesis. However, the role of ORMDL3 in synthesis of leukotrienes is not known. In this study, we used peritoneal-derived mast cells (PDMCs) isolated from mice with ORMDL3 knockout (KO) or control mice and examined their properties. We found that PDMCs with ORMDL3 KO exhibited increased calcium response and ß-glucuronidase release when activated with antigen. These events were accompanied by increased phosphorylation of IκB-α and TNF-α production. Lipid analysis showed that ORMDL3-deficient cells exhibited not only enhanced production of sphingolipids, but also increased production of leukotriene inflammatory mediators, such as LTB4 and LTC4. These data were supported by the finding that ORMDL3 physically interacts with 5-LO. Further studies showed that 5-LO interacts with the SPT long-chain (LC)1 and SPTLC2 subunits and decreases the ceramide levels. In line with these findings, 5-LO knockdown increased the ceramide levels, and silencing of SPTLC1 decreased transition of arachidonic acid to leukotrienes. These results demonstrate physical and functional crosstalk between leukotriene and sphingolipid metabolism pathways leading to production of lipid signaling mediators.

Human Keratinocytes Synthesize Ultra Long‐Chain Sphingoid Bases (Up To C28) That Are Abundant in Skin Stratum Corneum

Skin sphingolipids are unique and fulfill the important task of forming a hydrophobic barrier to limit water loss from the body but also to protect from allergen, bacterial and viral penetration through the skin. Skin sphingolipids are highly diverse in their combination of fatty acids and sphingoid bases, the presence of highly hydrophobic ω‐esterified ceramides (EOS‐CER), and the abundance of ultra long‐chain fatty acids (ULCFA, up to C36) within ceramide molecules. The chain length of sphingoid bases in ceramides is commonly attributed to C18 molecules but there are only a few reports demonstrating the presence of C20‐ and C22‐sphingoid bases in skin ceramides. As free sphingoid bases and ceramides are metabolically connected, it is assumed that similar to other tissues, C18‐sphingoid bases represent the majority of sphingoid bases within the total pool of free and ceramide‐linked molecules in the skin.Our sphingolipidomic work during the study of the mechanisms leading to the impairment of skin barrier function has revealed that ceramides with C18‐, C20‐, and C22‐ sphingoid bases are almost equally represented in human skin stratum corneum. Surprisingly, we found that free sphingoid bases in stratum corneum are highly abundant and composed primarily of ultra long‐chain (C24‐ and C26‐) dihydrosphingosines. This finding prompted us to investigate in details the ability of skin keratinocytes to synthesize ultra long‐chain length sphingoid bases. Using undifferentiated and Ca2+‐differentiated primary human keratinocytes, [U‐13C,15N]‐serine, and stable isotope pulse‐labeling approach in vitro, we have confirmed that all ceramides and free sphingoid base molecules do incorporate the labeled serine into their structure. Next, we explored the ability of serine palmitoyltransferase (SPT), the enzyme that initiates sphingolipid biosynthesis, to synthesize ultra long‐chain sphingoid bases in vitro. By comparing the activity of SPT in total cell lysates prepared from undifferentiated and Ca2+‐differentiated human keratinocytes, 16:0–24:0‐Coenzyme A molecules and [U‐13C,15N]‐serine, we confirmed the ability of SPT expressed in human keratinocytes to produce, albeit at low capacity, C24‐ and C26‐3‐keto‐dihydrosphingosines. As keratinocytes and skin stratum corneum do not have measurable amounts of ceramides with C24‐C26‐dihydrosphingosines or C24‐C26‐sphingosines, we conclude that keratinocyte ceramide synthases are unable to utilize dihydrosphingosines with carbon chain length more than C22, thus providing a foundation for the accumulation of C24‐C26‐dihydrosphingosine in human stratum corneum. The question of the possible special biological function of long‐chain dihydrosphingosines in human skin is currently under investigation.

Sphingolipid Signature of Human Feto-Placental Vasculature in Preeclampsia.

Bioactive sphingolipids are emerging as key regulators of vascular function and homeostasis. While most of the clinical studies have been devoted to profile circulating sphingolipids in maternal plasma, little is known about the role of the sphingolipid at the feto-placental vasculature, which is in direct contact with the offspring circulation. Our study aims to compare the sphingolipid profile of normal with preeclamptic (PE) placental chorionic arteries and isolated endothelial cells, with the goal of unveiling potential underlying pathomechanisms in the vasculature. Dihydrosphingosine and sphingomyelin (SM) concentrations (C16:0-, C18:0-, and C24:0- sphingomyelin) were significantly increased in chorionic arteries of preeclamptic placentas, whereas total ceramide, although showing a downward trend, were not statistically different. Moreover, RNA and immunofluorescence analysis showed impaired sphingosine-1-phosphate (S1P) synthesis and signaling in PE vessels. Our data reveal that the exposure to a deranged maternal intrauterine environment during PE alters the sphingolipid signature and gene expression on the fetal side of the placental vasculature. This pathological remodeling consists in increased serine palmitoyltransferase (SPT) activity and SM accrual in PE chorionic arteries, with concomitance impairment endothelial S1P signaling in the endothelium of these vessels. The increase of endothelial S1P phosphatase, lyase and S1PR2, and blunted S1PR1 expression support the onset of the pathological phenotype in chorionic arteries.

Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity

Serine palmitoyltranferase (SPT) is a pyridoxal 5' phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane proteins termed the ORMDLs. Here we describe a procedure to assay ORMDL dependent lipid inhibition of SPT activity. The assay of SPT activity using radiolabeled L-serine was developed from the procedure established by the Hornemann laboratory. The activity of SPT can also be measured using deuterated L-serine but it requires mass spectrometry, which consumes money, time and instrumentation. The ORMDL dependent lipid inhibition of SPT activity can be studied in both cells and in a cell free system. This assay procedure is applicable to any type of mammalian cell. Here we provide the detailed protocol to measure SPT activity in the presence of either short chain (C8-ceramide) or long chain ceramide (C24-ceramide). One of the greatest advantages of this protocol is the ability to test insoluble long chain ceramides. We accomplished this by generating long chain ceramide through endogenous ceramide synthase by providing exogenous sphingosine and 24:1 acyl CoA in HeLa cell membranes. This SPT assay procedure is simple and easy to perform and does not require sophisticated instruments.

Seipin negatively regulates sphingolipid production at the ER-LD contact site.

Seipin is known for its critical role in controlling lipid droplet (LD) assembly at the LD-forming subdomain of the endoplasmic reticulum (ER). Here, we identified a new function of seipin as a negative regulator for sphingolipid production. We show that yeast cells lacking seipin displayed altered sensitivity to sphingolipid inhibitors, accumulated sphingoid precursors and intermediates, and increased serine palmitoyltransferase (SPT) and fatty acid (FA) elongase activities. Seipin associated with SPT and FA elongase, and the interaction was reduced by inhibitors for sphingolipid synthesis in a concentration-dependent manner. We further show that the interactions of seipin with SPT and FA elongase occurred at ER-LD contacts and were likely regulated differentially. Further evidence indicated that LD biogenesis was intact when SPT activity was blocked, whereas excess sphingoid intermediates may affect LD morphology. Expression of human seipin rescued the altered sphingolipids in yeast seipin mutants, suggesting that the negative regulation of sphingolipid synthesis by seipin is likely an evolutionarily conserved process.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.