Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Krüppel-like factor 12 regulates aging ovarian granulosa cell apoptosis by repressing SPHK1 transcription and sphingosine-1-phosphate (S1P) production.

Oxidative stress triggered by aging, radiation, or inflammation impairs ovarian function by inducing granulosa cell (GC) apoptosis. However, the mechanism inducing GC apoptosis has not been characterized. Here, we found that ovarian GCs from aging patients showed increased oxidative stress, enhanced reactive oxygen species activity, and significantly decreased expression of the known antiapoptotic factor sphingosine-1-phosphate/sphingosine kinase 1 (SPHK1) in GCs. Interestingly, the expression of Krüppel-like factor 12 (KLF12) was significantly increased in the ovarian GCs of aging patients. Furthermore, we determined that KLF12 was significantly upregulated in hydrogen peroxide-treated GCs and a 3-nitropropionic acid-induced in vivo model of ovarian oxidative stress. This phenotype was further confirmed to result from inhibition of SPHK1 by KLF12. Interestingly, when endogenous KLF12 was knocked down, it rescued oxidative stress-induced apoptosis. Meanwhile, supplementation with SPHK1 partially reversed oxidative stress-induced apoptosis. However, this function was lost in SPHK1 with deletion of the binding region to the KLF12 promoter. SPHK1 reversed apoptosis caused by hydrogen peroxide-KLF12 overexpression, a result further confirmed in an in vitro ovarian culture model and an in vivo 3-nitropropionic acid-induced ovarian oxidative stress model. Overall, our study reveals that KLF12 is involved in regulating apoptosis induced by oxidative stress in aging ovarian GCs and that sphingosine-1-phosphate/SPHK1 can rescue GC apoptosis by interacting with KLF12 in negative feedback.

Endothelial Spns2 and ApoM Regulation of Vascular Tone and Hypertension Via Sphingosine-1-Phosphate.

Background Most of the circulating sphingosine-1-phosphate (S1P) is bound to ApoM (apolipoprotein M) of high-density lipoprotein (HDL) and mediates many beneficial effects of HDL on the vasculature via G protein-coupled S1P receptors. HDL-bound S1P is decreased in atherosclerosis, myocardial infarction, and diabetes mellitus. In addition to being the target, the endothelium is a source of S1P, which is transported outside of the cells by Spinster-2, contributing to circulating S1P as well as to local signaling. Mice lacking endothelial S1P receptor 1 are hypertensive, suggesting a vasculoprotective role of S1P signaling. This study investigates the role of endothelial-derived S1P and ApoM-bound S1P in regulating vascular tone and blood pressure. Methods and Results ApoM knockout (ApoM KO) mice and mice lacking endothelial Spinster-2 (ECKO-Spns2) were infused with angiotensin II for 28 days. Blood pressure, measured by telemetry and tail-cuff, was significantly increased in both ECKO-Spns2 and ApoM KO versus control mice, at baseline and following angiotensin II. Notably, ECKO-Spns2 presented an impaired vasodilation to flow and blood pressure dipping, which is clinically associated with increased risk for cardiovascular events. In hypertension, both groups presented reduced flow-mediated vasodilation and some degree of impairment in endothelial NO production, which was more evident in ECKO-Spns2. Increased hypertension in ECKO-Spns2 and ApoM KO mice correlated with worsened cardiac hypertrophy versus controls. Conclusions Our study identifies an important role for Spinster-2 and ApoM-HDL in blood pressure homeostasis via S1P-NO signaling and dissects the pathophysiological impact of endothelial-derived S1P and ApoM of HDL-bound S1P in hypertension and cardiac hypertrophy.

The structure of gangliosides hides a code for determining neuronal functions.

Gangliosides are particularly abundant in the central nervous system, where they are mainly associated with the synaptic membranes. Their structure underlies a specific role in determining several cell physiological processes of the nervous system. The high number of different gangliosides available in nature suggests that their structure, related to both the hydrophobic and hydrophilic portion of the molecule, defines a code, although not completely understood, that through hydrophobic interactions and hydrogen bonds allows the transduction of signals starting at the plasma membranes. In this short review, we describe some structural aspects responsible for the role played by gangliosides in maintaining and determining neuronal functions.

