Sphingomyelin Hydrolysis Research Articles

Mevalonate inhibits acid sphingomyelinase activity, increases sphingomyelin levels and inhibits cell proliferation of HepG2 and Caco-2 cells

BackgroundSphingomyelin (SM) and cholesterol are two types of lipid closely related biophysically. Treating the cells with exogenous sphingomyelinase (SMase) induces trafficking of cholesterol from membrane to intracellular pools and inhibition of cholesterol synthesis. In the present work, we address a question whether increased cholesterol synthesis affects hydrolysis of SM by endogenous SMases.MethodsBoth HepG2 and Caco-2 cells were incubated with mevalonate. The SMase activity was determined and its mRNA examined by qPCR. The cellular levels of cholesterol, SM, and phosphatidylcholine (PC) were determined and cell proliferation rate assayed.ResultsWe found that mevalonate dose-dependently decreased acid but not neutral SMase activity in both HepG2 and Caco-2 cells with HepG2 cells being more sensitive to mevalonate. Kinetic examination in HepG2 cells revealed that acid SMase activity was increasing with cell proliferation, and such an increase was reversed by mevalonate treatment. Acid SMase mRNA was not significantly decreased and Western blot showed signs of proteolysis of acid SMase by mevalonate. After mevalonate treatment, the levels of cholesterol were significantly increased associated with increases in SM and PC. The cell growth was retarded by mevalonate and the effect was more obvious in HepG2 cells than in Caco-2 cells.ConclusionMevalonate can trigger a mechanism to enhance SM levels by inhibition of acid SMase. The effect may ensure the coordinate changes of SM and cholesterol in the cells. Mevalonate also affects cell growth with mechanism required further characterization.

Surface hydrolysis of sphingomyelin by the outer membrane protein Rv0888 supports replication of Mycobacterium tuberculosis in macrophages.

Sphingomyelinases secreted by pathogenic bacteria play important roles in host-pathogen interactions ranging from interfering with phagocytosis and oxidative burst to iron acquisition. This study shows that the Mtb protein Rv0888 possesses potent sphingomyelinase activity cleaving sphingomyelin, a major lipid in eukaryotic cells, into ceramide and phosphocholine, which are then utilized by Mtb as carbon, nitrogen and phosphorus sources, respectively. An Mtb rv0888 deletion mutant did not grow on sphingomyelin as a sole carbon source anymore and replicated poorly in macrophages indicating that Mtb utilizes sphingomyelin during infection. Rv0888 is an unusual membrane protein with a surface-exposed C-terminal sphingomyelinase domain and a putative N-terminal channel domain that mediated glucose and phosphocholine uptake across the outer membrane in an M. smegmatis porin mutant. Hence, we propose to name Rv0888 as SpmT (sphingomyelinase of Mycobacterium tuberculosis). Erythrocyte membranes contain up to 27% sphingomyelin. The finding that Rv0888 accounts for half of Mtb's hemolytic activity is consistent with its sphingomyelinase activity and the observation that Rv0888 levels are increased in the presence of erythrocytes and sphingomyelin by 5- and 100-fold, respectively. Thus, Rv0888 is a novel outer membrane protein that enables Mtb to utilize sphingomyelin as a source of several essential nutrients during intracellular growth.

Epidermal Hydration Is Improved by Enhanced Ceramide Metabolism in Aged C57BL/6J Mice After Dietary Supplementation of Royal Jelly.

