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Sample records for regulated sphingolipid synthesis

  1. Regulation of sphingolipid synthesis in renal cells from normal subjects and familial hypercholesterolemic subjects

    SciTech Connect

    Chatterjee, S.; Clarke, K.S.; Kwiterovich, P.O. Jr.

    1986-05-01

    The authors have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic (FH) subjects (TB,BA,DD,VH) employing (/sup 3/H)serine and (/sup 3/H)glucose. The results were: normal cells - 1) LDL (25 ..mu..g/ml) decreased the incorporation of (/sup 3/H)glucose and (/sup 3/H)serine into ceramide and LacCer about 2-3 fold; 2) the incorporation of (/sup 3/H)serine into sphingomyelin was also reduced 2 fold by LDL; 3) LDL modified by reductive methylation of lysine residues (which is not recognized by the LDL receptor) did not decrease the incorporation of (/sup 3/H)glucose and (/sup 3/H)serine into sphingolipids; FH cells - In contrast to normal cells, LDL (100 ..mu..g/ml) stimulated both the incorporation of (/sup 3/H)glucose into LacCer and of (/sup 3/H)serine into ceramide, LacCer and sphingomyelin 2-3 fold in cells lacking LDL receptors. The authors conclude that the endogenous synthesis of sphingolipids in normal renal cells may be regulated by the LDL receptor. Lack of LDL receptor, as in FH cells, results in increased sphingolipid synthesis and storage of LacCer.

  2. Ethylene Modulates Sphingolipid Synthesis in Arabidopsis

    PubMed Central

    Wu, Jian-xin; Wu, Jia-li; Yin, Jian; Zheng, Ping; Yao, Nan

    2015-01-01

    Sphingolipids have essential structural and bioactive functions in membranes and in signaling. However, how plants regulate sphingolipid biosynthesis in the response to stress remains unclear. Here, we reveal that the plant hormone ethylene can modulate sphingolipid synthesis. The fungal toxin Fumonisin B1 (FB1) inhibits the activity of ceramide synthases, perturbing sphingolipid homeostasis, and thus inducing cell death. We used FB1 to test the role of ethylene signaling in sphingolipid synthesis in Arabidopsis thaliana. The etr1-1 and ein2 mutants, which have disrupted ethylene signaling, exhibited hypersensitivity to FB1; by contrast, the eto1-1 and ctr1-1 mutants, which have enhanced ethylene signaling, exhibited increased tolerance to FB1. Gene expression analysis showed that during FB1 treatment, transcripts of genes involved in de novo sphingolipid biosynthesis were down-regulated in ctr1-1 mutants but up-regulated in ein2 mutants. Strikingly, under normal conditions, ctr1-1 mutants contained less ceramides and hydroxyceramides, compared with wild type. After FB1 treatment, ctr1-1 and ein2 mutants showed a significant improvement in sphingolipid contents, except the ctr1-1 mutants showed little change in hydroxyceramide levels. Treatment of wild-type seedlings with the ethylene precursor 1-aminocyclopropane carboxylic acid down-regulated genes involved in the sphingolipid de novo biosynthesis pathway, thus reducing sphingolipid contents and partially rescuing FB1-induced cell death. Taking these results together, we propose that ethylene modulates sphingolipids by regulating the expression of genes related to the de novo biosynthesis of sphingolipids. PMID:26734030

  3. Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans.

    PubMed

    Cutler, Roy G; Thompson, Kenneth W; Camandola, Simonetta; Mack, Kendra T; Mattson, Mark P

    2014-12-15

    Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1-C24:1), gangliosides (e.g., GM1-C24:1), and sphingomyelins (e.g., dC18:1-C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides. PMID:25437839

  4. Sphingolipid metabolism regulates development and lifespan in Caenorhabditis elegans

    PubMed Central

    Cutler, Roy G.; Thompson, Kenneth W.; Camandola, Simonetta; Mack, Kendra T.; Mattson, Mark P.

    2015-01-01

    Sphingolipids are a highly conserved lipid component of cell membranes involved in the formation of lipid raft domains that house many of the receptors and cell-to-cell signaling factors involved in regulating cell division, maturation, and terminal differentiation. By measuring and manipulating sphingolipid metabolism using pharmacological and genetic tools in Caenorhabditis elegans, we provide evidence that the synthesis and remodeling of specific ceramides (e.g., dC18:1–C24:1), gangliosides (e.g., GM1–C24:1), and sphingomyelins (e.g., dC18:1–C18:1) influence development rate and lifespan. We found that the levels of fatty acid chain desaturation and elongation in many sphingolipid species increased during development and aging, with no such changes in developmentally-arrested dauer larvae or normal adults after food withdrawal (an anti-aging intervention). Pharmacological inhibitors and small interfering RNAs directed against serine palmitoyl transferase and glucosylceramide synthase acted to slow development rate, extend the reproductive period, and increase lifespan. In contrast, worms fed an egg yolk diet rich in sphingolipids exhibited accelerated development and reduced lifespan. Our findings demonstrate that sphingolipid accumulation and remodeling are critical events that determine development rate and lifespan in the nematode model, with both development rate and aging being accelerated by the synthesis of sphingomyelin, and its metabolism to ceramides and gangliosides. PMID:25437839

  5. Reducing sphingolipid synthesis orchestrates global changes to extend yeast lifespan.

    PubMed

    Liu, Jun; Huang, Xinhe; Withers, Bradley R; Blalock, Eric; Liu, Ke; Dickson, Robert C

    2013-10-01

    Studies of aging and longevity are revealing how diseases that shorten life can be controlled to improve the quality of life and lifespan itself. Two strategies under intense study to accomplish these goals are rapamycin treatment and calorie restriction. New strategies are being discovered including one that uses low-dose myriocin treatment. Myriocin inhibits the first enzyme in sphingolipid synthesis in all eukaryotes, and we showed recently that low-dose myriocin treatment increases yeast lifespan at least in part by down-regulating the sphingolipid-controlled Pkh1/2-Sch9 (ortholog of mammalian S6 kinase) signaling pathway. Here we show that myriocin treatment induces global effects and changes expression of approximately forty percent of the yeast genome with 1252 genes up-regulated and 1497 down-regulated (P < 0.05) compared with untreated cells. These changes are due to modulation of evolutionarily conserved signaling pathways including activation of the Snf1/AMPK pathway and down-regulation of the protein kinase A (PKA) and target of rapamycin complex 1 (TORC1) pathways. Many processes that enhance lifespan are regulated by these pathways in response to myriocin treatment including respiration, carbon metabolism, stress resistance, protein synthesis, and autophagy. These extensive effects of myriocin match those of rapamycin and calorie restriction. Our studies in yeast together with other studies in mammals reveal the potential of myriocin or related compounds to lower the incidence of age-related diseases in humans and improve health span. PMID:23725375

  6. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    SciTech Connect

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  7. TOR complex 2-Ypk1 signaling maintains sphingolipid homeostasis by sensing and regulating ROS accumulation.

    PubMed

    Niles, Brad J; Joslin, Amelia C; Fresques, Tara; Powers, Ted

    2014-02-13

    Reactive oxygen species (ROS) are produced during normal metabolism and can function as signaling molecules. However, ROS at elevated levels can damage cells. Here, we identify the conserved target of rapamycin complex 2 (TORC2)/Ypk1 signaling module as an important regulator of ROS in the model eukaryotic organism, S. cerevisiae. We show that TORC2/Ypk1 suppresses ROS produced both by mitochondria as well as by nonmitochondrial sources, including changes in acidification of the vacuole. Furthermore, we link vacuole-related ROS to sphingolipids, essential components of cellular membranes, whose synthesis is also controlled by TORC2/Ypk1 signaling. In total, our data reveal that TORC2/Ypk1 act within a homeostatic feedback loop to maintain sphingolipid levels and that ROS are a critical regulatory signal within this system. Thus, ROS sensing and signaling by TORC2/Ypk1 play a central physiological role in sphingolipid biosynthesis and in the maintenance of cell growth and viability. PMID:24462291

  8. An overview of sphingolipid metabolism: from synthesis to breakdown

    PubMed Central

    Gault, CR; Obeid, LM; Hannun, YA

    2011-01-01

    Sphingolipids constitute a class of lipids defined by their eighteen carbon amino-alcohol backbones which are synthesized in the ER from nonsphingolipid precursors. Modification of this basic structure is what gives rise to the vast family of sphingolipids that play significant roles in membrane biology and provide many bioactive metabolites that regulate cell function. Despite the diversity of structure and function of sphingolipids, their creation and destruction are governed by common synthetic and catabolic pathways. In this regard, sphingolipid metabolism can be imagined as an array of interconnected networks that diverge from a single common entry point and converge into a single common breakdown pathway. In their simplest forms, sphingosine, phytosphingosine and dihydrosphingosine serve as the backbones upon which further complexity is achieved. For example, phosphorylation of the C1 hydroxyl group yields the final breakdown products and/or the important signaling molecules sphingosine-1-phosphate, phytosphingosine-1-phosphate and dihydrosphingosine-1-phosphate, respectively. On the other hand, acylation of sphingosine, phytosphingosine, or dihydrosphingosine with one of several possible acyl CoA molecules through the action of distinct ceramide synthases produces the molecules defined as ceramide, phytoceramide, or dihydroceramide. Ceramide, due to the differing acyl CoAs that can be used to produce it, is technically a class of molecules rather than a single molecule and therefore may have different biological functions depending on the acyl chain it is composed of. At the apex of complexity is the group of lipids known as glycosphingolipids (GSL) which contain dozens of different sphingolipid species differing by both the order and type of sugar residues attached to their headgroups. Since these molecules are produced from ceramide precursors, they too may have differences in their acyl chain composition, revealing an additional layer of variation. The

  9. Regulation of PP2A by Sphingolipid Metabolism and Signaling

    PubMed Central

    Oaks, Joshua; Ogretmen, Besim

    2014-01-01

    Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that is a primary regulator of cellular proliferation through targeting of proliferative kinases, cell cycle regulators, and apoptosis inhibitors. It is through the regulation of these regulatory elements that gives PP2A tumor suppressor functions. In addition to mutations on the regulatory subunits, the phosphatase/tumor suppressing activity of PP2A is also inhibited in several cancer types due to overexpression or modification of the endogenous PP2A inhibitors such as SET/I2PP2A. This review focuses on the current literature regarding the interactions between the lipid signaling molecules, selectively sphingolipids, and the PP2A inhibitor SET for the regulation of PP2A, and the therapeutic potential of sphingolipids as PP2A activators for tumor suppression via targeting SET oncoprotein. PMID:25642418

  10. Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure.

    PubMed

    Cantalupo, Anna; Zhang, Yi; Kothiya, Milankumar; Galvani, Sylvain; Obinata, Hideru; Bucci, Mariarosaria; Giordano, Frank J; Jiang, Xian-Cheng; Hla, Timothy; Di Lorenzo, Annarita

    2015-09-01

    Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein-coupled receptor-dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II-induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II-induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis. PMID:26301690

  11. Nogo-B regulates endothelial sphingolipid homeostasis to control vascular function and blood pressure

    PubMed Central

    Kothiya, Milankumar; Galvani, Sylvain; Obinata, Hideru; Bucci, Mariarosaria; Giordano, Frank J; Jiang, Xian-Cheng; Hla, Timothy; Di Lorenzo, Annarita

    2015-01-01

    Endothelial dysfunction is a critical factor in many cardiovascular diseases, including hypertension. Although lipid signaling has been implicated in endothelial dysfunction and cardiovascular disease, specific molecular mechanisms are poorly understood. Here we report that Nogo-B, a membrane protein of the endoplasmic reticulum, regulates endothelial sphingolipid biosynthesis with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, the rate-limiting enzyme of the de novo sphingolipid biosynthetic pathway, thereby controlling production of endothelial sphingosine 1-phosphate and autocrine, G protein–coupled receptor–dependent signaling by this metabolite. Mice lacking Nogo-B either systemically or specifically in endothelial cells are hypotensive, resistant to angiotensin II–induced hypertension and have preserved endothelial function and nitric oxide release. In mice that lack Nogo-B, pharmacological inhibition of serine palmitoyltransferase with myriocin reinstates endothelial dysfunction and angiotensin II–induced hypertension. Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and shows that autocrine sphingolipid signaling within the endothelium is critical for vascular function and blood pressure homeostasis. PMID:26301690

  12. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

    PubMed

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D

    2016-01-29

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  13. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    PubMed Central

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  14. Sphingolipids: Key Regulators of Apoptosis and Pivotal Players in Cancer Drug Resistance

    PubMed Central

    Giussani, Paola; Tringali, Cristina; Riboni, Laura; Viani, Paola; Venerando, Bruno

    2014-01-01

    Drug resistance elicited by cancer cells still constitutes a huge problem that frequently impairs the efficacy of both conventional and novel molecular therapies. Chemotherapy usually acts to induce apoptosis in cancer cells; therefore, the investigation of apoptosis control and of the mechanisms used by cancer cells to evade apoptosis could be translated in an improvement of therapies. Among many tools acquired by cancer cells to this end, the de-regulated synthesis and metabolism of sphingolipids have been well documented. Sphingolipids are known to play many structural and signalling roles in cells, as they are involved in the control of growth, survival, adhesion, and motility. In particular, in order to increase survival, cancer cells: (a) counteract the accumulation of ceramide that is endowed with pro-apoptotic potential and is induced by many drugs; (b) increase the synthesis of sphingosine-1-phosphate and glucosylceramide that are pro-survivals signals; (c) modify the synthesis and the metabolism of complex glycosphingolipids, particularly increasing the levels of modified species of gangliosides such as 9-O acetylated GD3 (αNeu5Ac(2-8)αNeu5Ac(2-3)βGal(1-4)βGlc(1-1)Cer) or N-glycolyl GM3 (αNeu5Ac (2-3)βGal(1-4)βGlc(1-1)Cer) and de-N-acetyl GM3 (NeuNH(2)βGal(1-4)βGlc(1-1)Cer) endowed with anti-apoptotic roles and of globoside Gb3 related to a higher expression of the multidrug resistance gene MDR1. In light of this evidence, the employment of chemical or genetic approaches specifically targeting sphingolipid dysregulations appears a promising tool for the improvement of current chemotherapy efficacy. PMID:24625663

  15. The yeast model system as a tool towards the understanding of apoptosis regulation by sphingolipids.

    PubMed

    Rego, António; Trindade, Dário; Chaves, Susana R; Manon, Stéphen; Costa, Vítor; Sousa, Maria João; Côrte-Real, Manuela

    2014-02-01

    It has been established that sphingolipids are engaged in the regulation of apoptosis both as direct executors and as signalling molecules. However, the peculiarities of this class of bioactive lipids, namely the interconnectivity of their metabolic pathways, the specific subcellular localization where they are generated and the transport mechanisms involved, introduce a considerably high level of complexity in deciphering their role in the signalling and regulation of programmed cell death. Although yeast is undeniably a simple model, the conservation of the sphingolipid metabolism and of the core machinery engaged in regulated cell death has already provided valuable clues to the understanding of metabolic pathways involved in distinct cellular processes, including apoptosis. It can be anticipated that studies using this model system will further unravel mechanisms underlying the regulation of apoptosis by sphingolipids and contribute to novel therapeutic strategies against serious human diseases associated with dysfunction of sphingolipid-dependent cell death programmes. PMID:24103214

  16. Tamoxifen regulation of sphingolipid metabolism—therapeutic implications

    PubMed Central

    Morad, Samy A F; Cabot, Myles C

    2015-01-01

    Tamoxifen, a triphenylethylene antiestrogen and one of the first-line endocrine therapies used to treat estrogen receptor-positive breast cancer, has a number of interesting, off-target effects, and among these is the inhibition of sphingolipid metabolism. More specifically, tamoxifen inhibits ceramide glycosylation, and enzymatic step that can adventitiously support the influential tumor-suppressor properties of ceramide, the aliphatic backbone of sphingolipids. Additionally, tamoxifen and metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, have been shown to inhibit ceramide hydrolysis by the enzyme acid ceramidase. This particular intervention slows ceramide destruction and thereby depresses formation of sphingosine 1-phosphate, a mitogenic sphingolipid with cancer growth-promoting properties. As ceramide-centric therapies are becoming appealing clinical interventions in the treatment of cancer, agents like tamoxifen that can retard the generation of mitogenic sphingolipids and buffer ceramide clearance via inhibition of glycosylation, take on new importance. In this review, we present an abridged, lay introduction to sphingolipid metabolism, briefly chronicle tamoxifen’s history in the clinic, examine studies that demonstrate the impact of triphenylethylenes on sphingolipid metabolism in cancer cells, and canvass works relevant to the use of tamoxifen as adjuvant to drive ceramide-centric therapies in cancer treatment. The objective is to inform the readership of what could be a novel, off-label indication of tamoxifen and structurally-related triphenylethylenes, an indication divorced from estrogen receptor status and one with application in drug resistance. PMID:25964209

  17. Sphingolipids and cancer: ceramide and sphingosine-1-phosphate in the regulation of cell death and drug resistance

    PubMed Central

    Ponnusamy, Suriyan; Meyers-Needham, Marisa; Senkal, Can E; Saddoughi, Sahar A; Sentelle, David; Selvam, Shanmugam Panneer; Salas, Arelis; Ogretmen, Besim

    2011-01-01

    Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C16-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase 1-generated C18-ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate sphingosine-1-phosphate (S1P) by sphingosine kinases 1 and 2 mediates, with or without the involvement of G-protein-coupled S1P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S1P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer

  18. Sphingolipid synthesis and scavenging in the intracellular apicomplexan parasite, Toxoplasma gondii

    PubMed Central

    Pratt, Steven; Wansadhipathi-Kannangara, Nilu K.; Bruce, Catherine R.; Mina, John G.; Shams-Eldin, Hosam; Casas, Josefina; Hanada, Kentaro; Schwarz, Ralph T.; Sonda, Sabrina; Denny, Paul W.