Fecal g. Streptococcus and g. Eubacterium_coprostanoligenes_group combined with sphingosine to modulate the serum dyslipidemia in high-fat diet mice.

BACKGROUND & AIMS: Although high-fat diet (HFD) could impact the composition of fecal microbiome and their metabolites, it is still largely unknown which fecal bacteria and metabolites are relatively important in responding to the HFD. This study aimed to identify the crucial fecal bacteria and metabolites in the HFD mice using a microbial-metabolite network, and to investigate the synergistic mediation effect of the crucial fecal bacteria and metabolites on serum dyslipidemia induced by the HFD. METHODS: The 16srDNA sequencing and the ultra-performance liquid chromatography (UPLC/TOF MSMS) platform were performed to characterize the composition and function of fecal microbiome, and metabolites in the HFD. The microbial-metabolite network, correlation and mediation analyses were performed to examine the relationships among fecal microbiome, metabolites, and serum dyslipidemia indicators. Mice models were conducted to evaluate the effect of fecal metabolite on dyslipidemia. RESULTS: Compared to the control, 32 genera were altered in the HFD, including 26 up-regulated and 6 down-regulated. A total of 42 altered pathways were observed between the control and HFD, and the "Glycosphingolipid biosynthesis" was identified as the most significant pathway (fold change = 0.64; p < 0.001). Meanwhile, 49 fecal metabolites were altered in the HFD, and the fecal microbiome was associated with the fecal metabolism (M2 = 0.776, p = 0.008). Based on the microbial-metabolite network, two major hub genera were screened (HUB1: g. Streptococcus, HUB2: g. Eubacterium_coprostanoligenes_group), and one bacterial metabolite, sphingosine, was found in this study. Further, the HUB2 was positively associated with fecal sphingosine (r = 0.646, p = 0.001), and its downstream metabolic pathway, "Glycosphingolipid biosynthesis" pathway (r = 0.544, p = 0.009). The regulatory relationship between the HUB2 and sphingosine synergistically mediated the effect of HFD on TCHO (33.7%), HDL-C (37.3%), and bodyweight (36.7%). Besides, compared to the HFD, the HFD with sphingosine supplementation had lower bodyweight (35.12 ± 1.23 vs. 39.42 ± 1.25, p < 0.001), TG (0.44 ± 0.08 vs. 0.52 ± 0.05, p = 0.002), TCHO (3.81 ± 0.34 vs. 4.51 ± 0.38, p = 0.002), and LDL-c (0.82 ± 0.09 vs. 0.97 ± 0.15, p = 0.016). CONCLUSIONS: The g. Streptococcus and g. Eubacterium_coprostanoligenes are two hub genera in the fecal micro-ecosystem of the HFD, and the g. Eubacterium_coprostanoligenes mediates the effect of HFD on dyslipidemia through sphingosine. Sphingosine supplementation can improve dyslipidemia induced by HFD.

Engineering Yarrowia lipolytica for de novo production of tetraacetyl phytosphingosine.

AIMS: To genetically engineer the oleaginous yeast Yarrowia lipolytica for de novo production of tetraacetylphytosphingosine (TAPS), a precursor of phytosphingosine, and optimization of fermentation conditions for high yield. METHODS AND RESULTS: We successfully constructed a TAPS-producing Y. lipolytica CE3 strain by co-expression of Wickerhamomyces ciferrii-derived acetyl transferases, Sli1p and Atf2p. Next, we optimized several environmental factors including temperature, initial pH and C/N ratio for TAPS production in a shake culture. Deletion of LCB4 in CE3 strain increased the volumetric TAPS titre and cell-specific yield to 142·1 ± 10·7 mgTAPS  l-1 and 3·08 ± 0·11 mgTAPS  gDCW -1 , respectively, in a shake flask culture incubated for 120 h at 28°C with glycerol as the carbon source. Finally, we developed a 5-l fed-batch process with NaOH-mediated pH control and olive oil as a carbon source, exhibiting 650 ± 24 mgTAPS  l-1 of TAPS production within 56 h of the fermentation. CONCLUSIONS: The introduction of codon-optimized Sli1p and Atf2p, deletion of LCB4 gene and sexual hybridization, accompanied by specific fermentation conditions, enhanced TAPS yield in Y. lipolytica. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results highlight Y. lipolytica as a promising candidate for the industrial production of TAPS, an important component of cosmetic formulations.