Epidermal hydration is maintained by the epidermal lipid barrier, of which ceramide (Cer) is the major constituent. We examined the dietary effect of royal jelly (RJ) on epidermal hydration in aged mice. Altered Cer metabolism was further determined by measuring epidermal levels of individual Cer, glucosylceramide (GC), and sphingomyelin (SM) species, and of Cer-metabolizing enzymes. Aged C57BL/6J mice were fed a control diet (group AGED) or diets with 1% RJ harvested from two different areas (groups AGED+RJ1:AGED + RJ2) for 16 weeks. Aged C57BL/6J mice with no dietary intervention (the control group: group C) represented the onset of aging. In group AGED, epidermal levels of hydration, Cer1/2/5/6/7, GC-A/B/C/D, SM1/2/3, and β-glucocerebrosidase (GCase) protein, an enzyme of GC hydrolysis for Cer generation, were lower than in group C; these levels, as well as those of Cer3/4 and acidic sphingomyelinase (aSMase) protein, an enzyme of SM hydrolysis for Cer generation, were higher in group AGED + RJ1 than in group AGED. Despite increases in GC-B, SM1/2/3, and serine palmitoyltransferase2 protein, an enzyme of de novo Cer synthesis, in group AGED + RJ2 to levels higher than in group AGED, epidermal levels of hydration, Cer1-7, GC-A/C/D, GCase, and aSMase proteins were similar in these two groups. Expression of GCase and aSMase mRNAs, and of Cer synthase3 and ceramidase proteins, enzymes of de novo Cer synthesis and degradation, did not differ among groups. Dietary RJ1 improved epidermal hydration by enhancing Cer metabolism with increased levels of all Cer, GC, and SM species, and of GCase and aSMase proteins.

Cambinol, a novel inhibitor of neutral sphingomyelinase 2 shows neuroprotective properties.

Ceramide is a bioactive lipid that plays an important role in stress responses leading to apoptosis, cell growth arrest and differentiation. Ceramide production is due in part to sphingomyelin hydrolysis by sphingomyelinases. In brain, neutral sphingomyelinase 2 (nSMase2) is expressed in neurons and increases in its activity and expression have been associated with pro-inflammatory conditions observed in Alzheimer’s disease, multiple sclerosis and human immunodeficiency virus (HIV-1) patients. Increased nSMase2 activity translates into higher ceramide levels and neuronal cell death, which can be prevented by chemical or genetic inhibition of nSMase2 activity or expression. However, to date, there are no soluble, specific and potent small molecule inhibitor tool compounds for in vivo studies or as a starting point for medicinal chemistry optimization. Moreover, the majority of the known inhibitors were identified using bacterial, bovine or rat nSMase2. In an attempt to identify new inhibitor scaffolds, two activity assays were optimized as screening platform using the recombinant human enzyme. First, active hits were identified using a fluorescence-based high throughput compatible assay. Then, hits were confirmed using a 14C sphingomyelin-based direct activity assay. Pharmacologically active compounds and approved drugs were screened using this strategy which led to the identification of cambinol as a novel uncompetitive nSMase2 inhibitor (K i = 7 μM). The inhibitory activity of cambinol for nSMase2 was approximately 10-fold more potent than for its previously known target, silence information regulator 1 and 2 (SIRT1/2). Cambinol decreased tumor necrosis factor-α or interleukin-1 β-induced increases of ceramide and cell death in primary neurons. A preliminary study of cambinol structure and activity allowed the identification of the main structural features required for nSMase2 inhibition. Cambinol and its analogs may be useful as nSMase2 inhibitor tool compounds to prevent ceramide-dependent neurodegeneration.

Ceramide formation mediated by acid sphingomyelinase facilitates endosomal escape of caliciviruses

Our recent results demonstrated that bile acids facilitate virus escape from the endosomes into the cytoplasm for successful replication of porcine enteric calicivirus (PEC). We report a novel finding that bile acids can be substituted by cold treatment for endosomal escape and virus replication. This endosomal escape by cold treatment or bile acids is associated with ceramide formation by acid sphingomyelinase (ASM). ASM catalyzes hydrolysis of sphingomyelin into ceramide, which is known to destabilize lipid bilayer. Treatment of LLC-PK cells with bile acids or cold led to ceramide formation, and small molecule antagonists or siRNA of ASM blocked ceramide formation in the endosomes and significantly reduced PEC replication. Inhibition of ASM resulted in the retention of PEC, feline calicivirus or murine norovirus in the endosomes in correlation with reduced viral replication. These results suggest the importance of viral escape from the endosomes for the replication of various caliciviruses.

Sphingomyelinase D/ceramide 1-phosphate in cell survival and inflammation.

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions.

Secretory sphingomyelinase in health and disease.