    2013-01-01

    Sphingolipids are essential components of eukaryotic cell membranes, particularly the plasma membrane, and are involved in a diverse array of signal transduction pathways. Mammals produce sphingomyelin (SM) as the primary complex sphingolipid via the well characterised SM synthase. In contrast yeast, plants and some protozoa utilise an evolutionarily related inositol phosphorylceramide (IPC) synthase to synthesise IPC. This activity has no mammalian equivalent and IPC synthase has been proposed as a target for anti-fungals and anti-protozoals. However, detailed knowledge of the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites was lacking. In this study bioinformatic analyses indicated a single copy orthologue of the putative SM synthase from the apicomplexan Plasmodium falciparum (the causative agent of malaria) was a bona fide sphingolipid synthase in the related model parasite, Toxoplasma gondii (TgSLS). Subsequently, TgSLS was indicated, by complementation of a mutant cell line, to be a functional orthologue of the yeast IPC synthase (AUR1p), demonstrating resistance to the well characterised AUR1p inhibitor aureobasidin A. In vitro, recombinant TgSLS exhibited IPC synthase activity and, for the first time, the presence of IPC was demonstrated in T. gondii lipid extracts by mass spectrometry. Furthermore, host sphingolipid biosynthesis was indicated to influence, but be non-essential for, T. gondii proliferation, suggesting that whilst scavenging does take place de novo sphingolipid synthesis may be important for parasitism. PMID:23246819

  19. Sphingolipids as Regulators of the Phagocytic Response to Fungal Infections

    PubMed Central

    Bryan, Arielle M.; Del Poeta, Maurizio; Luberto, Chiara

    2015-01-01

    Fungal infections pose a significant risk for the increasing population of individuals who are immunocompromised. Phagocytes play an important role in immune defense against fungal pathogens, but the interactions between host and fungi are still not well understood. Sphingolipids have been shown to play an important role in many cell functions, including the function of phagocytes. In this review, we discuss major findings that relate to the importance of sphingolipids in macrophage and neutrophil function and the role of macrophages and neutrophils in the most common types of fungal infections, as well as studies that have linked these three concepts to show the importance of sphingolipid signaling in immune response to fungal infections. PMID:26688618

  20. Sphingolipids in High Fat Diet and Obesity-Related Diseases.

    PubMed

    Choi, Songhwa; Snider, Ashley J

    2015-01-01

    Nutrient oversupply associated with a high fat diet (HFD) significantly alters cellular metabolism, and specifically including sphingolipid metabolism. Sphingolipids are emerging as bioactive lipids that play key roles in regulating functions, in addition to their traditional roles as membrane structure. HFD enhances de novo sphingolipid synthesis and turnover of sphingolipids via the salvage pathway, resulting in the generation of ceramide, and more specifically long chain ceramide species. Additionally, HFD elevates sphingomyelin and sphingosine-1 phosphate (S1P) levels in several tissues including liver, skeletal muscle, adipose tissue, and cardiovascular tissues. HFD-stimulated sphingolipid generation contributes to systemic insulin resistance, dysregulated lipid accumulation, and cytokine expression and secretion from skeletal muscle and adipose tissues, exacerbating obesity-related conditions. Furthermore, altered sphingolipid levels, particularly ceramide and sphingomyelin, are involved in obesity-induced endothelial dysfunction and atherosclerosis. In this review, HFD-mediated sphingolipid metabolism and its impact on HFD-induced biology and pathobiology will be discussed. PMID:26648664

  1. Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload

    PubMed Central

    Zhang, Yi; Huang, Yan; Cantalupo, Anna; Azevedo, Paula S.; Siragusa, Mauro; Bielawski, Jacek; Giordano, Frank J.; Di Lorenzo, Annarita

    2016-01-01

    We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α–activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B–deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition. PMID:27158676

  2. TORC2-dependent protein kinase Ypk1 phosphorylates ceramide synthase to stimulate synthesis of complex sphingolipids.

    PubMed

    Muir, Alexander; Ramachandran, Subramaniam; Roelants, Françoise M; Timmons, Garrett; Thorner, Jeremy

    2014-01-01

    Plasma membrane lipid composition must be maintained during growth and under environmental insult. In yeast, signaling mediated by TOR Complex 2 (TORC2)-dependent protein kinase Ypk1 controls lipid abundance and distribution in response to membrane stress. Ypk1, among other actions, alleviates negative regulation of L-serine:palmitoyl-CoA acyltransferase, upregulating production of long-chain base precursors to sphingolipids. To explore other roles for TORC2-Ypk1 signaling in membrane homeostasis, we devised a three-tiered genome-wide screen to identify additional Ypk1 substrates, which pinpointed both catalytic subunits of the ceramide synthase complex. Ypk1-dependent phosphorylation of both proteins increased upon either sphingolipid depletion or heat shock and was important for cell survival. Sphingolipidomics, other biochemical measurements and genetic analysis demonstrated that these modifications of ceramide synthase increased its specific activity and stimulated channeling of long-chain base precursors into sphingolipid end-products. Control at this branch point also prevents accumulation of intermediates that could compromise cell growth by stimulating autophagy. PMID:25279700

  3. Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

    SciTech Connect

    Fujibayashi, S.; Wenger, D.A.

    1986-11-15

    Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When (/sup 35/S)methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the (/sup 35/S)methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.

  4. Hepatic fatty acid uptake is regulated by the sphingolipid acyl chain length.

    PubMed

    Park, Woo-Jae; Park, Joo-Won; Merrill, Alfred H; Storch, Judith; Pewzner-Jung, Yael; Futerman, Anthony H

    2014-12-01

    Ceramide synthase 2 (CerS2) null mice cannot synthesize very-long acyl chain (C22-C24) ceramides resulting in significant alterations in the acyl chain composition of sphingolipids. We now demonstrate that hepatic triacylglycerol (TG) levels are reduced in the liver but not in the adipose tissue or skeletal muscle of the CerS2 null mouse, both before and after feeding with a high fat diet (HFD), where no weight gain was observed and large hepatic nodules appeared. Uptake of both BODIPY-palmitate and [VH]-palmitate was also abrogated in the hepa- tocytes and liver. The role of a number of key proteins involved in fatty acid uptake was examined, including FATP5, CD36/FAT, FABPpm and cytoplasmic FABP1. Levels of FATP5 and FABP1 were decreased in the CerS2 null mouse liver, whereas CD36/FAT levels were significantly elevated and CD36/FAT was also mislocalized upon insulin treatment. Moreover, treatment of hepatocytes with C22-C24-ceramides down-regulated CD36/FAT levels. Infection of CerS2 null mice with recombinant adeno-associated virus (rAAV)-CerS2 restored normal TG levels and corrected the mislocalization of CD36/FAT, but had no effect on the intracellular localization or levels of FATP5 or FABP1. Together, these results demonstrate that hepatic fatty acid uptake via CD36/FAT can be regulated by altering the acyl chain composition of sphingolipids. PMID:25241943

  5. Arabidopsis mutants in sphingolipid synthesis as tools to understand the structure and function of membrane microdomains in plasmodesmata

    PubMed Central

    González-Solís, Ariadna; Cano-Ramírez, Dora L.; Morales-Cedillo, Francisco; Tapia de Aquino, Cinthya; Gavilanes-Ruiz, Marina

    2013-01-01

    Plasmodesmata—intercellular channels that communicate adjacent cells—possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures. PMID:24478783

  6. Sphingolipids in the Root Play an Important Role in Regulating the Leaf Ionome in Arabidopsis thaliana[W][OA

    PubMed Central

    Chao, Dai-Yin; Gable, Kenneth; Chen, Ming; Baxter, Ivan; Dietrich, Charles R.; Cahoon, Edgar B.; Guerinot, Mary Lou; Lahner, Brett; Lü, Shiyou; Markham, Jonathan E.; Morrissey, Joe; Han, Gongshe; Gupta, Sita D.; Harmon, Jeffrey M.; Jaworski, Jan G.; Dunn, Teresa M.; Salt, David E.

    2011-01-01

    Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis. PMID:21421810

  7. Sphingolipids in colon cancer

    PubMed Central

    García-Barros, Mónica; Coant, Nicolas; Truman, Jean-Philip; Snider, Ashley J.

    2013-01-01

    Colorectal cancer is one of the major causes of death in the western world. Despite increasing knowledge of the molecular signaling pathways implicated in colon cancer, therapeutic outcomes are still only moderately successful. Sphingolipids, a family of N-acyl linked lipids, have not only structural functions but are also implicated in important biological functions. Ceramide, sphingosine and sphingosine-1-phosphate are the most important bioactive lipids, and they regulate several key cellular functions. Accumulating evidence suggests that many cancers present alterations in sphingolipids and their metabolizing enzymes. The aim of this review is to discuss the emerging roles of sphingolipids, both endogenous and dietary, in colon cancer and the interaction of sphingolipids with WNT/β-catenin pathway, one of the most important signaling cascades that regulate development and homeostasis in intestine PMID:24060581

  8. Loss of Frataxin induces iron toxicity, sphingolipid synthesis, and Pdk1/Mef2 activation, leading to neurodegeneration

    PubMed Central

    Chen, Kuchuan; Lin, Guang; Haelterman, Nele A; Ho, Tammy Szu-Yu; Li, Tongchao; Li, Zhihong; Duraine, Lita; Graham, Brett H; Jaiswal, Manish; Yamamoto, Shinya; Rasband, Matthew N; Bellen, Hugo J

    2016-01-01

    Mutations in Frataxin (FXN) cause Friedreich’s ataxia (FRDA), a recessive neurodegenerative disorder. Previous studies have proposed that loss of FXN causes mitochondrial dysfunction, which triggers elevated reactive oxygen species (ROS) and leads to the demise of neurons. Here we describe a ROS independent mechanism that contributes to neurodegeneration in fly FXN mutants. We show that loss of frataxin homolog (fh) in Drosophila leads to iron toxicity, which in turn induces sphingolipid synthesis and ectopically activates 3-phosphoinositide dependent protein kinase-1 (Pdk1) and myocyte enhancer factor-2 (Mef2). Dampening iron toxicity, inhibiting sphingolipid synthesis by Myriocin, or reducing Pdk1 or Mef2 levels, all effectively suppress neurodegeneration in fh mutants. Moreover, increasing dihydrosphingosine activates Mef2 activity through PDK1 in mammalian neuronal cell line suggesting that the mechanisms are evolutionarily conserved. Our results indicate that an iron/sphingolipid/Pdk1/Mef2 pathway may play a role in FRDA. DOI: http://dx.doi.org/10.7554/eLife.16043.001 PMID:27343351

  9. Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids

    PubMed Central

    Mor, Visesato; Rella, Antonella; Farnoud, Amir M.; Singh, Ashutosh; Munshi, Mansa; Bryan, Arielle; Naseem, Shamoon; Konopka, James B.; Ojima, Iwao; Bullesbach, Erika; Ashbaugh, Alan; Linke, Michael J.; Cushion, Melanie; Collins, Margaret; Ananthula, Hari Krishna; Sallans, Larry; Desai, Pankaj B.; Wiederhold, Nathan P.; Fothergill, Annette W.; Kirkpatrick, William R.; Patterson, Thomas; Wong, Lai Hong; Sinha, Sunita; Giaever, Guri; Nislow, Corey; Flaherty, Patrick; Pan, Xuewen; Cesar, Gabriele Vargas; de Melo Tavares, Patricia; Frases, Susana; Miranda, Kildare; Rodrigues, Marcio L.; Luberto, Chiara; Nimrichter, Leonardo

    2015-01-01

    ABSTRACT Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. PMID:26106079

  10. Sphingolipid metabolites in inflammatory disease

    PubMed Central

    Maceyka, Michael; Spiegel, Sarah

    2015-01-01

    Sphingolipids are ubiquitous building blocks of eukaryotic cell membranes. Progress in our understanding of sphingolipid metabolism, state-of-the-art sphingolipidomic approaches and animal models have generated a large body of evidence demonstrating that sphingolipid metabolites, particularly ceramide and sphingosine-1-phosphate, are signalling molecules that regulate a diverse range of cellular processes that are important in immunity, inflammation and inflammatory disorders. Recent insights into the molecular mechanisms of action of sphingolipid metabolites and new perspectives on their roles in regulating chronic inflammation have been reported. The knowledge gained in this emerging field will aid in the development of new therapeutic options for inflammatory disorders. PMID:24899305

  11. Amyotrophic lateral sclerosis and denervation alter sphingolipids and up-regulate glucosylceramide synthase

    PubMed Central

    Henriques, Alexandre; Croixmarie, Vincent; Priestman, David A.; Rosenbohm, Angela; Dirrig-Grosch, Sylvie; D'Ambra, Eleonora; Huebecker, Mylene; Hussain, Ghulam; Boursier-Neyret, Claire; Echaniz-Laguna, Andoni; Ludolph, Albert C.; Platt, Frances M.; Walther, Bernard; Spedding, Michael; Loeffler, Jean-Philippe; Gonzalez De Aguilar, Jose-Luis

    2015-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset disease characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. Growing evidence suggests a link between changes in lipid metabolism and ALS. Here, we used UPLC/TOF-MS to survey the lipidome in SOD1(G86R) mice, a model of ALS. Significant changes in lipid expression were evident in spinal cord and skeletal muscle before overt neuropathology. In silico analysis also revealed appreciable changes in sphingolipids including ceramides and glucosylceramides (GlcCer). HPLC analysis showed increased amounts of GlcCer and downstream glycosphingolipids (GSLs) in SOD1(G86R) muscle compared with wild-type littermates. Glucosylceramide synthase (GCS), the enzyme responsible for GlcCer biosynthesis, was up-regulated in muscle of SOD1(G86R) mice and ALS patients, and in muscle of wild-type mice after surgically induced denervation. Conversely, inhibition of GCS in wild-type mice, following transient peripheral nerve injury, reversed the overexpression of genes in muscle involved in oxidative metabolism and delayed motor recovery. GCS inhibition in SOD1(G86R) mice also affected the expression of metabolic genes and induced a loss of muscle strength and morphological deterioration of the motor endplates. These findings suggest that GSLs may play a critical role in ALS muscle pathology and could lead to the identification of new therapeutic targets. PMID:26483191

  12. Substrate availability for long-chain base formation as a regulator of hepatic sphingolipid and cholesterol biosynthesis

    SciTech Connect

    Messmer, T.O.; Merrill, A.H. Jr.

    1986-03-05

    The de novo biosynthesis of the sphinganine and sphingosine backbones of sphingolipids was studied with isolated rat hepatocytes and established liver cell lines. The rate of incorporation of radiolabel from (/sup 14/C)-serine by intact cells was half maximal at 0.3 mM, which is similar to the K/sub m/ of the initial enzyme of this pathway and in vivo concentrations of this substrate. Long-chain base biosynthesis was stimulated by another precursor, palmitic acid, but other fatty acids were inhibitory. Hepatocytes isolated from fed and fasted rats had different rates of sphingolipid formation, which may also reflect the relative levels of palmitoyl-CoA. These results established that the availability of the precursors of long-chain base formation, serine and palmitic acid, is a major factor in the regulation of this pathway. Since sphingomyelin biosynthesis could be modified, its relationship to cholesterol metabolism was also examined. Both hepatocytes and cultured liver cells in high serine (0.6mM) had increased incorporation of (/sup 14/C)-acetate into cholesterol (13%, P < 0.05 and 50%, P < 0.01, respectively). These results indicate that sphingolipid and cholesterol biosynthesis are coordinately regulated, perhaps because these lipids are located in similar membranes and lipoproteins.

  13. Sng1 associates with Nce102 to regulate the yeast Pkh-Ypk signalling module in response to sphingolipid status.

    PubMed

    García-Marqués, Sara; Randez-Gil, Francisca; Dupont, Sebastien; Garre, Elena; Prieto, Jose A

    2016-06-01

    All cells are delimited by biological membranes, which are consequently a primary target of stress-induced damage. Cold alters membrane functionality by decreasing lipid fluidity and the activity of membrane proteins. In Saccharomyces cerevisiae, evidence links sphingolipid homeostasis and membrane phospholipid asymmetry to the activity of the Ypk1/2 proteins, the yeast orthologous of the mammalian SGK1-3 kinases. Their regulation is mediated by different protein kinases, including the PDK1 orthologous Pkh1/2p, and requires the function of protein effectors, among them Nce102p, a component of the sphingolipid sensor machinery. Nevertheless, the mechanisms and the actors involved in Pkh/Ypk regulation remain poorly defined. Here, we demonstrate that Sng1, a transmembrane protein, is an effector of the Pkh/Ypk module and identify the phospholipid asymmetry as key for yeast cold adaptation. Overexpression of SNG1 impairs phospholipid flipping, reduces reactive oxygen species (ROS) and improves, in a Pkh-dependent manner, yeast growth in myriocin-treated cells, suggesting that excess Sng1p stimulates the Pkh/Ypk signalling. Furthermore, we link these effects to the association of Sng1p with Nce102p. Indeed, we found that Sng1p interacts with Nce102p both physically and genetically. Moreover, mutant nce102∆ sng1∆ cells show features of impaired Pkh/Ypk signalling, including increased ROS accumulation, reduced life span and defects in Pkh/Ypk-controlled regulatory pathways. Finally, myriocin-induced hyperphosphorylation of Ypk1p and Orm2p, which controls sphingolipid homeostasis, does not occur in nce102∆ sng1∆ cells. Hence, both Nce102p and Sng1p participate in a regulatory circuit that controls the activity of the Pkh/Ypk module and their function is required in response to sphingolipid status. PMID:27033517

  14. Sphingolipids in parasitic protozoa

    PubMed Central

    Zhang, Kai; Bangs, James D.; Beverley, Stephen M.

    2009-01-01

    The surface of most protozoan parasites relies heavily upon lipid-anchored molecules, to form protective barriers and play critical functions required for infectivity. Sphingolipids (SLs) play important roles through their abundance and involvement in membrane microdomain formation, as well as serving as the lipid anchor for many of these molecules, and in some but possibly not all species, as important signaling molecules. Interactions of parasite sphingolipid metabolism with that of the host may potentially contribute to parasite survival and/or host defense. In this chapter we summarize current knowledge of SL structure, synthesis and function in several of the major parasitic protozoan groups. PMID:20919659

  15. The GARP complex is required for cellular sphingolipid homeostasis

    PubMed Central

    Fröhlich, Florian; Petit, Constance; Kory, Nora; Christiano, Romain; Hannibal-Bach, Hans-Kristian; Graham, Morven; Liu, Xinran; Ejsing, Christer S; Farese, Robert V; Walther, Tobias C

    2015-01-01

    Sphingolipids are abundant membrane components and important signaling molecules in eukaryotic cells. Their levels and localization are tightly regulated. However, the mechanisms underlying this regulation remain largely unknown. In this study, we identify the Golgi-associated retrograde protein (GARP) complex, which functions in endosome-to-Golgi retrograde vesicular transport, as a critical player in sphingolipid homeostasis. GARP deficiency leads to accumulation of sphingolipid synthesis intermediates, changes in sterol distribution, and lysosomal dysfunction. A GARP complex mutation analogous to a VPS53 allele causing progressive cerebello-cerebral atrophy type 2 (PCCA2) in humans exhibits similar, albeit weaker, phenotypes in yeast, providing mechanistic insights into disease pathogenesis. Inhibition of the first step of de novo sphingolipid synthesis is sufficient to mitigate many of the phenotypes of GARP-deficient yeast or mammalian cells. Together, these data show that GARP is essential for cellular sphingolipid homeostasis and suggest a therapeutic strategy for the treatment of PCCA2. DOI: http://dx.doi.org/10.7554/eLife.08712.001 PMID:26357016

  16. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  17. Sphingolipids in spinal cord injury.