The role of sphingosine 1-phosphate and its receptors in cardiovascular diseases.

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P-based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT-1303) and the non-selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.

Role of bioactive sphingolipids in physiology and pathology.

Sphingolipids are a class of complex lipids containing a backbone of sphingoid bases, namely the organic aliphatic amino alcohol sphingosine (Sph), that are essential constituents of eukaryotic cells. They were first described as major components of cell membrane architecture, but it is now well established that some sphingolipids are bioactive and can regulate key biological functions. These include cell growth and survival, cell differentiation, angiogenesis, autophagy, cell migration, or organogenesis. Furthermore, some bioactive sphingolipids are implicated in pathological processes including inflammation-associated illnesses such as atherosclerosis, rheumatoid arthritis, inflammatory bowel disease (namely Crohn's disease and ulcerative colitis), type II diabetes, obesity, and cancer. A major sphingolipid metabolite is ceramide, which is the core of sphingolipid metabolism and can act as second messenger, especially when it is produced at the plasma membrane of cells. Ceramides promote cell cycle arrest and apoptosis. However, ceramide 1-phosphate (C1P), the product of ceramide kinase (CerK), and Sph 1-phosphate (S1P), which is generated by the action of Sph kinases (SphK), stimulate cell proliferation and inhibit apoptosis. Recently, C1P has been implicated in the spontaneous migration of cells from some types of cancer, and can enhance cell migration/invasion of malignant cells through interaction with a Gi protein-coupled receptor. In addition, CerK and SphK are implicated in inflammatory responses, some of which are associated with cancer progression and metastasis. Hence, targeting these sphingolipid kinases to inhibit C1P or S1P production, or blockade of their receptors might contribute to the development of novel therapeutic strategies to reduce metabolic alterations and disease.

Sphingosine Kinases as Druggable Targets.

There is substantial evidence that the enzymes, sphingosine kinase 1 and 2, which catalyse the formation of the bioactive lipid sphingosine 1-phosphate, are involved in pathophysiological processes. In this chapter, we appraise the evidence that both enzymes are druggable and describe how isoform-specific inhibitors can be developed based on the plasticity of the sphingosine-binding site. This is contextualised with the effect of sphingosine kinase inhibitors in cancer, pulmonary hypertension, neurodegeneration, inflammation and sickling.

The Sphkl/SlP pathway regulates angiogenesis via NOS/NO synthesis following cerebral ischemia-reperfusion.

AIMS: Sphingosine kinase 1 (Sphk1) and the signaling molecule sphingosine-1-phosphate (S1P) are known to be key regulators of a variety of important biological processes, such as neovascularization. Nitric oxide (NO) is also known to play a role in vasoactive properties, whether Sphk1/S1P signaling is able to alter angiogenesis in the context of cerebral ischemia-reperfusion injury (IRI), and whether such activity is linked with NO production, however, remains uncertain. METHODS: We used immunofluorescence to detect the expression of Sphk1 and NOS in cerebral epithelial cells (EC) after IR or oxygen-glucose deprivation (OGDR). Western blotting was used to detect the Sphk1 and NOS protein levels in brain tissues or HBMECs. Adenovirus transfection was used to inhibit Sphk1 and NOS. An NO kit was used to detect NO contents in brain tissues and epithelial cells. Tube formation assays were conducted to measure angiogenesis. RESULTS: We determined that EC used in a model of cerebral IRI expressed Sphk1, and that inhibiting this expression led to decreased expression of two isoforms of NO synthase (eNOS and iNOS), as well as to decrease neovascularization density and NO production following injury. In HBMECs, knocking down Sphk1 markedly reduced NO production owing to reduced eNOS activity, and inhibiting eNOS directly similarly decreased NO production in a manner which could be reversed via exogenously treating cells with S1P. We further found that knocking down Sphk1 reduced HBMEC eNOS expression, in addition to decreasing the adhesion, migration, and tube formation abilities of these cells under OGDR conditions. CONCLUSIONS: Based on these results, we therefore postulate that Sphk1/S1P signaling is able to mediate angiogenesis following cerebral IRI via the regulation of eNOS activity and NO production. As such, targeting these pathways may potentially represent a novel means of improving patient prognosis in those suffering from cerebral IRI.