Acid sphingomyelinase (ASM), a key enzyme in sphingolipid metabolism, hydrolyzes sphingomyelin to ceramide and phosphorylcholine. In mammals, the expression of a single gene, SMPD1, results in two forms of the enzyme that differ in several characteristics. Lysosomal ASM (L-ASM) is located within the lysosome, requires no additional Zn2+ ions for activation and is glycosylated mainly with high-mannose oligosaccharides. By contrast, the secretory ASM (S-ASM) is located extracellularly, requires Zn2+ ions for activation, has a complex glycosylation pattern and has a longer in vivo half-life. In this review, we summarize current knowledge regarding the physiology and pathophysiology of S-ASM, including its sources and distribution, molecular and cellular mechanisms of generation and regulation and relevant in vitro and in vivo studies. Polymorphisms or mutations of SMPD1 lead to decreased S-ASM activity, as detected in patients with Niemann-Pick disease B. Thus, lower serum/plasma activities of S-ASM are trait markers. No genetic causes of increased S-ASM activity have been identified. Instead, elevated activity is the result of enhanced release (e.g., induced by lipopolysaccharide and cytokine stimulation) or increased enzyme activation (e.g., induced by oxidative stress). Increased S-ASM activity in serum or plasma is a state marker of a wide range of diseases. In particular, high S-ASM activity occurs in inflammation of the endothelium and liver. Several studies have demonstrated a correlation between S-ASM activity and mortality induced by severe inflammatory diseases. Serial measurements of S-ASM reveal prolonged activation and, therefore, the measurement of this enzyme may also provide information on past inflammatory processes. Thus, S-ASM may be both a promising clinical chemistry marker and a therapeutic target.

Pressure-temperature phase behavior of mixtures of natural sphingomyelin and ceramide extracts.

Ceramides are a group of sphingolipids that act as highly important signaling molecules in a variety of cellular processes including differentiation and apoptosis. The predominant in vivo synthetic pathway for ceramide formation is via sphingomyelinase catalyzed hydrolysis of sphingomyelin. The biochemistry of this essential pathway has been studied in detail; however, there is currently a lack of information on the structural behavior of sphingomyelin- and ceramide-rich model membrane systems, which is essential for developing a bottom-up understanding of ceramide signaling and platform formation. We have studied the lyotropic phase behavior of sphingomyelin-ceramide mixtures in excess water as a function of temperature (30-70 °C) and pressure (1-200 MPa) by small- and wide-angle X-ray scattering. At low ceramide concentrations the mixtures form the ripple gel phase (P(β)') below the gel transition temperature for sphingomyelin, and this observation has been confirmed by atomic force microscopy. Formation of the ripple gel phase can also be induced at higher temperatures via the application of hydrostatic pressure. At high ceramide concentration an inverse hexagonal phase (HII) is formed coexisting with a cubic phase.

GPR40/FFA1 and neutral sphingomyelinase are involved in palmitate-boosted inflammatory response of microvascular endothelial cells to LPS

ObjectivesIncreased levels of both saturated fatty acids (SFAs) and lipopolysaccharide (LPS) are associated with type 2 diabetes. However, it remains largely unknown how SFAs interact with LPS to regulate inflammatory responses in microvascular endothelial cells (MIC ECs) that are critically involved in atherosclerosis as a diabetic complication. In this study, we compared the effects of LPS, palmitic acid (PA), the most abundant saturated fatty acid, or the combination of LPS and PA on interleukin (IL)-6 expression by MIC ECs and explored the underlying mechanisms. MethodsHuman cardiac MIC ECs were treated with LPS, PA and LPS plus PA and the regulatory pathways including receptors, signal transduction, transcription and post-transcription, and sphingolipid metabolism for IL-6 expression were investigated. ResultsG protein-coupled receptor (GPR)40 or free fatty acid receptor 1 (FFA1), but not toll-like receptor 4, was involved in PA-stimulated IL-6 expression. PA not only stimulated IL-6 expression by itself, but also remarkably enhanced LPS-stimulated IL-6 expression via a cooperative stimulation on mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, and both transcriptional and post-transcriptional activation. Furthermore, PA induced a robust neutral sphingomyelinase (nSMase)-mediated sphingomyelin hydrolysis that was involved in PA-augmented IL-6 upregulation. ConclusionPA boosted inflammatory response of microvascular endothelial cells to LPS via GPR40 and nSMase.

Novel mechanisms of action of classical chemotherapeutic agents on sphingolipid pathways.