    PubMed

    Jones, Zachary B; Ren, Yi

    2016-01-01

    Spinal cord injury (SCI) is a debilitating condition that affects millions of individuals worldwide. Despite progress over the last few decades, the molecular mechanisms of secondary SCI that continue to occur days and weeks after the original trauma remain poorly understood. As a result, current therapies for SCI are only marginally effective. Sphingolipids, a diverse class of bioactive lipids, have been shown to regulate SCI repair and key secondary injury processes such as apoptosis, ischemia and inflammation. This review will discuss the numerous roles of sphingolipids and highlight the potential of sphingolipid-targeted therapies for SCI. PMID:27570580

  18. Sphingolipids in spinal cord injury

    PubMed Central

    Jones, Zachary B; Ren, Yi

    2016-01-01

    Spinal cord injury (SCI) is a debilitating condition that affects millions of individuals worldwide. Despite progress over the last few decades, the molecular mechanisms of secondary SCI that continue to occur days and weeks after the original trauma remain poorly understood. As a result, current therapies for SCI are only marginally effective. Sphingolipids, a diverse class of bioactive lipids, have been shown to regulate SCI repair and key secondary injury processes such as apoptosis, ischemia and inflammation. This review will discuss the numerous roles of sphingolipids and highlight the potential of sphingolipid-targeted therapies for SCI. PMID:27570580

  19. Sphingolipids as targets for treatment of fungal infections.

    PubMed

    Rollin-Pinheiro, Rodrigo; Singh, Ashutosh; Barreto-Bergter, Eliana; Del Poeta, Maurizio

    2016-08-01

    Invasive fungal infections have significantly increased in the last few decades. Three classes of drugs are commonly used to treat these infections: polyenes, azoles and echinocandins. Unfortunately each of these drugs has drawbacks; polyenes are toxic, resistance against azoles is emerging and echinocandins have narrow spectrum of activity. Thus, the development of new antifungals is urgently needed. In this context, fungal sphingolipids have emerged as a potential target for new antifungals, because their biosynthesis in fungi is structurally different than in mammals. Besides, some fungal sphingolipids play an important role in the regulation of virulence in a variety of fungi. This review aims to highlight the diverse strategies that could be used to block the synthesis or/and function of fungal sphingolipids. PMID:27502288

  20. Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins.

    PubMed

    Yamaji, Toshiyuki; Hanada, Kentaro

    2015-02-01

    In recent decades, many sphingolipid enzymes, sphingolipid-metabolism regulators and sphingolipid transfer proteins have been isolated and characterized. This review will provide an overview of the intracellular localization and topology of sphingolipid enzymes in mammalian cells to highlight the locations where respective sphingolipid species are produced. Interestingly, three sphingolipids that reside or are synthesized in cytosolic leaflets of membranes (ceramide, glucosylceramide and ceramide-1-phosphate) all have cytosolic lipid transfer proteins (LTPs). These LTPs consist of ceramide transfer protein (CERT), four-phosphate adaptor protein 2 (FAPP2) and ceramide-1-phosphate transfer protein (CPTP), respectively. These LTPs execute functions that affect both the location and metabolism of the lipids they bind. Molecular details describing the mechanisms of regulation of LTPs continue to emerge and reveal a number of critical processes, including competing phosphorylation and dephosphorylation reactions and binding interactions with regulatory proteins and lipids that influence the transport, organelle distribution and metabolism of sphingolipids. PMID:25382749

  1. Differential Regulation of Specific Sphingolipids in Colon Cancer Cells during Staurosporine-Induced Apoptosis.

    PubMed

    del Solar, Virginia; Lizardo, Darleny Y; Li, Nasi; Hurst, Jerod J; Brais, Christopher J; Atilla-Gokcumen, G Ekin

    2015-12-17

    Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells. PMID:26687483

  2. Assessment of crosstalks between the Snf1 kinase complex and sphingolipid metabolism in S. cerevisiae via systems biology approaches.

    PubMed

    Borklu Yucel, Esra; Ulgen, Kutlu O

    2013-11-01

    Sphingolipids are essential building blocks of the plasma membranes and are highly bioactive in the regulation of diverse cellular functions and pathological processes, a fact which renders the sphingolipid metabolism an important research area. In this study, a computational framework was recruited for the reconstruction of a functional interaction network for sphingolipid metabolism in Baker's yeast, SSN. Gene Ontology (GO) annotations were integrated with functional interaction data of the BIOGRID database and the reconstructed protein interaction network was subjected to topological and descriptive analyses. SSN was of a scale-free nature, following a power law model with γ=1.41. Prominent processes of SSN revealed that the reconstructed network encapsulated the involvement of sphingolipid metabolism in vital cellular processes such as energy homeostasis, cell growth and/or death and synthesis of building blocks. To investigate the potential of SSN for predicting signal transduction pathways regulating and/or being regulated by sphingolipid biosynthesis in yeast, a case study involving the S. cerevisiae counterpart of AMP-activated protein kinase, the Snf1 kinase complex, was conducted. The mutant strain lacking the catalytic α subunit, snf1Δ/snf1Δ, had elevated inositol phosphorylceramide and mannosyl-inositol phosphorylceramide levels, and decreased mannosyl-diinositol phosphorylceramide levels compared to the wild type strain, revealing that Snf1p has a regulatory role in the sphingolipid metabolism. Transcriptome data belonging to that strain available in the literature were mapped onto SSN and the correlated SSN was further investigated to evaluate the possible crosstalk machineries where sphingolipids and Snf1p function in coordination, in other words the crosstalk points between sphingolipid-mediated and Snf1 kinase signalling. The subsequent investigation of the discovered candidate crosstalk processes by performing sensitivity experiments imply a

  3. Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells.

    PubMed

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea; Nusshold, Christoph; Brunner, Anna Martina; Plastira, Ioanna; Rechberger, Gerald; Reicher, Helga; Wadsack, Christian; Zimmer, Andreas; Malle, Ernst; Sattler, Wolfgang

    2015-07-15

    Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53(wt) and p53(mut) glioma cells, and interfered with S1P signaling specifically in p53(wt) U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53(wt) but not p53(mut) GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation. PMID:26002572

  4. Cancer Treatment Strategies Targeting Sphingolipid Metabolism

    PubMed Central

    Oskouian, Babak; Saba, Julie D.

    2011-01-01

    Ceramide and sphingosine-1-phosphate are related sphingolipid metabolites that can be generated through a de novo biosynthetic route or derived from the recycling of membrane sphingomyelin. Both these lipids regulate cellular responses to stress, with generally opposing effects. Sphingosine-1-phosphate functions as a growth and survival factor, acting as a ligand for a family of G protein-coupled receptors, whereas ceramide activates intrinsic and extrinsic apoptotic pathways through receptor-independent mechanisms. A growing body of evidence has implicated ceramide, sphingosine-1-phosphate and the genes involved in their synthesis, catabolism and signaling in various aspects of oncogenesis, cancer progression and drug- and radiation resistance. This may be explained in part by the finding that both lipids impinge upon the PI3K/AKT pathway, which represses apoptosis and autophagy. In addition, sphingolipids influence cell cycle progression, telomerase function, cell migration and stem cell biology. Considering the central role of ceramide in mediating physiological as well as pharmacologically stimulated apoptosis, ceramide can be considered a tumor-suppressor lipid. In contrast, sphingosine-1-phosphate can be considered a tumor-promoting lipid, and the enzyme responsible for its synthesis functions as an oncogene. Not surprisingly, genetic mutations that result in reduced ceramide generation, increased sphingosine-1-phosphate synthesis or which reduce steady state ceramide levels and increase sphingosine-1-phosphate levels have been identified as mechanisms of tumor progression and drug resistance in cancer cells. Pharmacological tools for modulating sphingolipid pathways are being developed and represent novel therapeutic strategies for the treatment of cancer. PMID:20919655

  5. Sphingolipid Long-Chain Base Synthesis in Plants (Characterization of Serine Palmitoyltransferase Activity in Squash Fruit Microsomes).

    PubMed Central

    Lynch, D. V.; Fairfield, S. R.

    1993-01-01

    The activity of serine palmitoyltransferase (palmitoyl-coenzyme A [CoA]:L-serine [Ser]-C-palmitoyltransferase [decarboxylating], EC 2.3.1.50), the enzyme catalyzing the first step in the synthesis of the long-chain base required for sphingolipid assembly, has been characterized in a plant system. Enzyme activity in a microsomal membrane fraction from summer squash fruit (Cucurbita pepo L. cv Early Prolific Straightneck) was assayed by monitoring the incorporation of L-[3H]Ser into the chloroform-soluble product, 3-ketosphinganine. Addition of NADPH to the assay system resulted in the conversion of 3-ketosphinganine to sphinganine. The apparent Km for Ser was approximately 1.8 mM. The enzyme exhibited a strong preference for palmitoyl-CoA, with optimal activity at a substrate concentration of 200 [mu]M. Pyridoxal 5[prime]-phosphate was required as a coenzyme. The pH optimum was 7.6, and the temperature optimum was 36 to 40[deg]C. Enzyme activity was greatest in the microsomal fraction obtained by differential centrifugation and was localized to the endoplasmic reticulum using marker enzymes. Two known mechanism-based inhibitors of the mammalian enzyme, L-cycloserine and [beta]-chloro-L-alanine, were effective inhibitors of enzyme activity in squash microsomes. Changes in enzyme activity with size (age) of squash fruit were observed. The results from this study suggest that the properties and catalytic mechanism of Ser palmitoyltransferase from squash are similar to those of the animal, fungal, and bacterial enzyme in most respects. The specific activity of the enzyme in squash microsomes ranged from 0.57 to 0.84 nmol min-1 mg-1 of protein, values 2- to 20-fold higher than those previously reported for preparations from animal tissues. PMID:12232036

  6. Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism.

    PubMed

    Rolando, Monica; Escoll, Pedro; Buchrieser, Carmen

    2016-06-01

    Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the host's sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival. PMID:27191778

  7. [Sphingolipid-mediated apoptotic signaling pathways].

    PubMed

    Cuvillier, Olivier; Andrieu-Abadie, Nathalie; Ségui, Bruno; Malagarie-Cazenave, Sophie; Tardy, Claudine; Bonhoure, Elisabeth; Levade, Thierry

    2003-01-01

    Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate. PMID:14708343

  8. Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest*

    PubMed Central

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N.; Barny, Marie-Anne

    2014-01-01

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  9. Expression of the bacterial type III effector DspA/E in Saccharomyces cerevisiae down-regulates the sphingolipid biosynthetic pathway leading to growth arrest.

    PubMed

    Siamer, Sabrina; Guillas, Isabelle; Shimobayashi, Mitsugu; Kunz, Caroline; Hall, Michael N; Barny, Marie-Anne

    2014-06-27

    Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest. PMID:24828506

  10. Sphingolipid metabolism and obesity-induced inflammation.

    PubMed

    Kang, Se-Chan; Kim, Bo-Rahm; Lee, Su-Yeon; Park, Tae-Sik

    2013-01-01

    Obesity is a metabolic disorder developed by overnutrition and a major cause for insulin resistance and cardiovascular events. Since adipose tissue is one of the major sites for the synthesis and secretion of cytokines, enlarged adipose tissue in obese condition alters inflammatory state leading to pathophysiological conditions such as type 2 diabetes and increased cardiovascular risk. A plausible theory for development of metabolic dysregulation is that obesity increases secretion of inflammatory cytokines from adipose tissue and causes a chronic inflammation in the whole body. Additionally accumulation of lipids in non-adipose tissues elevates the cellular levels of bioactive lipids that inhibit the signaling pathways implicated in metabolic regulation together with activated inflammatory response. Recent findings suggest that obesity-induced inflammatory response leads to modulation of sphingolipid metabolism and these bioactive lipids may function as mediators for increased risk of metabolic dysfunction. Importantly, elucidation of mechanism regarding sphingolipid metabolism and inflammatory disease will provide crucial information to development of new therapeutic strategies for the treatment of obesity-induced pathological inflammation. PMID:23761785

  11. Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

    PubMed

    Olson, D K; Fröhlich, F; Farese, R V; Walther, T C

    2016-08-01

    Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26747648

  12. [Sphingolipid and apoptosis].

    PubMed

    Wang, Jing; Hu, Xiao-Song; Shi, Jie-Ping

    2003-07-01

    Over the last decade, considerable progress has been made in the study of sphingolipids with the development of biological techniques. Sphingolipids play important roles in diverse physiological process, including cytoskeleton migration, angiogenesis, embryonic development and signal transduction. Except for this, the lastest evidence has suggested that sphingolipids and their metabolite (ceramide, sphingosine, sphingosine 1-phosphate) can induce apoptosis in a wide variety of tumor cell lines such as LoVo HT29, Bel7402, A549, CNE2 cells. This paper is attempted to review the recent advances of investigation into the relationship between sphingolipids and apoptosis. PMID:14628466

  13. Effects of stereochemistry, saturation, and hydrocarbon chain length on the ability of synthetic constrained azacyclic sphingolipids to trigger nutrient transporter down-regulation, vacuolation, and cell death.

    PubMed

    Perryman, Michael S; Tessier, Jérémie; Wiher, Timothy; O'Donoghue, Heather; McCracken, Alison N; Kim, Seong M; Nguyen, Dean G; Simitian, Grigor S; Viana, Matheus; Rafelski, Susanne; Edinger, Aimee L; Hanessian, Stephen

    2016-09-15

    Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound. PMID:27475534

  14. Nuclear Sphingolipid Metabolism

    PubMed Central

    Lucki, Natasha C.; Sewer, Marion B.

    2014-01-01

    Nuclear lipid metabolism is implicated in various processes, including transcription, splicing, and DNA repair. Sphingolipids play roles in numerous cellular functions, and an emerging body of literature has identified roles for these lipid mediators in distinct nuclear processes. Different sphingolipid species are localized in various subnuclear domains, including chromatin, the nuclear matrix, and the nuclear envelope, where sphingolipids exert specific regulatory and structural functions. Sphingomyelin, the most abundant nuclear sphingolipid, plays both structural and regulatory roles in chromatin assembly and dynamics in addition to being an integral component of the nuclear matrix. Sphingosine-1-phosphate modulates histone acetylation, sphingosine is a ligand for steroidogenic factor 1, and nuclear accumulation of ceramide has been implicated in apoptosis. Finally, nuclear membrane–associated ganglioside GM1 plays a pivotal role in Ca2+ homeostasis. This review highlights research on the factors that control nuclear sphingolipid metabolism and summarizes the roles of these lipids in various nuclear processes. PMID:21888508

  15. Fostering Inflammatory Bowel Disease: Sphingolipid Strategies to Join Forces

    PubMed Central

    Abdel Hadi, Loubna; Di Vito, Clara; Riboni, Laura

    2016-01-01

    Complex sphingolipids are essential structural components of intestinal membranes, providing protection and integrity to the intestinal mucosa and regulating intestinal absorption processes. The role of sphingolipid signaling has been established in numerous cellular events, including intestinal cell survival, growth, differentiation, and apoptosis. A significant body of knowledge demonstrates that intestinal sphingolipids play a crucial role, as such and through their signaling pathways, in immunity and inflammatory disorders. In this review, we report on and discuss the current knowledge on the metabolism, signaling, and functional implications of sphingolipids in inflammatory bowel disease (IBD), focusing on the different aspects of sphingolipid actions on inflammatory responses and on the potential of sphingolipid-targeted molecules as anti-IBD therapeutic agents. PMID:26880864

  16. Sphingolipid mediators in cardiovascular cell biology and pathology.

    PubMed

    Levade, T; Augé, N; Veldman, R J; Cuvillier, O; Nègre-Salvayre, A; Salvayre, R

    2001-11-23

    Sphingolipids have emerged as a new class of lipid mediators. In response to various extracellular stimuli, sphingolipid turnover can be stimulated in vascular cells and cardiac myocytes. Subsequent generation of sphingolipid molecules such as ceramide, sphingosine, and sphingosine-1-phosphate, is followed by regulation of ion fluxes and activation of various signaling pathways leading to smooth muscle cell proliferation, endothelial cell differentiation or apoptotic cell death, cell contraction, retraction, or migration. The importance of sphingolipids in cardiovascular signaling is illustrated by recent observations implicating them in physiological processes such as vasculogenesis as well as in frequent pathological conditions, including atherosclerosis and its complications. PMID:11717151

  17. Human genetic disorders of sphingolipid biosynthesis.

    PubMed

    Astudillo, Leonardo; Sabourdy, Frédérique; Therville, Nicole; Bode, Heiko; Ségui, Bruno; Andrieu-Abadie, Nathalie; Hornemann, Thorsten; Levade, Thierry

    2015-01-01

    Monogenic defects of sphingolipid biosynthesis have been recently identified in human patients. These enzyme deficiencies affect the synthesis of sphingolipid precursors, ceramides or complex glycosphingolipids. They are transmitted as autosomal recessive or dominant traits, and their resulting phenotypes often replicate the abnormalities seen in murine models deficient for the corresponding enzymes. In quite good agreement with the known critical roles of sphingolipids in cells from the nervous system and the epidermis, these genetic defects clinically manifest as neurological disorders, including paraplegia, epilepsy or peripheral neuropathies, or present with ichthyosis. The present review summarizes the genetic alterations, biochemical changes and clinical symptoms of this new group of inherited metabolic disorders. Hypotheses regarding the molecular pathophysiology and potential treatments of these diseases are also discussed. PMID:25141825

  18. Rice ORMDL controls sphingolipid homeostasis affecting fertility resulting from abnormal pollen development.

    PubMed

    Chueasiri, Chutharat; Chunthong, Ketsuwan; Pitnjam, Keasinee; Chakhonkaen, Sriprapai; Sangarwut, Numphet; Sangsawang, Kanidta; Suksangpanomrung, Malinee; Michaelson, Louise V; Napier, Johnathan A; Muangprom, Amorntip

    2014-01-01

    The orosomucoids (ORM) are ER-resisdent polypeptides encoded by ORM and ORMDL (ORM-like) genes. In humans, ORMDL3 was reported as genetic risk factor associated to asthma. In yeast, ORM proteins act as negative regulators of sphingolipid synthesis. Sphingolipids are important molecules regulating several processes including stress responses and apoptosis. However, the function of ORM/ORMDL genes in plants has not yet been reported. Previously, we found that temperature sensitive genetic male sterility (TGMS) rice lines controlled by tms2 contain a deletion of about 70 kb in chromosome 7. We identified four genes expressed in panicles, including an ORMDL ortholog, as candidates for tms2. In this report, we quantified expression of the only two candidate genes normally expressed in anthers of wild type plants grown in controlled growth rooms for fertile and sterile conditions. We found that only the ORMDL gene (LOC_Os07g26940) showed differential expression under these conditions. To better understand the function of rice ORMDL genes, we generated RNAi transgenic rice plants suppressing either LOC_Os07g26940, or all three ORMDL genes present in rice. We found that the RNAi transgenic plants with low expression of either LOC_Os07g26940 alone or all three ORMDL genes were sterile, having abnormal pollen morphology and staining. In addition, we found that both sphingolipid metabolism and expression of genes involved in sphingolipid synthesis were perturbed in the tms2 mutant, analogous to the role of ORMs in yeast. Our results indicated that plant ORMDL proteins influence sphingolipid homeostasis, and deletion of this gene affected fertility resulting from abnormal pollen development. PMID:25192280