Ceramide and Sphingosine Regulation of Myelinogenesis: Targeting Serine Palmitoyltransferase Using microRNA in Multiple Sclerosis.

Ceramide and sphingosine display a unique profile during brain development, indicating their critical role in myelinogenesis. Employing advanced technology such as gas chromatography-mass spectrometry, high performance liquid chromatography, and immunocytochemistry, along with cell culture and molecular biology, we have found an accumulation of sphingosine in brain tissues of patients with multiple sclerosis (MS) and in the spinal cord of rats induced with experimental autoimmune encephalomyelitis. The elevated sphingosine leads to oligodendrocyte death and fosters demyelination. Ceramide elevation by serine palmitoyltransferse (SPT) activation was the primary source of the sphingosine elevation as myriocin, an inhibitor of SPT, prevented sphingosine elevation and protected oligodendrocytes. Supporting this view, fingolimod, a drug used for MS therapy, reduced ceramide generation, thus offering partial protection to oligodendrocytes. Sphingolipid synthesis and degradation in normal development is regulated by a series of microRNAs (miRNAs), and hence, accumulation of sphingosine in MS may be prevented by employing miRNA technology. This review will discuss the current knowledge of ceramide and sphingosine metabolism (synthesis and breakdown), and how their biosynthesis can be regulated by miRNA, which can be used as a therapeutic approach for MS.

Cytokine-Induced and Stretch-Induced Sphingosine 1-Phosphate Production by Enthesis Cells Could Favor Abnormal Ossification in Spondyloarthritis.

Spondyloarthritis (SpA) is a common rheumatic disease characterized by enthesis inflammation (enthesitis) and ectopic ossification (enthesophytes). The current pathogenesis model suggests that inflammation and mechanical stress are both strongly involved in SpA pathophysiology. We have previously observed that the levels of sphingosine 1-phosphate (S1P), a bone anabolic molecule, were particularly high in SpA patients' serum compared to healthy donors. Therefore, we wondered how this deregulation was related to SpA molecular mechanisms. Mouse primary osteoblasts, chondrocytes, and tenocytes were used as cell culture models. The sphingosine kinase 1 (Sphk1) gene expression and S1P secretion were significantly enhanced by cyclic stretch in osteoblasts and chondrocytes. Further, TNF-α and IL-17, cytokines implicated in enthesitis, increased Sphk1 mRNA in chondrocytes in an additive manner when combined to stretch. The immunochemistry on mouse ankles showed that sphingosine kinase 1 (SK1) was localized in some chondrocytes; the addition of a pro-inflammatory cocktail augmented Sphk1 expression in cultured ankles. Subsequently, fingolimod was used to block S1P metabolism in cell cultures. It inhibited S1P receptors (S1PRs) signaling and SK1 and SK2 activity in both osteoblasts and chondrocytes. Fingolimod also reduced S1PR-induced activation by SpA patients' synovial fluid (SF), demonstrating that the stimulation of chondrocytes by SFs from SpA patients involves S1P. In addition, when the osteogenic culture medium was supplemented with fingolimod, alkaline phosphatase activity, matrix mineralization, and bone formation markers were significantly reduced in osteoblasts and hypertrophic chondrocytes. Osteogenic differentiation was accompanied by an increase in S1prs mRNA, especially S1P1/3 , but their contribution to S1P-impact on mineralization seemed limited. Our results suggest that S1P might be overproduced in SpA enthesis in response to cytokines and mechanical stress, most likely by chondrocytes. Moreover, S1P could locally favor the abnormal ossification of the enthesis; therefore, blocking the S1P metabolic pathway could be a potential therapeutic approach for the treatment of SpA. © 2019 American Society for Bone and Mineral Research.

Tamoxifen acts on Trypanosoma cruzi sphingolipid pathway triggering an apoptotic death process.