The prevailing mechanisms of action of traditional chemotherapeutic agents have been challenged by sphingolipid cancer research. Many studies have shown that ceramide generation in response to cytotoxic agents is central to tumor cell death. Ceramide can be generated either via hydrolysis of cell-membrane sphingomyelin by sphingomyelinases, hydrolysis of cerebrosides, or via de novo synthesis by ceramide synthases. Ceramide can act as a second messenger for apoptosis, senescence or autophagy. Inherent or acquired alterations in the sphingolipid pathway can account for resistance to the classic chemotherapeutic agents. In particular, it has been shown that activation of the acid ceramidase can lead to the formation of sphingosine 1-phosphate, which then antagonizes ceramide signaling by initiating a pro-survival signaling pathway. Furthermore, ceramide glycosylation catalyzed by glucosylceramide synthase converts ceramide to glucosylceramide, thus eliminating ceramide and consequently protecting cancer cells from apoptosis. In this review, we describe the effects of some of the most commonly used chemotherapeutic agents on ceramide generation, with a particular emphasis on strategies used to enhance the efficacy of these agents.

Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases.

Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase.

Regulation of Mitochondrial Function by Bioactive Sphingolipids

Sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate [S1P] are key regulators of various cellular functions. Sphingolipids mediates of cell-stress responses and regulate mitochondrial function. Ceramide and its metabolites play an important role in the development and progression of mitochondria related disorders. Ceramide functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. S1P, a potent signaling sphingolipid exerts myriads of pathophysiological function, including lymphocyte trafficking, angiogenesis, vascular development and inflammation. This review is focused on the role of signaling sphingolipids, such as S1P, sphingosine, and ceramide-1 phosphate on mitochondrial function, particularly mitochondrial respiratory function, apoptosis and calcium homeostasis. Further, we discuss the role of sphingolipids in mitochondrial diseases and targeting them for drug development. This review article is a part of special issue on Mitochondria: Implications in human health and disease.

Neutral sphingomyelinase and breast cancer research.

Our understanding of the functions of neutral sphingomyelinase (N-SMase) signaling has advanced over the past decade. In this review, we focus on the roles and regulation of N-SMase 1, N-SMase 2, N-SMase 3, an enzyme that generates the bioactive lipid ceramide through the hydrolysis of the membrane lipid sphingomyelin. A large body of work has now implicated N-SMase 2 in a diverse set of cellular functions, physiological processes, and disease pathologies. We focus on different aspects of this enzyme's regulation from transcriptional, post-translational, and biochemical. Furthermore, we expected N-SMase involvement in cellular processes including inflammatory signaling, cell growth, apoptosis, and tumor necrosis factor which in turn play important roles in pathologies such as cancer metastasis, variable disease, and other organ system disorders. Lastly, we examine avenues where targeted N-SMase inhibition may be clinically beneficial in disease scenarios.

Comparative effect of dietary borage oil and safflower oil on anti-proliferation and ceramide metabolism in the epidermis of essential fatty acid deficient guinea pigs

Purpose: Borage oil (BO) and safflower oil (SO) are efficacious in reversing epidermal hyperproliferation, which is caused by the disruption of epidermal barrier. In this study, we compared the antiproliferative effect of dietary BO and SO. Altered metabolism of ceramide (Cer), the major lipid of epidermal barrier, was further determined by measurement of epidermal levels of individual Cer, glucosylceramide (GlcCer), and sphingomyelin (SM) species, and protein expression of Cer metabolizing enzymes. Methods: Epidermal hyperproliferation was induced in guinea pigs by a hydrogenated coconut diet (HCO) for 8 weeks. Subsequently, animals were fed diets of either BO (group HCO + BO) or SO (group HCO + SO) for 2 weeks. As controls, animals were fed BO (group BO) or HCO (group HCO) diets for 10 weeks. Results: Epidermal hyperproliferation was reversed in groups HCO + BO (67.6% of group HCO) and HCO + SO (84.5% of group HCO). Epidermal levels of Cer1/2, GlcCer-A/B, and β-glucocerebrosidase (GCase), an enzyme of GlcCer hydrolysis for Cer generation, were higher in group HCO + BO than in group HCO, and increased to levels similar to those of group BO. In addition, epidermal levels of SM1, serine palmitoyltransferase (SPT), and acidic sphingomyelinase (aSMase), enzymes of de novo Cer synthesis and SM hydrolysis for Cer generation, but not of Cer3-7, were higher in group HCO + BO than in group HCO. Despite an increase of SPT and aSMase in group HCO + SO to levels higher than in group HCO, epidermal levels of Cer1-7, GlcCer-A/B, and GCase were similar in these two groups. Notably, acidic ceramidase, an enzyme of Cer degradation, was highly expressed in group HCO + SO. Epidermal levels of GlcCer-C/D and SM-2/3 did not differ among groups. Conclusion: Dietary BO was more prominent for reversing epidermal hyperproliferation by enhancing Cer metabolism with increased levels of Cer1/2, GlcCer-A/B, and SM1 species, and of GCase proteins.

Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis.The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis.The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.

Roles and regulation of neutral sphingomyelinase-2 in cellular and pathological processes.

Our understanding of the functions of ceramide signaling has advanced tremendously over the past decade. In this review, we focus on the roles and regulation of neutral sphingomyelinase 2 (nSMase2), an enzyme that generates the bioactive lipid ceramide through the hydrolysis of the membrane lipid sphingomyelin. A large body of work has now implicated nSMase2 in a diverse set of cellular functions, physiological processes, and disease pathologies. We discuss different aspects of this enzyme's regulation from transcriptional, post-translational, and biochemical. Furthermore, we highlight nSMase2 involvement in cellular processes including inflammatory signaling, exosome generation, cell growth, and apoptosis, which in turn play important roles in pathologies such as cancer metastasis, Alzheimer's disease, and other organ systems disorders. Lastly, we examine avenues where targeted nSMase2-inhibition may be clinically beneficial in disease scenarios.

Crystallization and preliminary X-ray diffraction analysis of a novel sphingomyelinase D from Loxosceles gaucho venom.

Brown spider envenomation results in dermonecrosis, intravascular coagulation, haemolysis and renal failure, mainly owing to the action of sphingomyelinases D (SMases D), which catalyze the hydrolysis of sphingomyelin to produce ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidylcholine to produce lysophosphatidic acid. Here, the heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of LgRec1, a novel SMase D from Loxosceles gaucho venom, are reported. The crystals belonged to space group P21212, with unit-cell parameters a = 52.98, b = 62.27, c = 84.84 Å and diffracted to a maximum resolution of 2.6 Å.

Inhibition of acid sphingomyelinase by tricyclic antidepressants and analogons.

Amitriptyline, a tricyclic antidepressant, has been used in the clinic to treat a number of disorders, in particular major depression and neuropathic pain. In the 1970s the ability of tricyclic antidepressants to inhibit acid sphingomyelinase (ASM) was discovered. The enzyme ASM catalyzes the hydrolysis of sphingomyelin to ceramide. ASM and ceramide were shown to play a crucial role in a wide range of diseases, including cancer, cystic fibrosis, diabetes, Alzheimer's disease, and major depression, as well as viral (e.g., measles virus) and bacterial (e.g., Staphylococcus aureus, Pseudomonas aeruginosa) infections. Ceramide molecules may act in these diseases by the alteration of membrane biophysics, the self-association of ceramide molecules within the cell membrane and the ultimate formation of larger ceramide-enriched membrane domains/platforms. These domains were shown to serve the clustering of certain receptors such as CD95 and may also act in the above named diseases. The potential to block the generation of ceramide by inhibiting the ASM has opened up new therapeutic approaches for the treatment of these conditions. Since amitriptyline is one of the longest used clinical drugs and side effects are well studied, it could potentially become a cheap and easily accessible medication for patients suffering from these diseases. In this review, we aim to provide an overview of current in vitro and in vivo studies and clinical trials utilizing amitriptyline to inhibit ASM and contemplate possible future applications of the drug.

Identification and biochemical characterization of an acid sphingomyelinase-like protein from the bacterial plant pathogen Ralstonia solanacearum that hydrolyzes ATP to AMP but not sphingomyelin to ceramide.