  19. Following the flux of long-chain bases through the sphingolipid pathway in vivo using mass spectrometry.

    PubMed

    Martínez-Montañés, Fernando; Schneiter, Roger

    2016-05-01

    Sphingolipids are essential components of the plasma membrane. Their synthesis is tightly controlled by regulatory proteins, which impinge on the rate-limiting step of the pathway, the condensation of serine and palmitoyl-CoA to long-chain base (LCB). The subsequent conversion of LCB to ceramide by ceramide synthase (CerS) is also tightly regulated, because both the accumulation of LCB as well as an excess of ceramide is toxic. Here we describe an in vivo assay to monitor the flux of LCB through the sphingolipid pathway in yeast. Cells are provided with nonnatural odd-chain sphingosine analogs, C17-dihydrosphingosine or C17-phytosphingosine (PHS), and their incorporation into ceramide and more complex sphingolipids is monitored by mass spectrometry. Incorporation of C17-PHS is time and concentration dependent, is inhibited by fumonisin B1, an inhibitor of CerS, and greatly reduced in double mutant cells lacking components of the CerS, Lac1 and Lag1. The resulting C17-ceramides are further metabolized to more complex sphingolipids, inositol phosphorylceramide and mannosylinositol phosphorylceramide), indicating that the tracer can be used to decipher the regulation of later steps of the pathway. In support of this notion, we show that mutants lacking the Orm proteins, regulators of the rate-limiting step of the pathway, display increased steady-state levels of these intermediates without affecting their rate of synthesis. PMID:26977056

  20. The yeast PH domain proteins Slm1 and Slm2 are targets of sphingolipid signaling during the response to heat stress.

    PubMed

    Daquinag, Alexes; Fadri, Maria; Jung, Sung Yun; Qin, Jun; Kunz, Jeannette

    2007-01-01

    The PH domain-containing proteins Slm1 and Slm2 were previously identified as effectors of the phosphatidylinositol-4,5-bisphosphate (PI4,5P(2)) and TORC2 signaling pathways. Here, we demonstrate that Slm1 and Slm2 are also targets of sphingolipid signaling during the heat shock response. We show that upon depletion of cellular sphingolipid levels, Slm1 function becomes essential for survival under heat stress. We further demonstrate that Slm proteins are regulated by a phosphorylation/dephosphorylation cycle involving the sphingolipid-activated protein kinases Pkh1 and Pkh2 and the calcium/calmodulin-dependent protein phosphatase calcineurin. By using a combination of mass spectrometry and mutational analysis, we identified serine residue 659 in Slm1 as a site of phosphorylation. Characterization of Slm1 mutants that mimic dephosphorylated and phosphorylated states demonstrated that phosphorylation at serine 659 is vital for survival under heat stress and promotes the proper polarization of the actin cytoskeleton. Finally, we present evidence that Slm proteins are also required for the trafficking of the raft-associated arginine permease Can1 to the plasma membrane, a process that requires sphingolipid synthesis and actin polymerization. Together with previous work, our findings suggest that Slm proteins are subject to regulation by multiple signals, including PI4,5P(2), TORC2, and sphingolipids, and may thus integrate inputs from different signaling pathways to temporally and spatially control actin polarization. PMID:17101780

  1. Sphingolipids from Conyza canadensis.

    PubMed

    Mukhtar, Naveen; Iqbal, Kiran; Anis, Itrat; Malik, Abdul

    2002-12-01

    Sphingolipid 1 and its corresponding beta-D-glucopyranoside derivative 2 have been isolated from the ethylacetate fraction of Conyza canadensis along with beta-sitosterol 3, stigmasterol 4, beta-sitosterol 3-O-beta-D-glucoside 5 and harmine 6, reported for the first time from this species. The structures of 1 and 2 were elucidated through spectroscopy including two-dimensional NMR. PMID:12453535

  2. Multiple sphingolipid abnormalities following cerebral microendothelial hypoxia

    PubMed Central

    Testai, Fernando D.; Kilkus, John P.; Berdyshev, Evgeny; Gorshkova, Irina; Natarajan, Viswanathan; Dawson, Glyn

    2015-01-01

    Hypoxia has been previously shown to inhibit the dihydroceramide (DHC) desaturase, leading to the accumulation of DHC. In this study, we used metabolic labeling with [3H]-palmitate, HPLC/MS/MS analysis, and specific inhibitors to show numerous sphingolipid changes after oxygen deprivation in cerebral microendothelial cells. The increased DHC, particularly long-chain forms, was observed in both whole cells and detergent-resistant membranes. This was reversed by reoxygenation and blocked by the de novo sphingolipid synthesis inhibitor myriocin, but not by the neutral sphingomyelinase inhibitor GW-4869. Furthermore, oxygen deprivation of microendothelial cells increased levels of dihydro-sphingosine (DH-Sph), DH-sphingosine1-phosphate (DH-S1P), DH-sphingomyelin (DH-SM), DH-glucosylceramide (DH-GlcCer), and S1P levels. In vitro assays revealed no changes in the activity of sphingomyelinases or sphingomyelin synthase, but resulted in reduced S1P lyase activity and 40% increase in glucosylceramide synthase (GCS) activity, which was reversed by reoxygenation. Inhibition of the de novo sphingolipid pathway (myriocin) or GCS (EtPoD4) induced endothelial barrier dysfunction and increased caspase 3-mediated cell death in response to hypoxia. Our findings suggest that hypoxia induces synthesis of S1P and multiple dihydrosphingolipids, including DHC, DH-SM, DH-GlcCer, DH-Sph and DH-S1P, which may be involved in ameliorating the effects of stroke. PMID:25060904

  3. Sphingolipid signaling and hematopoietic malignancies: to the rheostat and beyond.

    PubMed

    Loh, Kenneth C; Baldwin, Dianna; Saba, Julie D

    2011-11-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid with diverse functions including the promotion of cell survival, proliferation and migration, as well as the regulation of angiogenesis, inflammation, immunity, vascular permeability and nuclear mechanisms that control gene transcription. S1P is derived from metabolism of ceramide, which itself has diverse and generally growth-inhibitory effects through its impact on downstream targets involved in regulation of apoptosis, senescence and cell cycle progression. Regulation of ceramide, S1P and the biochemical steps that modulate the balance and interconversion of these two lipids are major determinants of cell fate, a concept referred to as the "sphingolipid rheostat." There is abundant evidence that the sphingolipid rheostat plays a role in the origination, progression and drug resistance patterns of hematopoietic malignancies. The pathway has also been exploited to circumvent the problem of chemotherapy resistance in leukemia and lymphoma. Given the broad effects of sphingolipids, targeting multiple steps in the metabolic pathway may provide possible therapeutic avenues. However, new observations have revealed that sphingolipid signaling effects are more complex than previously recognized, requiring a revision of the sphingolipid rheostat model. Here, we summarize recent insights regarding the sphingolipid metabolic pathway and its role in hematopoietic malignancies. PMID:21707493

  4. Role of Sphingolipids in the Pathobiology of Lung Inflammation

    PubMed Central

    Ghidoni, Riccardo; Caretti, Anna; Signorelli, Paola

    2015-01-01

    Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease. PMID:26770018

  5. The role of epidermal sphingolipids in dermatologic diseases.

    PubMed

    Borodzicz, Sonia; Rudnicka, Lidia; Mirowska-Guzel, Dagmara; Cudnoch-Jedrzejewska, Agnieszka

    2016-01-01

    Sphingolipids, a group of lipids containing the sphingoid base, have both structural and biological functions in human epidermis. Ceramides, as a part of extracellular lipids in the stratum corneum, are important elements of the skin barrier and are involved in the prevention of transepidermal water loss. In addition, ceramides regulate such processes as proliferation, differentiation and apoptosis of keratinocytes. Another important sphingolipid, sphingosine-1-phosphate (S1P), inhibits proliferation and induces differentiation of keratinocytes. A recent clinical study of the efficacy and safety of ponesimod (a selective modulator of the S1P receptor 1) suggested that sphingolipid metabolism may become a new target for the pharmacological treatment of psoriasis. The role of sphingolipids in some dermatologic diseases, including psoriasis, atopic dermatitis and ichthyoses was summarized in this article. PMID:26786937

  6. The Yeast Sphingolipid Signaling Landscape

    PubMed Central

    Montefusco, David J.; Matmati, Nabil

    2014-01-01

    Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field. PMID:24220500

  7. Involvement of Sphingolipids in Ethanol Neurotoxicity in the Developing Brain

    PubMed Central

    Saito, Mariko; Saito, Mitsuo

    2013-01-01

    Ethanol-induced neuronal death during a sensitive period of brain development is considered one of the significant causes of fetal alcohol spectrum disorders (FASD). In rodent models, ethanol triggers robust apoptotic neurodegeneration during a period of active synaptogenesis that occurs around the first two postnatal weeks, equivalent to the third trimester in human fetuses. The ethanol-induced apoptosis is mitochondria-dependent, involving Bax and caspase-3 activation. Such apoptotic pathways are often mediated by sphingolipids, a class of bioactive lipids ubiquitously present in eukaryotic cellular membranes. While the central role of lipids in ethanol liver toxicity is well recognized, the involvement of sphingolipids in ethanol neurotoxicity is less explored despite mounting evidence of their importance in neuronal apoptosis. Nevertheless, recent studies indicate that ethanol-induced neuronal apoptosis in animal models of FASD is mediated or regulated by cellular sphingolipids, including via the pro-apoptotic action of ceramide and through the neuroprotective action of GM1 ganglioside. Such sphingolipid involvement in ethanol neurotoxicity in the developing brain may provide unique targets for therapeutic applications against FASD. Here we summarize findings describing the involvement of sphingolipids in ethanol-induced apoptosis and discuss the possibility that the combined action of various sphingolipids in mitochondria may control neuronal cell fate. PMID:24961420

  8. Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow.

    PubMed

    Watanabe, Hiroshi; Okahara, Kyohei; Naito-Matsui, Yuko; Abe, Mitsuhiro; Go, Shinji; Inokuchi, Jinichi; Okazaki, Toshiro; Kobayashi, Toshihide; Kozutsumi, Yasunori; Oka, Shogo; Takematsu, Hiromu

    2016-07-01

    Endomitosis is a special type of mitosis in which only cytokinesis-the final step of the cell division cycle-is defective, resulting in polyploid cells. Although endomitosis is biologically important, its regulatory aspects remain elusive. Psychosine, a lysogalactosylceramide, prevents proper cytokinesis when supplemented to proliferating cells. Cytokinetic inhibition by psychosine does not inhibit genome duplication. Consequently cells undergo multiple rounds of endomitotic cell cycles, resulting in the formation of giant multiploid cells. Here we successfully quantified psychosine-triggered multiploid cell formation, showing that membrane sphingolipids ratios modulate psychosine-triggered polyploidy in Namalwa cells. Among enzymes that experimentally remodel cellular sphingolipids, overexpression of glucosylceramide synthase to biosynthesize glycosylsphingolipids (GSLs) and neutral sphingomyelinase 2 to hydrolyze sphingomyelin (SM) additively enhanced psychosine-triggered multiploidy; almost all of the cells became polyploid. In the presence of psychosine, Namalwa cells showed attenuated cell surface SM clustering and suppression of phosphatidylinositol 4,5-bisphosphate production at the cleavage furrow, both important processes for cytokinesis. Depending on the sphingolipid balance between GSLs and SM, Namalwa cells could be effectively converted to viable multiploid cells with psychosine. PMID:27170180

  9. Disruption of sphingolipid biosynthesis in Nicotiana benthamiana activates salicylic acid-dependent responses and compromises resistance to Alternaria alternata f. sp. lycopersici.

    PubMed

    Rivas-San Vicente, Mariana; Larios-Zarate, Guadalupe; Plasencia, Javier

    2013-01-01

    Sphingolipids play an important role in signal transduction pathways that regulate physiological functions and stress responses in eukaryotes. In plants, recent evidence suggests that their metabolic precursors, the long-chain bases (LCBs) act as bioactive molecules in the immune response. Interestingly, the virulence of two unrelated necrotrophic fungi, Fusarium verticillioides and Alternaria alternata, which are pathogens of maize and tomato plants, respectively, depends on the production of sphinganine-analog mycotoxins (SAMs). These metabolites inhibit de novo synthesis of sphingolipids in their hosts causing accumulation of LCBs, which are key regulators of programmed cell death. Therefore, to gain more insight into the role of sphingolipids in plant immunity against SAM-producing necrotrophic fungi, we disrupted sphingolipid metabolism in Nicotiana benthamiana through virus-induced gene silencing (VIGS) of the serine palmitoyltransfersase (SPT). This enzyme catalyzes the first reaction in LCB synthesis. VIGS of SPT profoundly affected N. benthamiana development as well as LCB composition of sphingolipids. While total levels of phytosphingosine decreased, sphinganine and sphingosine levels increased in SPT-silenced plants, compared with control plants. Plant immunity was also affected as silenced plants accumulated salicylic acid (SA), constitutively expressed the SA-inducible NbPR-1 gene and showed increased susceptibility to the necrotroph A. alternata f. sp. lycopersici. In contrast, expression of NbPR-2 and NbPR-3 genes was delayed in silenced plants upon fungal infection. Our results strongly suggest that LCBs modulate the SA-dependent responses and provide a working model of the potential role of SAMs from necrotrophic fungi to disrupt the plant host response to foster colonization. PMID:22990908

  10. SPHINGOLIPIDOMICS: METHODS FOR THE COMPREHENSIVE ANALYSIS OF SPHINGOLIPIDS

    PubMed Central

    Haynes, Christopher A.; Allegood, Jeremy C.; Park, Hyejung; Sullards, M. Cameron

    2009-01-01

    Sphingolipids comprise a highly diverse and complex class of molecules that serve as both structural components of cellular membranes and signaling molecules capable of eliciting apoptosis, differentiation, chemotaxis, and other responses in mammalian cells. Comprehensive or “sphingolipidomic” analyses (structure specific, quantitative analyses of all sphingolipids, or at least all members of a critical subset) are required in order to elucidate the role(s) of sphingolipids in a given biological context because so many of the sphingolipids in a biological system are inter-converted structurally and metabolically. Despite the experimental challenges posed by the diversity of sphingolipid-regulated cellular responses, the detection and quantitation of multiple sphingolipids in a single sample has been made possible by combining classical analytical separation techniques such as high-performance liquid chromatography (HPLC) with state-of-the-art tandem mass spectrometry (MS/MS) techniques. As part of the Lipid MAPS consortium an internal standard cocktail was developed that comprises the signaling metabolites (i.e. sphingoid bases, sphingoid base-1-phosphates, ceramides, and ceramide-1-phosphates) as well as more complex species such as mono- and di-hexosylceramides and sphingomyelin. Additionally, the number of species that can be analyzed is growing rapidly with the addition of fatty acyl Co-As, sulfatides, and other complex sphingolipids as more internal standards are becoming available. The resulting LC-MS/MS analyses are one of the most analytically rigorous technologies that can provide the necessary sensitivity, structural specificity, and quantitative precision with high-throughput for “sphingolipidomic” analyses in small sample quantities. This review summarizes historical and state-of-the-art analytical techniques used for the for the identification, structure determination, and quantitation of sphingolipids from free sphingoid bases through more

  11. Deciphering the link between salicylic acid signaling and sphingolipid metabolism

    PubMed Central

    Sánchez-Rangel, Diana; Rivas-San Vicente, Mariana; de la Torre-Hernández, M. Eugenia; Nájera-Martínez, Manuela; Plasencia, Javier

    2015-01-01

    The field of plant sphingolipid biology has evolved in recent years. Sphingolipids are abundant in cell membranes, and genetic analyses revealed essential roles for these lipids in plant growth, development, and responses to abiotic and biotic stress. Salicylic acid (SA) is a key signaling molecule that is required for induction of defense-related genes and rapid and localized cell death at the site of pathogen infection (hypersensitive response) during incompatible host–pathogen interactions. Conceivably, while levels of SA rapidly increase upon pathogen infection for defense activation, they must be tightly regulated during plant growth and development in the absence of pathogens. Genetic and biochemical evidence suggest that the sphingolipid intermediates, long-chain sphingoid bases, and ceramides, play a role in regulating SA accumulation in plant cells. However, how signals generated from the perturbation of these key sphingolipid intermediates are transduced into the activation of the SA pathway has long remained to be an interesting open question. At least four types of molecules – MAP kinase 6, reactive oxygen species, free calcium, and nitric oxide – could constitute a mechanistic link between sphingolipid metabolism and SA accumulation and signaling. PMID:25806037

  12. A systematic simulation of the effect of salicylic acid on sphingolipid metabolism

    PubMed Central

    Shi, Chao; Yin, Jian; Liu, Zhe; Wu, Jian-Xin; Zhao, Qi; Ren, Jian; Yao, Nan

    2015-01-01

    The phytohormone salicylic acid (SA) affects plant development and defense responses. Recent studies revealed that SA also participates in the regulation of sphingolipid metabolism, but the details of this regulation remain to beexplored. Here, we use in silico Flux Balance Analysis (FBA) with published microarray data to construct a whole-cell simulation model, including 23 pathways, 259 reactions, and 172 metabolites, to predict the alterations in flux of major sphingolipid species after treatment with exogenous SA. This model predicts significant changes in fluxes of certain sphingolipid species after SA treatment, changes that likely trigger downstream physiological and phenotypic effects. To validate the simulation, we used 15N-labeled metabolic turnover analysis to measure sphingolipid contents and turnover rate in Arabidopsis thaliana seedlings treated with SA or the SA analog benzothiadiazole (BTH). The results show that both SA and BTH affect sphingolipid metabolism, altering the concentrations of certain species and also changing the optimal flux distribution and turnover rate of sphingolipids. Our strategy allows us to estimate sphingolipid fluxes on a short time scale and gives us a systemic view of the effect of SA on sphingolipid homeostasis. PMID:25859253

  13. Novel sphingolipid derivatives promote keratinocyte differentiation.

    PubMed

    Paragh, György; Schling, Petra; Ugocsai, Peter; Kel, Alexander E; Liebisch, Gerhard; Heimerl, Susanne; Moehle, Christoph; Schiemann, Yvonne; Wegmann, Michael; Farwick, Mike; Wikonkál, Norbert M; Mandl, József; Langmann, Thomas; Schmitz, Gerd

    2008-12-01

    Sphingolipids are important components of the water permeability barrier of the skin. Moreover, ceramides were also shown to influence keratinocyte differentiation and regulate cellular signalling. A confluence-induced differentiation model of normal human keratinocytes was established to allow evaluation of pro- and anti-differentiation effects of exogenous compounds. The effects of phytosphingosine (PS), sphingosine (SO), sphinganine (SA) and their hexanoyl (-C6), stearoyl (-C18) and salicyl (-SLC) derivatives, C12-alkylamine-salicylate (C12-SLC), salicylate (SLC) along with vitamin D3 (VD3) and retinol as control substances were tested in this system. Cytotoxicity assays were carried out to optimize the incubation conditions of compounds and whole genome expression changes were monitored by DNA-microarray on days 0, 1 and 4. Geometric means of gene expression levels of a subset of known keratinocyte differentiation-related genes were calculated from the microarray data to compare effects of the sphingolipid derivatives. Compound treatment-induced transcriptional changes were analysed by the ExPlain software (BIOBASE GmbH). Five of the assayed substances (SA, SO-C6, PS-C6, SO-SLC, PS-SLC) were found to be potent promoters of keratinocyte differentiation compared with VD3, and C12-SLC revealed potential anti-differentiation properties. ExPlain analysis found a different regulatory profile in the computed transcriptional networks of the sphingoid bases versus their -C6 and especially -SLC derivatives suggesting that the change in their keratinocyte differentiation modifying potential is due to a unique effect of the covalent attachment of the salicylic acid. Taken together, these results demonstrate the gene regulatory potential of sphingolipid species that could be valuable for dermatological or cosmetic applications. PMID:18631249