This study shows the effects of tamoxifen, a known estrogen receptor antagonist used in the treatment of breast cancer, on the sphingolipid pathway of Trypanosoma cruzi, searching for potential chemotherapeutic targets. A dose-dependent epimastigote growth inhibition at increasing concentration of tamoxifen was determined. In blood trypomastigotes, treatment with 10 µM showed 90% lysis, while 86% inhibition of intracellular amastigote development was obtained using 50 µM. Lipid extracts from treated and non-treated metabolically labelled epimastigotes evidenced by thin layer chromatography different levels of sphingolipids and MALDI-TOF mass spectrometry analysis assured the identity of the labelled species. Comparison by HPLC-ESI mass spectrometry of lipids, notably exhibited a dramatic increase in the level of ceramide in tamoxifen-treated parasites and a restrained increase of ceramide-1P and sphingosine, indicating that the drug is acting on the enzymes involved in the final breakdown of ceramide. The ultrastructural analysis of treated parasites revealed characteristic morphology of cells undergoing an apoptotic-like death process. Flow cytometry confirmed cell death by an apoptotic-like machinery indicating that tamoxifen triggers this process by acting on the parasitic sphingolipid pathway.

Light Stress-Induced Increase of Sphingosine 1-Phosphate in Photoreceptors and Its Relevance to Retinal Degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Inhibiting Sphingosine Kinase 2 Derived-sphingosine-1-phosphate Ameliorates Psoriasis-like Skin Disease via Blocking Th17 Differentiation of Naïve CD4 T Lymphocytes in Mice.

Sphingosine-1-phosphate (S1P) is a signalling sphingolipid metabolite that regulates important cell processes, including cell proliferation and apoptosis. Circulating S1P levels have been reported to be increased in patients with psoriasis relative to healthy patients. The aim of this study was to examine the potency of S1P inhibition using an imiquimod-induced psoriasis mouse model. Both topical ceramidase and sphingosine kinase 1/2 inhibition, which blocks S1P generation, alleviated imiquimod-induced skin lesions and reduced the serum interleukin 17-A levels induced by application of imiquimod. These treatments also normalized skin mRNA levels of genes associated with inflammation and keratinocyte differentiation. Inhibition of sphingosine kinase 2, but not sphingosine kinase 1, diminished levels of suppressor of cytokine signalling 1 and blocked T helper type 17 differentiation of naïve CD4+ T cells; imiquimod-induced psoriasis-like skin symptoms were also ameliorated. These results indicate the distinct effects of sphingosine kinase 1 and sphingosine kinase 2 inhibition on T helper type 17 generation and suggest molecules that inhibit S1P formation, including ceramidase and sphingosine kinase 2 inhibitors, as novel therapeutic candidates for psoriasis.

Exosomal sphingosine 1-phosphate secreted by mesenchymal stem cells regulated Treg/Th17 balance in aplastic anemia.

This study was designed to explore whether exosomal sphingosine 1-phosphate (S1P) from mesenchymal stem cells (MSCs) regulate the Treg/Th17 balance in aplastic anemia (AA) patients and to validate the underlying mechanism. To address this, exosomes from human bone marrow MSCs (MSCs-Exos) were co-cultured with CD4+ T cells from AA patients (AA CD4+ T cells), which were transfected with si-S1PR1, si-S1PR3, or not. The proportion of Th17 and Treg was evaluated by flow cytometry. The levels of Th17-associated interleukin-17 (IL-17), Treg-associated IL-10, and transforming growth factor-ß were determined by ELISA. S1P content in MSCs-Exos isolated from control, si-SphK1, or si-SphK2 transfected MSCs was examined by LC-MS/MS. Hematoxylin and eosin staining of bone marrow tissues was performed to evaluate the effect of MSCs-Exos in AA mice. Our results showed that MSCs-Exos reversed the increased Th17/Treg in AA through SphK1-mediated exosomal S1P enrichment. Furthermore, the promotion of Treg differentiation by exosomal S1P from MSCs was mediated through the receptor S1PR1 expressed on CD4+ T cells. Further in vivo experiments showed that MSCs-Exos reversed the increased Th17/Treg and alleviated AA progression in AA mice. In summary, SphK1-mediated enrichment of exosomal S1P secreted by MSCs reversed the increased Treg/Th17 ratio via the receptor S1PR1 in AA patients. © 2019 IUBMB Life, 71(9):1284-1292, 2019.