Acid sphingomyelinase (aSMase) is a human enzyme that catalyzes the hydrolysis of sphingomyelin to generate the bioactive lipid ceramide and phosphocholine. ASMase deficiency is the underlying cause of the genetic diseases Niemann-Pick Type A and B and has been implicated in the onset and progression of a number of other human diseases including cancer, depression, liver, and cardiovascular disease. ASMase is the founding member of the aSMase protein superfamily, which is a subset of the metallophosphatase (MPP) superfamily. To date, MPPs that share sequence homology with aSMase, termed aSMase-like proteins, have been annotated and presumed to function as aSMases. However, none of these aSMase-like proteins have been biochemically characterized to verify this. Here we identify RsASML, previously annotated as RSp1609: acid sphingomyelinase-like phosphodiesterase, as the first bacterial aSMase-like protein from the deadly plant pathogen Ralstonia solanacearum based on sequence homology with the catalytic and C-terminal domains of human aSMase. A biochemical characterization of RsASML does not support a role in sphingomyelin hydrolysis but rather finds RsASML capable of acting as an ATP diphosphohydrolase, catalyzing the hydrolysis of ATP and ADP to AMP. In addition, RsASML displays a neutral, not acidic, pH optimum and prefers Ni2+ or Mn2+, not Zn2+, for catalysis. This alters the expectation that all aSMase-like proteins function as acid SMases and expands the substrate possibilities of this protein superfamily to include nucleotides. Overall, we conclude that sequence homology with human aSMase is not sufficient to predict substrate specificity, pH optimum for catalysis, or metal dependence. This may have implications to the biochemically uncharacterized human aSMase paralogs, aSMase-like 3a (aSML3a) and aSML3b, which have been implicated in cancer and kidney disease, respectively, and assumed to function as aSMases.

The composition of West Nile virus lipid envelope unveils a role of sphingolipid metabolism in flavivirus biogenesis.

West Nile virus (WNV) is an emerging zoonotic mosquito-borne flavivirus responsible for outbreaks of febrile illness and meningoencephalitis. The replication of WNV takes place on virus-modified membranes from the endoplasmic reticulum of the host cell, and virions acquire their envelope by budding into this organelle. Consistent with this view, the cellular biology of this pathogen is intimately linked to modifications of the intracellular membranes, and the requirement for specific lipids, such as cholesterol and fatty acids, has been documented. In this study, we evaluated the impact of WNV infection on two important components of cellular membranes, glycerophospholipids and sphingolipids, by mass spectrometry of infected cells. A significant increase in the content of several glycerophospholipids (phosphatidylcholine, plasmalogens, and lysophospholipids) and sphingolipids (ceramide, dihydroceramide, and sphingomyelin) was noticed in WNV-infected cells, suggesting that these lipids have functional roles during WNV infection. Furthermore, the analysis of the lipid envelope of WNV virions and recombinant virus-like particles revealed that their envelopes had a unique composition. The envelopes were enriched in sphingolipids (sphingomyelin) and showed reduced levels of phosphatidylcholine, similar to sphingolipid-enriched lipid microdomains. Inhibition of neutral sphingomyelinase (which catalyzes the hydrolysis of sphingomyelin into ceramide) by either pharmacological approaches or small interfering RNA-mediated silencing reduced the release of flavivirus virions as well as virus-like particles, suggesting a role of sphingomyelin-to-ceramide conversion in flavivirus budding and confirming the importance of sphingolipids in the biogenesis of WNV. Importance: West Nile virus (WNV) is a neurotropic flavivirus spread by mosquitoes that can infect multiple vertebrate hosts, including humans. There is no specific vaccine or therapy against this pathogen licensed for human use. Since the multiplication of this virus is associated with rearrangements of host cell membranes, we analyzed the effect of WNV infection on different cellular lipids that constitute important membrane components. The levels of multiple lipid species were increased in infected cells, pointing to the induction of major alterations of cellular lipid metabolism by WNV infection. Interestingly, certain sphingolipids, which were increased in infected cells, were also enriched in the lipid envelope of the virus, thus suggesting a potential role during virus assembly. We further verified the role of sphingolipids in the production of WNV by means of functional analyses. This study provides new insight into the formation of flavivirus infectious particles and the involvement of sphingolipids in the WNV life cycle.