  14. Exploring the role of sphingolipid machinery during the epithelial to mesenchymal transition program using an integrative approach

    PubMed Central

    Meshcheryakova, Anastasia; Köfeler, Harald C.; Triebl, Alexander; Mungenast, Felicitas; Heinze, Georg; Gerner, Christopher; Zimmermann, Philip; Jaritz, Markus; Mechtcheriakova, Diana

    2016-01-01

    The epithelial to mesenchymal transition (EMT) program is activated in epithelial cancer cells and facilitates their ability to metastasize based on enhanced migratory, proliferative, anti-apoptotic, and pluripotent capacities. Given the fundamental impact of sphingolipid machinery to each individual process, the sphingolipid-related mechanisms might be considered among the most prominent drivers/players of EMT; yet, there is still limited knowledge. Given the complexity of the interconnected sphingolipid system, which includes distinct sphingolipid mediators, their synthesizing enzymes, receptors and transporters, we herein apply an integrative approach for assessment of the sphingolipid-associated mechanisms underlying EMT program. We created the sphingolipid-/EMT-relevant 41-gene/23-gene signatures which were applied to denote transcriptional events in a lung cancer cell-based EMT model. Based on defined 35-gene sphingolipid/EMT-attributed signature of regulated genes, we show close associations between EMT markers, genes comprising the sphingolipid network at multiple levels and encoding sphingosine 1-phosphate (S1P)-/ceramide-metabolizing enzymes, S1P and lysophosphatidic acid (LPA) receptors and S1P transporters, pluripotency genes and inflammation-related molecules, and demonstrate the underlying biological pathways and regulators. Mass spectrometry-based sphingolipid analysis revealed an EMT-attributed shift towards increased S1P and LPA accompanied by reduced ceramide levels. Notably, using transcriptomics data across various cell-based perturbations and neoplastic tissues (24193 arrays), we identified the sphingolipid/EMT signature primarily in lung adenocarcinoma tissues; besides, bladder, colorectal and prostate cancers were among the top-ranked. The findings also highlight novel regulatory associations between influenza virus and the sphingolipid/EMT-associated mechanisms. In sum, data propose the multidimensional contribution of sphingolipid machinery

  15. Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies

    PubMed Central

    Kitatani, Kazuyuki; Taniguchi, Makoto; Okazaki, Toshiro

    2015-01-01

    Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma. PMID:25997737

  16. Role of Sphingolipids and Metabolizing Enzymes in Hematological Malignancies.

    PubMed

    Kitatani, Kazuyuki; Taniguchi, Makoto; Okazaki, Toshiro

    2015-06-01

    Sphingolipids such as ceramide, sphingosine-1-phosphate and sphingomyelin have been emerging as bioactive lipids since ceramide was reported to play a role in human leukemia HL-60 cell differentiation and death. Recently, it is well-known that ceramide acts as an inducer of cell death, that sphingomyelin works as a regulator for microdomain function of the cell membrane, and that sphingosine-1-phosphate plays a role in cell survival/proliferation. The lipids are metabolized by the specific enzymes, and each metabolite could be again returned to the original form by the reverse action of the different enzyme or after a long journey of many metabolizing/synthesizing pathways. In addition, the metabolites may serve as reciprocal bio-modulators like the rheostat between ceramide and sphingosine-1-phosphate. Therefore, the change of lipid amount in the cells, the subcellular localization and the downstream signal in a specific subcellular organelle should be clarified to understand the pathobiological significance of sphingolipids when extracellular stimulation induces a diverse of cell functions such as cell death, proliferation and migration. In this review, we focus on how sphingolipids and their metabolizing enzymes cooperatively exert their function in proliferation, migration, autophagy and death of hematopoetic cells, and discuss the way developing a novel therapeutic device through the regulation of sphingolipids for effectively inhibiting cell proliferation and inducing cell death in hematological malignancies such as leukemia, malignant lymphoma and multiple myeloma. PMID:25997737

  17. Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

    PubMed

    Tani, Motohiro; Kuge, Osamu

    2012-12-01

    Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. PMID:23062277

  18. Sphingolipids and Plant Defense/Disease: The “Death” Connection and Beyond

    PubMed Central

    Berkey, Robert; Bendigeri, Dipti; Xiao, Shunyuan

    2012-01-01

    Sphingolipids comprise a major class of structural materials and lipid signaling molecules in all eukaryotic cells. Over the past two decades, there has been a phenomenal growth in the study of sphingolipids (i.e., sphingobiology) at an average rate of ∼1000 research articles per year. Sphingolipid studies in plants, though accounting for only a small fraction (∼6%) of the total number of publications, have also enjoyed proportionally rapid growth in the past decade. Concomitant with the growth of sphingobiology, there has also been tremendous progress in our understanding of the molecular mechanisms of plant innate immunity. In this review, we (i) cross examine and analyze the major findings that establish and strengthen the intimate connections between sphingolipid metabolism and plant programmed cell death (PCD) associated with plant defense or disease; (ii) highlight and compare key bioactive sphingolipids involved in the regulation of plant PCD and possibly defense; (iii) discuss the potential role of sphingolipids in polarized membrane/protein trafficking and formation of lipid rafts as subdomains of cell membranes in relation to plant defense; and (iv) where possible, attempt to identify potential parallels for immunity-related mechanisms involving sphingolipids across kingdoms. PMID:22639658

  19. On-tissue localization of ceramides and other sphingolipids by MALDI mass spectrometry imaging.

    PubMed

    Jones, E Ellen; Dworski, Shaalee; Canals, Daniel; Casas, Josefina; Fabrias, Gemma; Schoenling, Drew; Levade, Thierry; Denlinger, Chadrick; Hannun, Yusuf A; Medin, Jeffrey A; Drake, Richard R

    2014-08-19

    A novel MALDI-FTICR imaging mass spectrometry (MALDI-IMS) workflow is described for on-tissue detection, spatial localization, and structural confirmation of low abundance bioactive ceramides and other sphingolipids. Increasingly, altered or elevated levels of sphingolipids, sphingolipid metabolites, and sphingolipid metabolizing enzymes have been associated with a variety of disorders such as diabetes, obesity, lysosomal storage disorders, and cancer. Ceramide, which serves as a metabolic hub in sphingolipid metabolism, has been linked to cancer signaling pathways and to metabolic regulation with involvement in autophagy, cell-cycle arrest, senescence, and apoptosis. Using kidney tissues from a new Farber disease mouse model in which ceramides of all acyl chain lengths and other sphingolipid metabolites accumulate in tissues, specific ceramides and sphingomyelins were identified by on-tissue isolation and fragmentation, coupled with an on-tissue digestion by ceramidase or sphingomyelinase. Multiple glycosphingolipid species were also detected. The newly generated library of sphingolipid ions was then applied to MALDI-IMS of human lung cancer tissues. Multiple tumor specific ceramide and sphingomyelin species were detected and confirmed by on-tissue enzyme digests and structural confirmation. High-resolution MALDI-IMS in combination with novel on-tissue ceramidase and sphingomyelinase enzyme digestions makes it now possible to rapidly visualize the distribution of bioactive ceramides and sphingomyelin in tissues. PMID:25072097

  20. On-Tissue Localization of Ceramides and Other Sphingolipids by MALDI Mass Spectrometry Imaging

    PubMed Central

    2015-01-01

    A novel MALDI-FTICR imaging mass spectrometry (MALDI-IMS) workflow is described for on-tissue detection, spatial localization, and structural confirmation of low abundance bioactive ceramides and other sphingolipids. Increasingly, altered or elevated levels of sphingolipids, sphingolipid metabolites, and sphingolipid metabolizing enzymes have been associated with a variety of disorders such as diabetes, obesity, lysosomal storage disorders, and cancer. Ceramide, which serves as a metabolic hub in sphingolipid metabolism, has been linked to cancer signaling pathways and to metabolic regulation with involvement in autophagy, cell-cycle arrest, senescence, and apoptosis. Using kidney tissues from a new Farber disease mouse model in which ceramides of all acyl chain lengths and other sphingolipid metabolites accumulate in tissues, specific ceramides and sphingomyelins were identified by on-tissue isolation and fragmentation, coupled with an on-tissue digestion by ceramidase or sphingomyelinase. Multiple glycosphingolipid species were also detected. The newly generated library of sphingolipid ions was then applied to MALDI-IMS of human lung cancer tissues. Multiple tumor specific ceramide and sphingomyelin species were detected and confirmed by on-tissue enzyme digests and structural confirmation. High-resolution MALDI-IMS in combination with novel on-tissue ceramidase and sphingomyelinase enzyme digestions makes it now possible to rapidly visualize the distribution of bioactive ceramides and sphingomyelin in tissues. PMID:25072097

  1. SEPARATION AND IDENTIFICATION OF MAJOR PLANT SPHINGOLIPID CLASSES FROM LEAVES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sphingolipids are major components of the plasma membrane, tonoplast and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphi...

  2. Cellulose Synthesis and Its Regulation

    PubMed Central

    Li, Shundai; Bashline, Logan; Lei, Lei; Gu, Ying

    2014-01-01

    Cellulose, the most abundant biopolymer synthesized on land, is made of linear chains of ß (1–4) linked D-glucose. As a major structural component of the cell wall, cellulose is important not only for industrial use but also for plant growth and development. Cellulose microfibrils are tethered by other cell wall polysaccharides such as hemicellulose, pectin, and lignin. In higher plants, cellulose is synthesized by plasma membrane-localized rosette cellulose synthase complexes. Despite the recent advances using a combination of molecular genetics, live cell imaging, and spectroscopic tools, many aspects of the cellulose synthesis remain a mystery. In this chapter, we highlight recent research progress towards understanding the mechanism of cellulose synthesis in Arabidopsis. PMID:24465174

  3. Protein Synthesis Initiation Factors: Phosphorylation and Regulation

    SciTech Connect

    Karen S. Browning

    2009-06-15

    The initiation of the synthesis of proteins is a fundamental process shared by all living organisms. Each organism has both shared and unique mechanisms for regulation of this vital process. Higher plants provide for a major amount of fixation of carbon from the environment and turn this carbon into food and fuel sources for our use. However, we have very little understanding of how plants regulate the synthesis of the proteins necessary for these metabolic processes. The research carried out during the grant period sought to address some of these unknowns in the regulation of protein synthesis initiation. Our first goal was to determine if phosphorylation plays a significant role in plant initiation of protein synthesis. The role of phosphorylation, although well documented in mammalian protein synthesis regulation, is not well studied in plants. We showed that several of the factors necessary for the initiation of protein synthesis were targets of plant casein kinase and showed differential phosphorylation by the plant specific isoforms of this kinase. In addition, we identified and confirmed the phosphorylation sites in five of the plant initiation factors. Further, we showed that phosphorylation of one of these factors, eIF5, affected the ability of the factor to participate in the initiation process. Our second goal was to develop a method to make initiation factor 3 (eIF3) using recombinant methods. To date, we successfully cloned and expressed 13/13 subunits of wheat eIF3 in E. coli using de novo gene construction methods. The final step in this process is to place the subunits into three different plasmid operons for co-expression. Successful completion of expression of eIF3 will be an invaluable tool to the plant translation community.

  4. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

    PubMed

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C; Suttorp, Norbert; Proia, Richard L; Witzenrath, Martin; Kuebler, Wolfgang M

    2015-03-31

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  5. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

    PubMed Central

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M.; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C.; Suttorp, Norbert; Proia, Richard L.; Witzenrath, Martin; Kuebler, Wolfgang M.

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca2+ mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca2+ mobilization. Ca2+ mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  6. Interdiction of Sphingolipid Metabolism to Improve Standard Cancer Therapies

    PubMed Central

    Beckham, Thomas H.; Cheng, Joseph C.; Marrison, S. Tucker; Norris, James S.; Liu, Xiang

    2014-01-01

    Non-surgical therapies for human malignancies must negotiate complex cell signaling pathways to impede cancer cell growth, ideally promoting death of cancer cells while sparing healthy tissue. For most of the past half century, medical approaches for treating cancer have relied primarily on cytotoxic chemotherapeutics that interfere with DNA replication and cell division, susceptibilities of rapidly dividing cancer cells. As a consequence, these therapies exert considerable cell stress, promoting the generation of ceramide through de novo synthesis and recycling of complex glycosphingolipids and sphingomyelin into apoptotic ceramide. Radiotherapy of cancer exerts similar geno- and cytotoxic cell stresses, and generation of ceramide following ionizing radiation therapy is a well-described feature of radiation-induced cell death. Emerging evidence now describes sphingolipids as mediators of death in response to newer targeted therapies, cementing ceramide generation as a common mechanism of cell death in response to cancer therapy. Many studies have now shown that dysregulation of ceramide accumulation—whether by reduced generation or accelerated metabolism—is a common mechanism of resistance to standard cancer therapies. The aims of this chapter will be to discuss described mechanisms of cancer resistance to therapy related to dysregulation of sphingolipid metabolism and to explore clinical and preclinical approaches to interdict sphingolipid metabolism to improve outcomes of standard cancer therapies. PMID:23290775

  7. Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

    PubMed

    Veldman, Robert Jan; Klappe, Karin; Hinrichs, John; Hummel, Ina; van der Schaaf, Gieta; Sietsma, Hannie; Kok, Jan Willem

    2002-07-01

    Multidrug-resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug-resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug-resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell-free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells. PMID:12039850

  8. A method for analysis and design of metabolism using metabolomics data and kinetic models: Application on lipidomics using a novel kinetic model of sphingolipid metabolism.

    PubMed

    Savoglidis, Georgios; da Silveira Dos Santos, Aline Xavier; Riezman, Isabelle; Angelino, Paolo; Riezman, Howard; Hatzimanikatis, Vassily

    2016-09-01

    We present a model-based method, designated Inverse Metabolic Control Analysis (IMCA), which can be used in conjunction with classical Metabolic Control Analysis for the analysis and design of cellular metabolism. We demonstrate the capabilities of the method by first developing a comprehensively curated kinetic model of sphingolipid biosynthesis in the yeast Saccharomyces cerevisiae. Next we apply IMCA using the model and integrating lipidomics data. The combinatorial complexity of the synthesis of sphingolipid molecules, along with the operational complexity of the participating enzymes of the pathway, presents an excellent case study for testing the capabilities of the IMCA. The exceptional agreement of the predictions of the method with genome-wide data highlights the importance and value of a comprehensive and consistent engineering approach for the development of such methods and models. Based on the analysis, we identified the class of enzymes regulating the distribution of sphingolipids among species and hydroxylation states, with the D-phospholipase SPO14 being one of the most prominent. The method and the applications presented here can be used for a broader, model-based inverse metabolic engineering approach. PMID:27113440

  9. Regulation of Flavivirus RNA synthesis and replication.

    PubMed

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-12-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  10. Regulation of Flavivirus RNA synthesis and replication

    PubMed Central

    Selisko, Barbara; Wang, Chunling; Harris, Eva; Canard, Bruno

    2014-01-01

    RNA synthesis and replication of the members of the Flavivirus genus (including dengue, West Nile and Japanese encephalitis viruses) is regulated by a wide variety of mechanisms and actors. These include the sequestration of the RNA-dependent RNA polymerase (RdRp) for functions other than RNA synthesis, regulatory interactions with other viral and host proteins within the replication complex (RC), and regulatory elements within the RNA genome itself. In this review, we discuss our current knowledge of the multiple levels at which Flavivirus RNA synthesis is controlled. We aim to bring together two active research fields: the structural and functional biology of individual proteins of the RC and the impressive wealth of knowledge acquired regarding the viral genomic RNA. PMID:25462437

  11. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia.

    PubMed

    Melland-Smith, Megan; Ermini, Leonardo; Chauvin, Sarah; Craig-Barnes, Hayley; Tagliaferro, Andrea; Todros, Tullia; Post, Martin; Caniggia, Isabella

    2015-04-01

    Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder. PMID:25853898

  12. Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia

    PubMed Central

    Melland-Smith, Megan; Ermini, Leonardo; Chauvin, Sarah; Craig-Barnes, Hayley; Tagliaferro, Andrea; Todros, Tullia; Post, Martin; Caniggia, Isabella

    2015-01-01

    Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder. PMID:25853898

  13. A genome-wide screen for genes affecting eisosomes reveals Nce102 function in sphingolipid signaling

    PubMed Central

    Fröhlich, Florian; Moreira, Karen; Aguilar, Pablo S.; Hubner, Nina C.; Mann, Matthias; Walter, Peter

    2009-01-01

    The protein and lipid composition of eukaryotic plasma membranes is highly dynamic and regulated according to need. The sphingolipid-responsive Pkh kinases are candidates for mediating parts of this regulation, as they affect a diverse set of plasma membrane functions, such as cortical actin patch organization, efficient endocytosis, and eisosome assembly. Eisosomes are large protein complexes underlying the plasma membrane and help to sort a group of membrane proteins into distinct domains. In this study, we identify Nce102 in a genome-wide screen for genes involved in eisosome organization and Pkh kinase signaling. Nce102 accumulates in membrane domains at eisosomes where Pkh kinases also localize. The relative abundance of Nce102 in these domains compared with the rest of the plasma membrane is dynamically regulated by sphingolipids. Furthermore, Nce102 inhibits Pkh kinase signaling and is required for plasma membrane organization. Therefore, Nce102 might act as a sensor of sphingolipids that regulates plasma membrane function. PMID:19564405

  14. Vascular sphingolipids in physiological and pathological adaptation.

    PubMed

    Bao, Jun-Xiang; Su, Yu-Ting; Cheng, Yao-Ping; Zhang, Hai-Jun; Xie, Xiao-Ping; Chang, Yao-Ming

    2016-01-01

    Sphingolipids (SLs) are compounds containing a long-chain fatty alcohol amine called sphingosine which exists in cellular membranes, cytoplasm, nucleus, interstitial fluid, blood and lymphatic circulation. SLs act as essential constituents of membranes of eukaryotic cells, so the seesaw of SLs will lead to structural alteration of membranes instigating cellular functional change. SLs also act as crucial signaling molecules taking effect intracellularly or extracellularly which regulates activity of downstream molecules determining cellular adaptation to numerous stimulus. This review aims to highlight the contribution of SLs to physiological and pathophysiological remodeling of vasculature. We will first provide a short overview on metabolism, trafficking and compartmentalization of SLs. Then the regulation of SLs on reactive oxygen species (ROS) formation, vascular tone modulation, endothelial barrier integrity, apoptosis and autophagy are summarized. Finally, we will discuss how the SLs are modulated contributing to vascular development, angiogenesis and vascular remodeling in pathological situations as hypertension, atherosclerosis, and aging. The compellingly regulative actions of SLs bring about copious therapeutic targets for potential pharmacological intervention on the diseases involving vascular maladaptation. PMID:27100498

  15. Sphingolipids in Human Synovial Fluid - A Lipidomic Study

    PubMed Central

    Kosinska, Marta Krystyna; Liebisch, Gerhard; Lochnit, Guenter; Wilhelm, Jochen; Klein, Heiko; Kaesser, Ulrich; Lasczkowski, Gabriele; Rickert, Markus; Schmitz, Gerd; Steinmeyer, Juergen