Endogenous pore-forming protein complex targets acidic glycosphingolipids in lipid rafts to initiate endolysosome regulation.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. ßγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of ßγ-CAT remain elusive. Here, the actions of ßγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of ßγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of ßγ-CAT. Finally, the ability of ßγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.

C16­ceramide and sphingosine 1­phosphate/S1PR2 have opposite effects on cell growth through mTOR signaling pathway regulation.

Recently, sphingolipid derivatives, such as ceramide and sphingosine­1­phosphate (S1P), have emerged as key modulators in apoptotic cell death and cell proliferation. This study aimed to clarify the underlying signaling pathways of ceramide and S1P involved in breast cancer cell proliferation. Ceramide acyl chain length is determined by six mammalian ceramide synthases (CerS). We overexpressed CerS1 to 6 in MCF­7 cells to examine whether ceramide signaling propagation varies as a function of acyl chain length. Among the six CerS, only CerS6 overexpression reduced phosphorylation of Akt, S6 kinase (S6K), and extracellular signal­regulated kinases (ERK) as shown by western blotting. In addition, CerS6 overexpression reduced MCF­7 cell proliferation. This effect was partially reversed by co­treatment with MHY1485, an activator of mammalian target of rapamycin (mTOR), demonstrating an important role for the mTOR pathway in the CerS6­mediated decrease in MCF­7 cell proliferation. ERK inhibition, but not Akt inhibition, along with mTOR inhibition synergistically reduced MCF­7 cell proliferation as measured by MTT assay. Notably, the expression of CerS6 and S1P receptor 2 (S1PR2), or CerS6 and sphingosine kinase 1 (SphK1), were negatively correlated according to the invasive breast carcinoma patient cohort in The Cancer Genome Atlas database. In addition, both SphK1 overexpression and S1P addition increased mTOR phosphorylation as shown by ELISA, while S1PR2 inhibition had the inverse effect. These data suggest that CerS6 and SphK1 regulate mTOR signaling in breast cancer cell proliferation. Moreover, mTOR activity can be regulated by the balance between S1P and C16­ceramide, which is generated by CerS6.

LPS and palmitate synergistically stimulate sphingosine kinase 1 and increase sphingosine 1 phosphate in RAW264.7 macrophages.

It has been well established that patients with diabetes or metabolic syndrome (MetS) have increased prevalence and severity of periodontitis, an oral infection initiated by bacteria and characterized by tissue inflammation and destruction. To understand the underlying mechanisms, we have shown that saturated fatty acid (SFA), which is increased in patients with type 2 diabetes or MetS, and LPS, an important pathogenic factor for periodontitis, synergistically stimulate expression of proinflammatory cytokines in macrophages by increasing ceramide production. However, the mechanisms by which increased ceramide enhances proinflammatory cytokine expression have not been well understood. Since sphingosine 1 phosphate (S1P) is a metabolite of ceramide and a bioactive lipid, we tested our hypothesis that stimulation of ceramide production by LPS and SFA facilitates S1P production, which contributes to proinflammatory cytokine expression. Results showed that LPS and palmitate, a major SFA, synergistically increased not only ceramide, but also S1P, and stimulated sphingosine kinase (SK) expression and membrane translocation in RAW264.7 macrophages. Results also showed that SK inhibition attenuated the stimulatory effect of LPS and palmitate on IL-6 secretion. Moreover, results showed that S1P enhanced the stimulatory effect of LPS and palmitate on IL-6 secretion. Finally, results showed that targeting S1P receptors using either S1P receptor antagonists or small interfering RNA attenuated IL-6 upregulation by LPS and palmitate. Taken together, this study demonstrated that LPS and palmitate synergistically stimulated S1P production and S1P in turn contributed to the upregulation of proinflammatory cytokine expression in macrophages by LPS and palmitate.

An intrinsic lipid-binding interface controls sphingosine kinase 1 function.

Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes, including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen/deuterium exchange MS to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.