    2014-01-01

    Articular synovial fluid (SF) is a complex mixture of components that regulate nutrition, communication, shock absorption, and lubrication. Alterations in its composition can be pathogenic. This lipidomic investigation aims to quantify the composition of sphingolipids (sphingomyelins, ceramides, and hexosyl- and dihexosylceramides) and minor glycerophospholipid species, including (lyso)phosphatidic acid, (lyso)phosphatidylglycerol, and bis(monoacylglycero)phosphate species, in the SF of knee joints from unaffected controls and from patients with early (eOA) and late (lOA) stages of osteoarthritis (OA), and rheumatoid arthritis (RA). SF without cells and cellular debris from 9 postmortem donors (control), 18 RA, 17 eOA, and 13 lOA patients were extracted to measure lipid species using electrospray ionization tandem mass spectrometry - directly or coupled with hydrophilic interaction liquid chromatography. We provide a novel, detailed overview of sphingolipid and minor glycerophospholipid species in human SF. A total of 41, 48, and 50 lipid species were significantly increased in eOA, lOA, and RA SF, respectively when compared with normal SF. The level of 21 lipid species differed in eOA SF versus SF from lOA, an observation that can be used to develop biomarkers. Sphingolipids can alter synovial inflammation and the repair responses of damaged joints. Thus, our lipidomic study provides the foundation for studying the biosynthesis and function of lipid species in health and most prevalent joint diseases. PMID:24646942

  16. Novel sphingolipids from Conyza canadensis.

    PubMed

    Mukhtar, Naveen; Iqbal, Kiran; Malik, Abdul

    2002-12-01

    New sphingolipids, 1,3,5-trihydroxy-2-hexadecanoylamino-(6E,9E)-heptacosdiene (1). 1,3,5-trihydroxy-2-hexadecanoylamino-(6E,9E)-heptacosdiene-1-O-glucopyranoside (2). 1,3-dihydroxy-2-hexanoylamino-(4E)-heptadecene (3). have been isolated from Conyza canadensis, along with five known compounds, p-hydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 3,5-dimethoxybenzoic acid, 3beta-hydroxyolean-12-en-28-oic acid, and 3beta-erythrodiol, isolated for the first time from this species. Their structures were determined by spectroscopic methods ((1)H- and (13)C-NMR, IR and MS) and two dimensional (2D)-NMR experiments. PMID:12499589

  17. Use of Bodipy-labeled sphingolipid and cholesterol analogs to examine membrane microdomains in cells

    PubMed Central

    Marks, David L.; Bittman, Robert

    2014-01-01

    Much evidence has accumulated to show that cellular membranes such as the plasma membrane, contain multiple “microdomains” of differing lipid and protein composition and function. These domains are sometimes enriched in cholesterol and sphingolipids and are believed to be important structures for the regulation of many biological and pathological processes. This review focuses on the use of fluorescent (Bodipy) labeled analogs of sphingolipids and cholesterol to study such domains. We discuss the similarities between the behavior of Bodipy-cholesterol and natural cholesterol in artificial bilayers and in cultured cells, and the use of Bodipy-sphingolipid analogs to visualize membrane domains in living cells based on the concentration-dependent monomer-excimer fluorescence properties of the Bodipy-fluorophore. The use of Bodipy-d-erythro-lactosylceramide is highlighted for detection of domains on the plasma membrane and endosome membranes, and the importance of the sphingolipid stereochemistry in modulating domain formation is discussed. Finally, we suggest that Bodipy-sphingolipids may be useful in future studies to examine the relationship between membrane domains at the cell surface and domains enriched in other lipids and proteins on the inner leaflet of the plasma membrane. PMID:18820942

  18. Characterization of AnNce102 and its role in eisosome stability and sphingolipid biosynthesis

    PubMed Central

    Athanasopoulos, Alexandros; Gournas, Christos; Amillis, Sotiris; Sophianopoulou, Vicky

    2015-01-01

    The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid biosynthesis, since their deletions partially suppress the thermosensitivity of basA mutant encoding sphingolipid C4-hydroxylase and the growth defects observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or pharmacological depletion of sphingolipids, conditions that induce the production of Reactive Oxygen Species (ROS), and can be partially overcome by deletion of pilA and/or annce102 at high temperatures. Consistent with these findings, pilAΔ and annce102Δ also show differential sensitivity to various oxidative agents, while AnNce102 overexpression can bypass sphingolipid depletion regarding the PilA/SurG foci number and organization, also leading to the mislocalization of PilA to septa. PMID:26468899

  19. The pleiotropic roles of sphingolipid signaling in autophagy

    PubMed Central

    Li, Y; Li, S; Qin, X; Hou, W; Dong, H; Yao, L; Xiong, L

    2014-01-01

    The autophagic process involves encompassing damaged proteins and organelles within double- or multi-membraned structures and delivering these molecules to the lytic compartments of vacuoles. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including rafts, caveolae, and cytosolic vesicles. SLs are a complex family of molecules that have a growing number of members, including ceramide, sphingosine-1-phosphate, and dihydroceramide, which have been associated with the essential cellular process of autophagy. This review highlights recent studies focusing on the regulation and function of SL-associated autophagy and its role in cell fate, diseases, and therapeutic interventions. PMID:24853423

  20. Maternal disturbance in activated sphingolipid metabolism causes pregnancy loss in mice

    PubMed Central

    Mizugishi, Kiyomi; Li, Cuiling; Olivera, Ana; Bielawski, Jacek; Bielawska, Alicja; Deng, Chu-Xia; Proia, Richard L.

    2007-01-01

    Uterine decidualization, a process that occurs in response to embryo implantation, is critical for embryonic survival and thus is a key event for successful pregnancy. Here we show that the sphingolipid metabolic pathway is highly activated in the deciduum during pregnancy and disturbance of the pathway by disruption of sphingosine kinase (Sphk) genes causes defective decidualization with severely compromised uterine blood vessels, leading to early pregnancy loss. Sphk-deficient female mice (Sphk1–/–Sphk2+/–) exhibited both an enormous accumulation of dihydrosphingosine and sphingosine and a reduction in phosphatidylethanolamine levels in pregnant uteri. These mice also revealed increased cell death in decidual cells, decreased cell proliferation in undifferentiated stromal cells, and massive breakage of decidual blood vessels, leading to uterine hemorrhage and early embryonic lethality. Thus, sphingolipid metabolism regulates proper uterine decidualization and blood vessel stability. Our findings also suggest that disturbance in sphingolipid metabolism may be considered as a cause of pregnancy loss in humans. PMID:17885683

  1. Organization and Regulation of Mitochondrial Protein Synthesis.

    PubMed

    Ott, Martin; Amunts, Alexey; Brown, Alan

    2016-06-01

    Mitochondria are essential organelles of endosymbiotic origin that are responsible for oxidative phosphorylation within eukaryotic cells. Independent evolution between species has generated mitochondrial genomes that are extremely diverse, with the composition of the vestigial genome determining their translational requirements. Typically, translation within mitochondria is restricted to a few key subunits of the oxidative phosphorylation complexes that are synthesized by dedicated ribosomes (mitoribosomes). The dramatically rearranged mitochondrial genomes, the limited set of transcripts, and the need for the synthesized proteins to coassemble with nuclear-encoded subunits have had substantial consequences for the translation machinery. Recent high-resolution cryo-electron microscopy has revealed the effect of coevolution on the mitoribosome with the mitochondrial genome. In this review, we place the new structural information in the context of the molecular mechanisms of mitochondrial translation and focus on the novel ways protein synthesis is organized and regulated in mitochondria. PMID:26789594

  2. The role of sphingolipids in neuronal plasticity of the brain.

    PubMed

    Sonnino, Sandro; Prinetti, Alessandro

    2016-05-01

    This Editorial highlights a study by Müller et al. in which the authors suggest a new sphingolipid-dependent mechanism for behavioral extinction. Their study should be considered in the broad perspective of sphingolipid metabolic pathways and traffic (depicted in the graphic). Read the highlighted article 'A sphingolipid mechanism for behavioral extinction' on page 589. PMID:26990419

  3. The sphingolipid salvage pathway in ceramide metabolism and signaling

    PubMed Central

    Kitatani, Kazuyuki; Idkowiak-Baldys, Jolanta; Hannun, Yusuf A.

    2008-01-01

    Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Of these, ceramide is a central metabolite and plays key roles in a variety of cellular responses, including regulation of cell growth, viability, differentiation, and senescence. Ceramide is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups. Sphingosine serves as the product of sphingolipid catabolism, and it is mostly salvaged through re-acylation, resulting in the generation of ceramide or its derivatives. This recycling of sphingosine is termed the “salvage pathway”, and recent evidence points to important roles for this pathway in ceramide metabolism and function. A number of enzymes are involved in the salvage pathway, and these include sphingomyelinases, cerebrosidases, ceramidases, and ceramide synthases. Recent studies suggest that the salvage pathway is not only subject to regulation, but it also modulates the formation of ceramide and subsequent ceramide-dependent cellular signals. This review focuses on the salvage pathway in ceramide metabolism, its regulation, its experimental analysis, and emerging biological functions. PMID:18191382

  4. Functional characterization of a higher plant sphingolipid Delta4-desaturase: defining the role of sphingosine and sphingosine-1-phosphate in Arabidopsis.

    PubMed

    Michaelson, Louise V; Zäuner, Simone; Markham, Jonathan E; Haslam, Richard P; Desikan, Radhika; Mugford, Sarah; Albrecht, Sandra; Warnecke, Dirk; Sperling, Petra; Heinz, E; Napier, Johnathan A

    2009-01-01

    The role of Delta4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Delta4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Delta4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Delta4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Delta4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Delta4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Delta4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Delta4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Delta4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides. PMID:18978071

  5. Functional Characterization of a Higher Plant Sphingolipid Δ4-Desaturase: Defining the Role of Sphingosine and Sphingosine-1-Phosphate in Arabidopsis1[W][OA

    PubMed Central

    Michaelson, Louise V.; Zäuner, Simone; Markham, Jonathan E.; Haslam, Richard P.; Desikan, Radhika; Mugford, Sarah; Albrecht, Sandra; Warnecke, Dirk; Sperling, Petra; Heinz, E.; Napier, Johnathan A.

    2009-01-01

    The role of Δ4-unsaturated sphingolipid long-chain bases such as sphingosine was investigated in Arabidopsis (Arabidopsis thaliana). Identification and functional characterization of the sole Arabidopsis ortholog of the sphingolipid Δ4-desaturase was achieved by heterologous expression in Pichia pastoris. A P. pastoris mutant disrupted in the endogenous sphingolipid Δ4-desaturase gene was unable to synthesize glucosylceramides. Synthesis of glucosylceramides was restored by the expression of Arabidopsis gene At4g04930, and these sphingolipids were shown to contain Δ4-unsaturated long-chain bases, confirming that this open reading frame encodes the sphingolipid Δ4-desaturase. At4g04930 has a very restricted expression pattern, transcripts only being detected in pollen and floral tissues. Arabidopsis insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930 were isolated and found to be phenotypically normal. Sphingolipidomic profiling of a T-DNA insertion mutant indicated the absence of Δ4-unsaturated sphingolipids in floral tissue, also resulting in the reduced accumulation of glucosylceramides. No difference in the response to drought or water loss was observed between wild-type plants and insertion mutants disrupted in the sphingolipid Δ4-desaturase At4g04930, nor was any difference observed in stomatal closure after treatment with abscisic acid. No differences in pollen viability between wild-type plants and insertion mutants were detected. Based on these observations, it seems unlikely that Δ4-unsaturated sphingolipids and their metabolites such as sphingosine-1-phosphate play a significant role in Arabidopsis growth and development. However, Δ4-unsaturated ceramides may play a previously unrecognized role in the channeling of substrates for the synthesis of glucosylceramides. PMID:18978071

  6. Sphingolipids mediate differential echinocandin susceptibility in Candida albicans and Aspergillus nidulans.

    PubMed

    Healey, Kelley R; Challa, Krishna K; Edlind, Thomas D; Katiyar, Santosh K

    2015-01-01

    The cell wall synthesis-inhibiting echinocandins, including caspofungin and micafungin, play important roles in the treatment of candidiasis and aspergillosis. Previous studies revealed that, in the haploid yeast Candida glabrata, sphingolipid biosynthesis pathway mutations confer caspofungin reduced susceptibility (CRS) but micafungin increased susceptibility (MIS). Here, we describe one Candida albicans strain (of 10 tested) that similarly yields CRS-MIS mutants at relatively high frequency. Mutants demonstrated increased levels of long-chain bases (sphingolipid pathway intermediates) and, unique to this strain, loss of His104/Pro104 heterozygosity in the TSC13-encoded enoyl reductase. CRS-MIS was similarly observed in a C. albicans homozygous fen1Δ fen12Δ laboratory strain and in diverse wild-type strains following exogenous long-chain-base treatment. Analogous to these results, CRS-MIS was demonstrated in an Aspergillus nidulans basA mutant encoding defective sphingolipid C4-hydroxylase and in its wild-type parent exposed to long-chain bases. Sphingolipids likely modulate echinocandin interaction with their Fks membrane target in all susceptible fungi, with potential implications for optimizing therapy with existing antifungals and the development of novel agents. PMID:25824222

  7. Sphingolipids in neuroblastoma: their role in drug resistance mechanisms.

    PubMed

    Sietsma, Hannie; Dijkhuis, Anne Jan; Kamps, Willem; Kok, Jan Willem

    2002-08-01

    Disseminated neuroblastoma usually calls for chemotherapy as the primary approach for treatment. Treatment failure is often attributable to drug resistance. This involves a variety of cellular mechanisms, including increased drug efflux through expression of ATP-binding cassette transporters (e.g., P-glycoprotein) and the inability of tumor cells to activate or propagate the apoptotic response. In recent years it has become apparent that sphingolipid metabolism and the generation of sphingolipid species, such as ceramide, also play a role in drug resistance. This may involve an autonomous mechanism, related to direct effects of sphingolipids on the apoptotic response, but also a subtle interplay between sphingolipids and ATP-binding cassette transporters. Here, we present an overview of the current understanding of the multiple levels at which sphingolipids function in drug resistance, with an emphasis on sphingolipid function in neuroblastoma and how modulation of sphingolipid metabolism may be used as a novel treatment paradigm. PMID:12374201

  8. The involvement of sphingolipids in multidrug resistance.

    PubMed

    Sietsma, H; Veldman, R J; Kok, J W

    2001-06-01

    Administration of most chemotherapeutic agents eventually results in the onset of apoptosis, despite the agents' variety in structure and molecular targets. Ceramide, the central molecule in cellular glycosphingolipid metabolism, has recently been identified as an important mediator of this process. Indeed, one of the events elicited by application of many cytotoxic drugs is an accumulation of this lipid. Treatment failure in cancer chemotherapy is largely attributable to multidrug resistance, in which tumor cells are typically cross-resistant to multiple chemotherapeutic agents. Different cellular mechanisms underlying this phenomenon have been described. Of these the drug efflux pump activity of P-glycoprotein and the multidrug resistance-associated proteins are the most extensively studied examples. Recently, an increased cellular capacity for ceramide glycosylation has been recognized as a novel multidrug resistance mechanism. Indeed, virtually all multidrug-resistant cells exhibit a deviating sphingolipid composition, most typically, increased levels of glucosylceramide. On the other hand, several direct molecular interactions between sphingolipids and drug efflux proteins have been described. Therefore, in addition to a role in the multidrug resistance phenotype by which ceramide accumulation and, thus, the onset of apoptosis are prevented, an indirect role for sphingolipids might be envisaged, by which the activity of these efflux proteins is modulated. In this review, we present an overview of the current understanding of the interesting relations that exist between sphingolipid metabolism and multidrug resistance. PMID:11420602

  9. Sphingolipid signalling: molecular basis and role in TNF-alpha-induced cell death.

    PubMed

    Malagarie-Cazenave, Sophie; Andrieu-Abadie, Nathalie; Ségui, Bruno; Gouazé, Valérie; Tardy, Claudine; Cuvillier, Olivier; Levade, Thierry

    2002-12-01

    Various lipidic molecules serve as second messengers for transducing signals from the cell surface to the cell interior and trigger specific cellular responses. Sphingolipids represent a complex group of lipids that have recently emerged as new transducers in eukaryotic cells. Several sphingolipid molecules are able to modulate cell growth, differentiation and death. This review summarises current knowledge of the signalling functions of sphingolipids, especially in the regulation of tumour necrosis factor [alpha] (TNF-[alpha])-mediated cytotoxic effects. TNF-[alpha] is a multifaceted cytokine that controls a wide range of immune responses in mammals, including induction of programmed cell death (also called apoptosis). On the basis of recent observations, a working model is proposed for the molecular mechanisms underlying regulation of sphingolipid generation following TNF-[alpha] receptor 1 activation. The implications of these findings for the development of future pharmacological strategies to prevent the cytotoxic TNF-[alpha] response and subsequent cellular dysfunctions (as seen in various human diseases) are discussed. PMID:14987386

  10. IKK NBD peptide inhibits LPS induced pulmonary inflammation and alters sphingolipid metabolism in a murine model.

    PubMed

    von Bismarck, Philipp; Winoto-Morbach, Supandi; Herzberg, Mona; Uhlig, Ulrike; Schütze, Stefan; Lucius, Ralph; Krause, Martin F

    2012-06-01

    Airway epithelial NF-κB is a key regulator of host defence in bacterial infections and has recently evolved as a target for therapeutical approaches. Evidence is accumulating that ceramide, generated by acid sphingomyelinase (aSMase), and sphingosine-1-phosphate (S1-P) are important mediators in host defence as well as in pathologic processes of acute lung injury. Little is known about the regulatory mechanisms of pulmonary sphingolipid metabolism in bacterial infections of the lung. The objective of this study was to evaluate the influence of NF-κB on sphingolipid metabolism in Pseudomonas aeruginosa LPS-induced pulmonary inflammation. In a murine acute lung injury model with intranasal Pseudomonas aeruginosa LPS we investigated TNF-α, KC (murine IL-8), IL-6, MCP-1 and neutrophilic infiltration next to aSMase activity and ceramide and S1-P lung tissue concentrations. Airway epithelial NF-κB was inhibited by topically applied IKK NBD, a cell penetrating NEMO binding peptide. This treatment resulted in significantly reduced inflammation and suppression of aSMase activity along with decreased ceramide and S1-P tissue concentrations down to levels observed in healthy animals. In conclusion our results confirm that changes in sphingolipid metabolim due to Pseudomonas aeruginosa LPS inhalation are regulated by NF-κB translocation. This confirms the critical role of airway epithelial NF-κB pathway for the inflammatory response to bacterial pathogens and underlines the impact of sphingolipids in inflammatory host defence mechanisms. PMID:22469869

  11. Plant sphingolipids: decoding the enigma of the Sphinx

    PubMed Central

    Pata, Mickael O.; Hannun, Yusuf A.; Ng, Carl K.-Y.

    2009-01-01

    Summary Sphingolipids are a ubiquitous class of lipids present in a variety of organisms including eukaryotes and bacteria. In the last two decades, research has focused on characterizing the individual species of this complex family of lipids, leading to a new field of research called sphingolipidomics. There are at least 500 (and perhaps thousands) different molecular species of sphingolipids in cells, and in Arabidopsis alone, it has been reported that there are at least 168 different sphingolipids. Plant sphingolipids can be divided into four classes: glycosyl inositol phosphoceramides (GIPCs), glycosylceramides, ceramides, and free long chain bases (LCBs). Numerous enzymes involved in plant sphingolipid metabolism have now been cloned and characterized, and, in general, there is broad conservation in the way sphingolipids are metabolized in animals, yeast and plants. Here, we review the diversity of sphingolipids reported in the literature, some of the recent advances in our understanding of sphingolipid metabolism in plants, and the physiological roles that sphingolipids and sphingolipid metabolites play in plant physiology. PMID:20028469

  12. Disruption of Sphingolipid Metabolism Elicits Apoptosis-Associated Reproductive Defects in Drosophila

    PubMed Central

    Phan, Van H.; Herr, Deron R.; Panton, Dionne; Fyrst, Henrik; Saba, Julie D.; Harris, Greg L.

    2007-01-01

    Sphingolipid signaling is thought to regulate apoptosis via mechanisms that are dependent on the concentration of ceramide relative to that of sphingosine-1-phosphate (S1P). This study reports defects in reproductive structures and function that are associated with enhanced apoptosis in Drosophila Sply05091 mutants that lack functional S1P lyase and thereby accumulate sphingolipid long chain base metabolites. Analyses of reproductive structures in these adult mutants unmasked multiple abnormalities, including supernumerary spermathecae, degenerative ovaries, and severely reduced testes. TUNEL assessment revealed increased cell death in mutant egg chambers at most oogenic stages and in affected mutant testes. These reproductive abnormalities and elevated gonadal apoptosis were also observed, to varying degrees, in other mutants affecting sphingolipid metabolism. Importantly, the reproductive defects seen in the Sply05091 mutants were ameliorated both by a second site mutation in the lace gene that restores long chain base levels towards normal and by genetic disruption of the proapoptotic genes reaper, hid and grim. These data thus provide the first evidence in Drosophila that accumulated sphingolipids trigger elevated levels of apoptosis via the modulation of known signaling pathways. PMID:17706961

  13. A sphingolipid mechanism for behavioral extinction.

    PubMed

    Huston, Joseph P; Kornhuber, Johannes; Mühle, Christiane; Japtok, Lukasz; Komorowski, Mara; Mattern, Claudia; Reichel, Martin; Gulbins, Erich; Kleuser, Burkhard; Topic, Bianca; De Souza Silva, Maria A; Müller, Christian P

    2016-05-01

    Reward-dependent instrumental behavior must continuously be re-adjusted according to environmental conditions. Failure to adapt to changes in reward contingencies may incur psychiatric disorders like anxiety and depression. When an expected reward is omitted, behavior undergoes extinction. While extinction involves active re-learning, it is also accompanied by emotional behaviors indicative of frustration, anxiety, and despair (extinction-induced depression). Here, we report evidence for a sphingolipid mechanism in the extinction of behavior. Rapid extinction, indicating efficient re-learning, coincided with a decrease in the activity of the enzyme acid sphingomyelinase (ASM), which catalyzes turnover of sphingomyelin to ceramide, in the dorsal hippocampus of rats. The stronger the decline in ASM activity, the more rapid was the extinction. Sphingolipid-focused lipidomic analysis showed that this results in a decline of local ceramide species in the dorsal hippocampus. Ceramides shape the fluidity of lipid rafts in synaptic membranes and by that way can control neural plasticity. We also found that aging modifies activity of enzymes and ceramide levels in selective brain regions. Aging also changed how the chronic treatment with corticosterone (stress) or intranasal dopamine modified regional enzyme activity and ceramide levels, coinciding with rate of extinction. These data provide first evidence for a functional ASM-ceramide pathway in the brain involved in the extinction of learned behavior. This finding extends the known cellular mechanisms underlying behavioral plasticity to a new class of membrane-located molecules, the sphingolipids, and their regulatory enzymes, and may offer new treatment targets for extinction- and learning-related psychopathological conditions. Sphingolipids are common lipids in the brain which form lipid domains at pre- and postsynaptic membrane compartments. Here we show a decline in dorsal hippocampus ceramide species together with a

  14. Deregulation of sphingolipid metabolism in Alzheimer's disease

    PubMed Central

    He, Xingxuan; Huang, Yu; Li, Bin; Gong, Cheng-Xing; Schuchman, Edward H.

    2010-01-01

    Abnormal sphingolipid metabolism has been previously reported in Alzheimer's disease (AD). To extend these findings, several sphingolipids and sphingolipid hydrolases were analyzed in brain samples from AD patients and age-matched normal individuals. We found a pattern of elevated acid sphingomyelinase (ASM) and acid ceramidase (AC) expression in AD, leading to a reduction in sphingomyelin and elevation of ceramide. More sphingosine also was found in the AD brains, although sphingosine-1-phosphate (S1P) levels were reduced. Notably, significant correlations were observed between the brain ASM and S1P levels and the levels of amyloid beta peptide (Aβ) and phosphorylated tau protein. Based on these findings, neuronal cell cultures were treated with Aβ oligomers, which were found to activate ASM, increase ceramide, and induce apoptosis. Pre-treatment of the neurons with purified, recombinant AC prevented the cells from undergoing Aβ-induced apoptosis. We propose that ASM activation is an important pathological event leading to AD, perhaps due to Aβ deposition. The downstream consequences of ASM activation are elevated ceramide, activation of ceramidases, and production of sphingosine. The reduced levels of S1P in the AD brain, together with elevated ceramide, likely contribute to the disease pathogenesis. PMID:18547682

  15. Network-based analysis of the sphingolipid metabolism in hypertension

    PubMed Central

    Fenger, Mogens; Linneberg, Allan; Jeppesen, Jørgen

    2015-01-01

    Common diseases like essential hypertension or diabetes mellitus are complex as they are polygenic in nature, such that each genetic variation only has a small influence on the disease. Genes operates in integrated networks providing the blue-print for all biological processes and conditional of the complex genotype determines the state and dynamics of any trait, which may be modified to various extent by non-genetic factors. Thus, diseases are heterogenous ensembles of conditions with a common endpoint. Numerous studies have been performed to define genes of importance for a trait or disease, but only a few genes with small effect have been identified. The major reasons for this modest progress is the unresolved heterogeneity of the regulation of blood pressure and the shortcomings of the prevailing monogenic approach to capture genetic effects in a polygenic condition. Here, a two-step procedure is presented in which physiological heterogeneity is disentangled and genetic effects are analyzed by variance decomposition of genetic interactions and by an information theoretical approach including 162 single nucleotide polymorphisms (SNP) in 84 genes in the sphingolipid metabolism and related networks in blood pressure regulation. As expected, almost no genetic main effects were detected. In contrast, two-gene interactions established the entire sphingolipid metabolic and related genetic network to be highly involved in the regulation of blood pressure. The pattern of interaction clearly revealed that epistasis does not necessarily reflects the topology of the metabolic pathways i.e., the flow of metabolites. Rather, the enzymes and proteins are integrated in complex cellular substructures where communication flows between the components of the networks, which may be composite in structure. The heritabilities for diastolic and systolic blood pressure were estimated to be 0.63 and 0.01, which may in fact be the maximum heritabilities of these traits. This procedure

  16. Regulation of Phospholipid Synthesis in Escherichia coli by Guanosine Tetraphosphate

    PubMed Central

    Merlie, John P.; Pizer, Lewis I.

    1973-01-01

    Phospholipid synthesis has been reported to be subject to stringent control in Escherichia coli. We present evidence that demonstrates a strict correlation between guanosine tetraphosphate accumulation and inhibition of phospholipid synthesis. In vivo experiments designed to examine the pattern of phospholipid labeling with 32P-inorganic phosphate and 32P-sn-glycerol-3-phosphate suggest that regulation must occur at the glycerol-3-phosphate acyltransferase step. Assay of phospholipid synthesis by cell-free extracts and semipurified preparations revealed that guanosine tetraphosphate inhibits at least two enzymes specific for the biosynthetic pathway, sn-glycerol-3-phosphate acyltransferase as well as sn-glycerol-3-phosphate phosphatidyl transferase. These findings provide a biochemical basis for the stringent control of lipid synthesis as well as regulation of steady-state levels of phospholipid in growing cells. Images PMID:4583220

  17. Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

    PubMed Central

    Wang, Kelong; Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.

    2012-01-01

    Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18 to 10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and

  18. Fatty Acid 2-Hydroxylation in Mammalian Sphingolipid Biology

    PubMed Central

    Hama, Hiroko

    2010-01-01

    2-Hydroxy fatty acids (hFA) are important components of a subset of mammalian sphingolipids. The presence of hFA in sphingolipids is best described in the nervous system, epidermis, and kidney. However, the literature also indicates that various hFA-sphingolipids are present in additional tissues and cell types, as well as in tumors. Biosynthesis of hFA-sphingolipids requires fatty acid 2-hydroyxlase, and degradation of hFA-sphingolipids depends, at least in part, on lysosomal acid ceramidase and the peroxisomal fatty acid α-oxidation pathway. Mutations in the fatty acid 2-hydroxylase gene, FA2H, have been associated with leukodystrophy and spastic paraparesis in humans, underscoring the importance of hFA-sphingolipids in the nervous system. In the epidermis, hFA-ceramides are essential for the permeability barrier function. Physiological function of hFA-sphingolipids in other organs remains largely unknown. Recent evidence indicates that hFA-sphingolipids have specific roles in cell signaling. PMID:20026285

  19. Sphingolipids and Membrane Biology as Determined from Genetic Models

    PubMed Central

    Rao, Raghavendra Pralhada; Acharya, Jairaj K

    2008-01-01

    The importance of sphingolipids in membrane biology was appreciated early in the twentieth century when several human inborn errors of metabolism were linked to defects in sphingolipid degradation. The past two decades have seen an explosion of information linking sphingolipids with cellular processes. Studies have unraveled mechanistic details of the sphingolipid metabolic pathways, and these findings are being exploited in the development of novel therapies, some now in clinical trials. Pioneering work in yeast has laid the foundation for identifying genes encoding the enzymes of the pathways. The advent of the era of genomics and bioinformatics has led to the identification of homologous genes in other species and the subsequent creation of animal knock-out lines for these genes. Discoveries from these efforts have re-kindled interest in the role of sphingolipids in membrane biology. This review highlights some of the recent advances in understanding sphingolipids’ roles in membrane biology as determined from genetic models. PMID:18035569

  20. Age- and sex-dependent change in stratum corneum sphingolipids.

    PubMed

    Denda, M; Koyama, J; Hori, J; Horii, I; Takahashi, M; Hara, M; Tagami, H

    1993-01-01

    We measured six stratum corneum sphingolipid species (ceramides 1-6) in 26 males and 27 females, and found a significant change in their percentage composition only among female subjects of different age groups. There was a significant increase in ceramide 1 and 2 with a corresponding decrease in ceramide 3 and 6 from prepubertal age to adulthood. Thereafter the ratio of ceramide 2 to total sphingolipids decreased with age in contrast to ceramide 3 which showed an increase. Such a pattern of change in the aging population is different from that observed in scaly skin experimentally induced by tape stripping. The present results suggest a significant influence of female hormones on the composition of stratum corneum sphingolipids. Moreover, the different patterns of change in sphingolipid composition of stratum corneum lipids between scales from inflammatory skin and those from aged skin also suggest that epidermal biosynthesis of sphingolipids is influenced by epidermal proliferative activity. PMID:8304781

  1. A Sphingolipid Inhibitor Induces a Cytokinesis Arrest and Blocks Stage Differentiation in Giardia lamblia▿

    PubMed Central

    Sonda, Sabrina; Štefanić, Saša; Hehl, Adrian B.

    2008-01-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 μM. The inhibition of parasite replication was irreversible at 10 μM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  2. A sphingolipid inhibitor induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.

    PubMed

    Sonda, Sabrina; Stefanic, Sasa; Hehl, Adrian B

    2008-02-01

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-phenyl-2-palmitoylamino-3-morpholino-1-propanol). In this study, we investigated whether PPMP inhibits the proliferation and differentiation of the pathogenic parasite Giardia lamblia, the major cause of parasite-induced diarrhea worldwide. PPMP was found to block in vitro parasite replication in a dose-dependent manner, with a 50% inhibitory concentration of 3.5 muM. The inhibition of parasite replication was irreversible at 10 muM PPMP, a concentration that did not affect mammalian cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. Microscopic analysis of cells incubated with PPMP revealed the aberrant accumulation of cellular membranes belonging to the endoplasmic reticulum network in the caudal area of the parasites. Finally, PPMP induced a 90% reduction in G. lamblia differentiation into cysts, the parasite stage responsible for the transmission of the disease. These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as-yet-uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents. PMID:18086854

  3. Regulation of starch synthesis in potato tubers

    SciTech Connect

    Davies, H.; Oparka, K.; Viola, R.; Wright, K.; Ross, H. )

    1990-05-01

    Following tuber excision from the mother plant sucrose synthase activity fell from 3,120 to 960 nmol/g.f. wt./h within 7 days and starch synthesis ({sup 14}C sucrose incorporated into isolated discs) from 23 to 7 nmol/g.f. wt./h. While the maximum catalytic activity of sucrose synthase was more than sufficient to account for the observed rate of starch synthesis a maximum of 27% of sucrose incorporated by discs was converted into starch within 3 h. This compared with 80% conversion of {sup 14}C glucose incorporated. Tuber excision also reduced the rate of starch biosynthesis with glucose as a substrate (from 206 to 64 nmol/g.f. wt./h). The activities of UDPG-pyrophosphorylase, PPi-PFK, ATP-PFK, starch synthase and hexokinase (glucose or fructose substrates) were unaffected by tuber removal. ADPG pyrophosphorylase activity was reduced from 8,000 to 4,500 nmol/g.f. wt./h. Preliminary experiments indicate that the decline in sucrose synthease activity is prevented by maintaining sucrose flux into tubers through the cut stolon.

  4. The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma.

    PubMed

    Paulenda, T; Draber, P

    2016-07-01

    A family of widely expressed ORM-like (ORMDL) proteins has been recently linked to asthma in genomewide association studies in humans and extensively explored in in vivo studies in mice. ORMDL proteins are key regulators of serine palmitoyltransferase, an enzyme catalyzing the initial step of sphingolipid biosynthesis. Sphingolipids play prominent roles in cell signaling and response to stress, and they affect the mechanistic properties of cellular membranes. Deregulation of sphingolipid biosynthesis and their recycling has been proven to support and even cause several diseases including allergy, inflammation, and asthma. ORMDL3, the most extensively studied member of the ORMDL family, has been shown to be important for endoplasmic reticulum homeostasis by regulating the unfolded protein response and calcium response. In immune cells, ORMDL3 is involved in migration and in the production of proinflammatory cytokines. Furthermore, changes in the expression level of ORMDL3 are important in allergen-induced asthma pathologies. This review focuses on functional aspects of the ORMDL family proteins, which may serve as new therapeutic targets for the treatment of asthma and some other life-threatening diseases. PMID:26969910

  5. Analysis of Mammalian Sphingolipids by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Tissue Imaging Mass Spectrometry (TIMS)

    PubMed Central

    Sullards, M. Cameron; Liu, Ying; Chen, Yanfeng; Merrill, Alfred H.

    2011-01-01

    Sphingolipids are a highly diverse category of molecules that serve not only as components of biological structures but also as regulators of numerous cell functions. Because so many of the structural features of sphingolipids give rise to their biological activity, there is a need for comprehensive or “sphingolipidomic” methods for identification and quantitation of as many individual subspecies as possible. This review defines sphingolipids as a class, briefly discusses classical methods for their analysis, and focuses primarily on liquid chromatography tandem mass spectrometry (LC-MS/MS) and tissue imaging mass spectrometry (TIMS). Recently, a set of evolving and expanding methods have been developed and rigorously validated for the extraction, identification, separation, and quantitation of sphingolipids by LC-MS/MS. Quantitation of these biomolecules is made possible via the use of an internal standard cocktail. The compounds that can be readily analyzed are free long-chain (sphingoid) bases, sphingoid base 1-phosphates, and more complex species such as ceramides, ceramide 1-phosphates, sphingomyelins, mono- and di-hexosylceramides sulfatides, and novel compounds such as the 1-deoxy- and 1-(deoxymethyl)-sphingoid bases and their N-acyl-derivatives. These methods can be altered slightly to separate and quantitate isomeric species such as glucosyl/galactosylceramide. Because these techniques require the extraction of sphingolipids from their native environment, any information regarding their localization in histological slices is lost. Therefore, this review also describes methods for TIMS. This technique has been shown to be a powerful tool to determine the localization of individual molecular species of sphingolipids directly from tissue slices. PMID:21749933

  6. Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens.

    PubMed

    Quelas, J I; Mesa, S; Mongiardini, E J; Jendrossek, D; Lodeiro, A R

    2016-07-15

    Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4 Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2 IMPORTANCE: In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer

  7. Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2005-01-01

    Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family. PMID:16212488

  8. Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes

    PubMed Central

    Shah, Mithun Vinod; Zhang, Ranran; Irby, Rosalyn; Kothapalli, Ravi; Liu, Xin; Arrington, Ty; Frank, Bryan; Lee, Norman H.

    2008-01-01

    T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+CD8+ cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P5 is the predominant S1P receptor in leukemic LGLs, whereas S1P1 is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia. PMID:18477771

  9. Thematic Review Series: Sphingolipids. Biodiversity of sphingoid bases (“sphingosines”) and related amino alcohols*

    PubMed Central

    Pruett, Sarah T.; Bushnev, Anatoliy; Hagedorn, Kerri; Adiga, Madhura; Haynes, Christopher A.; Sullards, M. Cameron; Liotta, Dennis C.; Merrill, Alfred H.

    2008-01-01

    “Sphingosin” was first described by J. L. W. Thudichum in 1884 and structurally characterized as 2S,3R,4E-2-aminooctadec-4-ene-1,3-diol in 1947 by Herb Carter, who also proposed the designation of “lipides derived from sphingosine as sphingolipides.” This category of amino alcohols is now known to encompass hundreds of compounds that are referred to as sphingoid bases and sphingoid base-like compounds, which vary in chain length, number, position, and stereochemistry of double bonds, hydroxyl groups, and other functionalities. Some have especially intriguing features, such as the tail-to-tail combination of two sphingoid bases in the α,ω-sphingoids produced by sponges. Most of these compounds participate in cell structure and regulation, and some (such as the fumonisins) disrupt normal sphingolipid metabolism and cause plant and animal disease. Many of the naturally occurring and synthetic sphingoid bases are cytotoxic for cancer cells and pathogenic microorganisms or have other potentially useful bioactivities; hence, they offer promise as pharmaceutical leads. This thematic review gives an overview of the biodiversity of the backbones of sphingolipids and the broader field of naturally occurring and synthetic sphingoid base-like compounds. PMID:18499644

  10. Roles of Sphingolipid Metabolism in Pancreatic β Cell Dysfunction Induced by Lipotoxicity

    PubMed Central

    Véret, Julien; Bellini, Lara; Giussani, Paola; Ng, Carl; Magnan, Christophe; Le Stunff, Hervé

    2014-01-01

    Pancreatic β cells secrete insulin in order to maintain glucose homeostasis. However, various environmental stresses such as obesity have been shown to induce loss of secretory responsiveness in pancreatic β cells and pancreatic β cell apoptosis which can favor the development of type 2 diabetes (T2D). Indeed, elevated levels of free fatty acids (FFAs) have been shown to induce β cell apoptosis. Importantly, the chronic adverse effects of FFAs on β cell function and viability are potentiated in the presence of hyperglycaemia, a phenomenon that has been termed gluco-lipotoxicity. The molecular mechanisms underlying the pathogenesis of gluco-lipotoxicity in pancreatic β cells are not completely understood. Recent studies have shown that sphingolipid metabolism plays a key role in gluco-lipotoxicity induced apoptosis and loss of function of pancreatic β cells. The present review focuses on how the two main sphingolipid mediators, ceramides and sphingoid base-1-phosphates, regulate the deleterious effects of gluco-lipotoxicity on pancreatic β cells. The review highlights the role of a sphingolipid biostat on the dysregulation of β cell fate and function induced by gluco-lipotoxicity, offering the possibility of new therapeutic targets to prevent the onset of T2D. PMID:26237395

  11. Roles of Sphingolipid Metabolism in Pancreatic β Cell Dysfunction Induced by Lipotoxicity.

    PubMed

    Véret, Julien; Bellini, Lara; Giussani, Paola; Ng, Carl; Magnan, Christophe; Le Stunff, Hervé

    2014-01-01

    Pancreatic β cells secrete insulin in order to maintain glucose homeostasis. However, various environmental stresses such as obesity have been shown to induce loss of secretory responsiveness in pancreatic β cells and pancreatic β cell apoptosis which can favor the development of type 2 diabetes (T2D). Indeed, elevated levels of free fatty acids (FFAs) have been shown to induce β cell apoptosis. Importantly, the chronic adverse effects of FFAs on β cell function and viability are potentiated in the presence of hyperglycaemia, a phenomenon that has been termed gluco-lipotoxicity. The molecular mechanisms underlying the pathogenesis of gluco-lipotoxicity in pancreatic β cells are not completely understood. Recent studies have shown that sphingolipid metabolism plays a key role in gluco-lipotoxicity induced apoptosis and loss of function of pancreatic β cells. The present review focuses on how the two main sphingolipid mediators, ceramides and sphingoid base-1-phosphates, regulate the deleterious effects of gluco-lipotoxicity on pancreatic β cells. The review highlights the role of a sphingolipid biostat on the dysregulation of β cell fate and function induced by gluco-lipotoxicity, offering the possibility of new therapeutic targets to prevent the onset of T2D. PMID:26237395

  12. Temperature-Regulated Protein Synthesis by Leptospira interrogans

    PubMed Central

    Nally, Jarlath E.; Timoney, John F.; Stevenson, Brian

    2001-01-01

    Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection with L. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection. PMID:11119530

  13. Regulation of protein synthesis during sea urchin early development

    SciTech Connect

    Kelso, L.C.

    1989-01-01

    Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. ({sup 32}P) labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development.

  14. Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.

    PubMed

    Connan, Chloé; Denève, Cécile; Mazuet, Christelle; Popoff, Michel R

    2013-12-01

    Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. PMID:23769754

  15. Overexpression of Arabidopsis Ceramide Synthases Differentially Affects Growth, Sphingolipid Metabolism, Programmed Cell Death, and Mycotoxin Resistance.

    PubMed

    Luttgeharm, Kyle D; Chen, Ming; Mehra, Amit; Cahoon, Rebecca E; Markham, Jonathan E; Cahoon, Edgar B

    2015-10-01

    Ceramide synthases catalyze an N-acyltransferase reaction using fatty acyl-coenzyme A (CoA) and long-chain base (LCB) substrates to form the sphingolipid ceramide backbone and are targets for inhibition by the mycotoxin fumonisin B1 (FB1). Arabidopsis (Arabidopsis thaliana) contains three genes encoding ceramide synthases with distinct substrate specificities: LONGEVITY ASSURANCE GENE ONE HOMOLOG1 (LOH1; At3g25540)- and LOH3 (At1g19260)-encoded ceramide synthases use very-long-chain fatty acyl-CoA and trihydroxy LCB substrates, and LOH2 (At3g19260)-encoded ceramide synthase uses palmitoyl-CoA and dihydroxy LCB substrates. In this study, complementary DNAs for each gene were overexpressed to determine the role of individual isoforms in physiology and sphingolipid metabolism. Differences were observed in growth resulting from LOH1 and LOH3 overexpression compared with LOH2 overexpression. LOH1- and LOH3-overexpressing plants had enhanced biomass relative to wild-type plants, due in part to increased cell division, suggesting that enhanced synthesis of very-long-chain fatty acid/trihydroxy LCB ceramides promotes cell division and growth. Conversely, LOH2 overexpression resulted in dwarfing. LOH2 overexpression also resulted in the accumulation of sphingolipids with C16 fatty acid/dihydroxy LCB ceramides, constitutive induction of programmed cell death, and accumulation of salicylic acid, closely mimicking phenotypes observed previously in LCB C-4 hydroxylase mutants defective in trihydroxy LCB synthesis. In addition, LOH2- and LOH3-overexpressing plants acquired increased resistance to FB1, whereas LOH1-overexpressing plants showed no increase in FB1 resistance, compared with wild-type plants, indicating that LOH1 ceramide synthase is most strongly inhibited by FB1. Overall, the findings described here demonstrate that overexpression of Arabidopsis ceramide synthases results in strongly divergent physiological and metabolic phenotypes, some of which have significance

  16. Regulation of polyamine synthesis in plants. Annual progress report

    SciTech Connect

    Malmberg, R.L.

    1993-02-09

    Polyamines are small positively charged compounds that have been hypothesized to be involved in a wide variety of plant physiological and development functions. The regulation of the polyamine synthesis pathway is uniquely interesting because of the existence of two pathways to putrescine synthesis, and the consequent questions of how these two pathways are compartmentalized and how they interact with each other. The specific directions our research is taking are: (1) A characterization of arginine decarboxylase regulation; we have discovered two post-translational mechanisms for regulating arginine decarboxylase activity. One of these is a novel protease that clips the arginine decarboxylase pre-protein to activate it. We would like to understand this activating protease better, determine its mechanism of action, and determine its importance in the overall scheme of arginine decarboxylase regulation. (2) We have begun a similar characterization of ornithine decarboxylase by purifying it from plants. (3) We are characterizing the polyamine mutant collection we have developed. (4) Finally, we have begun to characterize the evolution of arginine decarboxylase, as an additional approach that could shed light on its functions in plants. Our intent is to understand arginine decarboxylase structure and regulation in detail, and then to further explore regulatory differences between ornithine and arginine decarboxylases.

  17. Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis

    PubMed Central

    Arendt, Kristin L.; Zhang, Zhenjie; Ganesan, Subhashree; Hintze, Maik; Shin, Maggie M.; Tang, Yitai; Cho, Ahryon; Graef, Isabella A.; Chen, Lu

    2015-01-01

    Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca2+ levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca2+-levels to RA synthesis remains unknown. Here we identify the Ca2+-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca2+-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity. PMID:26443861

  18. Calcineurin mediates homeostatic synaptic plasticity by regulating retinoic acid synthesis.

    PubMed

    Arendt, Kristin L; Zhang, Zhenjie; Ganesan, Subhashree; Hintze, Maik; Shin, Maggie M; Tang, Yitai; Cho, Ahryon; Graef, Isabella A; Chen, Lu

    2015-10-20

    Homeostatic synaptic plasticity is a form of non-Hebbian plasticity that maintains stability of the network and fidelity for information processing in response to prolonged perturbation of network and synaptic activity. Prolonged blockade of synaptic activity decreases resting Ca(2+) levels in neurons, thereby inducing retinoic acid (RA) synthesis and RA-dependent homeostatic synaptic plasticity; however, the signal transduction pathway that links reduced Ca(2+)-levels to RA synthesis remains unknown. Here we identify the Ca(2+)-dependent protein phosphatase calcineurin (CaN) as a key regulator for RA synthesis and homeostatic synaptic plasticity. Prolonged inhibition of CaN activity promotes RA synthesis in neurons, and leads to increased excitatory and decreased inhibitory synaptic transmission. These effects of CaN inhibitors on synaptic transmission are blocked by pharmacological inhibitors of RA synthesis or acute genetic deletion of the RA receptor RARα. Thus, CaN, acting upstream of RA, plays a critical role in gating RA signaling pathway in response to synaptic activity. Moreover, activity blockade-induced homeostatic synaptic plasticity is absent in CaN knockout neurons, demonstrating the essential role of CaN in RA-dependent homeostatic synaptic plasticity. Interestingly, in GluA1 S831A and S845A knockin mice, CaN inhibitor- and RA-induced regulation of synaptic transmission is intact, suggesting that phosphorylation of GluA1 C-terminal serine residues S831 and S845 is not required for CaN inhibitor- or RA-induced homeostatic synaptic plasticity. Thus, our study uncovers an unforeseen role of CaN in postsynaptic signaling, and defines CaN as the Ca(2+)-sensing signaling molecule that mediates RA-dependent homeostatic synaptic plasticity. PMID:26443861

  19. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions

    PubMed Central

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G.; Módos, Károly; Kardos, József; Liliom, Károly

    2010-01-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin–sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well.—Kovacs, E., Harmat, V., Tóth, J., Vértessy, B. G., Módos, K., Kardos, J., Liliom, K. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions. PMID:20522785

  20. Toxin Synthesis by Clostridium difficile Is Regulated through Quorum Signaling

    PubMed Central

    DuPont, Herbert L.; Norris, Steven J.; Kaplan, Heidi B.

    2015-01-01

    ABSTRACT Clostridium difficile infection (CDI) is dramatically increasing as a cause of antibiotic- and hospital-associated diarrhea worldwide. C. difficile, a multidrug-resistant pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Consequently, it produces toxins A and B that directly cause disease. Despite the enormous public health problem posed by this pathogen, the molecular mechanisms that regulate production of the toxins, which are directly responsible for disease, remained largely unknown until now. Here, we show that C. difficile toxin synthesis is regulated by an accessory gene regulator quorum-signaling system, which is mediated through a small (<1,000-Da) thiolactone that can be detected directly in stools of CDI patients. These findings provide direct evidence of the mechanism of regulation of C. difficile toxin synthesis and offer exciting new avenues both for rapid detection of C. difficile infection and development of quorum-signaling-based non-antibiotic therapies to combat this life-threatening emerging pathogen. PMID:25714717

  1. Fumonisin- and AAL-Toxin-Induced Disruption of Sphingolipid Metabolism with Accumulation of Free Sphingoid Bases.

    PubMed Central

    Abbas, H. K.; Tanaka, T.; Duke, S. O.; Porter, J. K.; Wray, E. M.; Hodges, L.; Sessions, A. E.; Wang, E.; Merrill, A. H.; Riley, R. T.

    1994-01-01

    Fumonisins (FB) and AAL-toxin are sphingoid-like compounds produced by several species of fungi associated with plant diseases. In animal cells, both fumonisins produced by Fusarium moniliforme and AAL-toxin produced by Alternaria alternata f. sp. lycopersici inhibit ceramide synthesis, an early biochemical event in the animal diseases associated with consumption of F. moniliforme-contaminated corn. In duckweed (Lemna pausicostata Heglem. 6746), tomato plants (Lycopersicon esculentum Mill), and tobacco callus (Nicotiana tabacum cv Wisconsin), pure FB1 or AAL-toxin caused a marked elevation of phytosphingosine and sphinganine, sphingoid bases normally present in low concentrations. The relative increases were quite different in the three plant systems. Nonetheless, disruption of sphingolipid metabolism was clearly a common feature in plants exposed to FB1 or AAL-toxin. Resistant varieties of tomato (Asc/Asc) were much less sensitive to toxin-induced increases in free sphinganine. Because free sphingoid bases are precursors to plant "ceramides," their accumulation suggests that the primary biochemical lesion is inhibition of de novo ceramide synthesis and reacylation of free sphingoid bases. Thus, in plants the disease symptoms associated with A. alternata and F. moniliforme infection may be due to disruption of sphingolipid metabolism. PMID:12232389

  2. The conserved global regulator VeA is necessary for symptom production and mycotoxin synthesis in maize seedlings by Fusarium verticillioides

    PubMed Central

    Myung, K.; Zitomer, N. C.; Duvall, M.; Glenn, A. E.; Riley, R. T.; Calvo, A. M.

    2011-01-01

    The veA or velvet gene is necessary for biosynthesis of mycotoxins and other secondary metabolites in Aspergillus species. In addition, veA has also been demonstrated to be necessary for normal seed colonization in Aspergillus flavus and Aspergillus parasiticus. The present study shows that veA homologues are broadly distributed in fungi, particularly in Ascomycetes. The Fusarium verticillioides veA orthologue, FvVE1, is also required for the synthesis of several secondary metabolites, including fumonisin and fusarins. This study also shows that maize plants grown from seeds inoculated with FvVE1 deletion mutants did not show disease symptoms, while plants grown from seeds inoculated with the F. verticillioides wildtype and complementation strains clearly showed disease symptoms under the same experimental conditions. In this latter case, the presence of lesions coincided with accumulation of fumonisins in the plant tissues, and only these plant tissues had elevated levels of sphingoid bases and their 1-phosphate derivatives, indicating inhibition of ceramide synthase and disruption of sphingolipid metabolism. The results strongly suggest that FvVE1 is necessary for pathogenicity by F. verticillioides against maize seedlings. The conservation of veA homologues among ascomycetes suggests that veA could play a pivotal role in regulating secondary metabolism and associated pathogenicity in other fungi. PMID:22247572

  3. STEROIDOGENIC FACTOR-1 IS A SPHINGOLIPID BINDING PROTEIN

    PubMed Central

    Urs, Aarti N.; Dammer, Eric; Kelly, Samuel; Wang, Elaine; Merrill, Alfred H.; Sewer, Marion B.

    2007-01-01

    Steroidogenic factor (SF1, NR5A1, Ad4BP) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Studies have found that the ability of this receptor to increase target gene expression can be regulated by post-translational modification, subnuclear localization, and protein-protein interactions. Recent crystallographic studies and our mass spectrometric analyses of the endogenous receptor have demonstrated an integral role for ligand-binding in the control of SF1 transactivation activity. Herein, we discuss our findings that sphingosine is an endogenous ligand for SF1. These studies and the structural findings of others have demonstrated that the receptor can bind both sphingolipids and phospholipids. Thus, it is likely that multiple bioactive lipids are ligands for SF1 and that these lipids will differentially act to control SF1 activity in a context-dependent manner. Finally, these findings highlight a central role for bioactive lipids as mediators of trophic-hormone stimulated steroid hormone biosynthesis. PMID:17196738

  4. Regulation of lung surfactant phospholipid synthesis and metabolism.

    PubMed

    Goss, Victoria; Hunt, Alan N; Postle, Anthony D

    2013-02-01

    The alveolar type II epithelial (ATII) cell is highly specialised for the synthesis and storage, in intracellular lamellar bodies, of phospholipid destined for secretion as pulmonary surfactant into the alveolus. Regulation of the enzymology of surfactant phospholipid synthesis and metabolism has been extensively characterised at both molecular and functional levels, but understanding of surfactant phospholipid metabolism in vivo in either healthy or, especially, diseased lungs is still relatively poorly understood. This review will integrate recent advances in the enzymology of surfactant phospholipid metabolism with metabolic studies in vivo in both experimental animals and human subjects. It will highlight developments in the application of stable isotope-labelled precursor substrates and mass spectrometry to probe lung phospholipid metabolism in terms of individual molecular lipid species and identify areas where a more comprehensive metabolic model would have considerable potential for direct application to disease states. PMID:23200861

  5. Transcriptional regulation of decreased protein synthesis during skeletal muscle unloading

    NASA Technical Reports Server (NTRS)

    Howard, G.; Steffen, J. M.; Geoghegan, T. E.

    1989-01-01

    The regulatory role of transcriptional alterations in unloaded skeletal muscles was investigated by determining levels of total muscle RNA and mRNA fractions in soleus, gastrocnemius, and extensor digitorum longus (EDL) of rats subjected to whole-body suspension for up to 7 days. After 7 days, total RNA and mRNA contents were lower in soleus and gastrocnemius, compared with controls, but the concentrations of both RNAs per g muscle were unaltered. Alpha-actin mRNA (assessed by dot hybridization) was significantly reduced in soleus after 1, 3, and 7 days of suspension and in gastrocnemius after 3 and 7 days, but was unchanged in EDL. Protein synthesis directed by RNA extracted from soleus and EDL indicated marked alteration in mRNAs coding for several small proteins. Results suggest that altered transcription and availability of specific mRNAs contribute significantly to the regulation of protein synthesis during skeletal muscle unloading.

  6. An oxygen-regulated switch in the protein synthesis machinery

    PubMed Central

    Uniacke, James; Holterman, Chet E.; Lachance, Gabriel; Franovic, Aleksandra; Jacob, Mathieu D.; Fabian, Marc R.; Payette, Josianne; Holcik, Martin; Pause, Arnim; Lee, Stephen

    2016-01-01

    SUMMARY Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis consists of the eukaryotic translation initiation factor 4E (eIF4E) binding to the 7-methylguanosine (m7-GpppG) 5′cap of mRNAs1,2. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms3–6. While the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells7,8. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition9. Here, we uncover an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. Hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. PAR-CLIP10 analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array mRNAs, including the epidermal growth factor receptor (EGFR). Once assembled at the rHRE, HIF-2α/RBM4/eIF4E2 captures the 5′cap and targets mRNAs to polysomes for active translation thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program whereby oxygen tension switches the basic translation initiation machinery. PMID:22678294

  7. Podocyte pathology and nephropathy - sphingolipids in glomerular diseases.

    PubMed

    Merscher, Sandra; Fornoni, Alessia

    2014-01-01

    Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay-Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann-Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in post-reperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed. PMID:25126087

  8. Podocyte Pathology and Nephropathy – Sphingolipids in Glomerular Diseases

    PubMed Central

    Merscher, Sandra; Fornoni, Alessia

    2014-01-01

    Sphingolipids are components of the lipid rafts in plasma membranes, which are important for proper function of podocytes, a key element of the glomerular filtration barrier. Research revealed an essential role of sphingolipids and sphingolipid metabolites in glomerular disorders of genetic and non-genetic origin. The discovery that glucocerebrosides accumulate in Gaucher disease in glomerular cells and are associated with clinical proteinuria initiated intensive research into the function of other sphingolipids in glomerular disorders. The accumulation of sphingolipids in other genetic diseases including Tay–Sachs, Sandhoff, Fabry, hereditary inclusion body myopathy 2, Niemann–Pick, and nephrotic syndrome of the Finnish type and its implications with respect to glomerular pathology will be discussed. Similarly, sphingolipid accumulation occurs in glomerular diseases of non-genetic origin including diabetic kidney disease (DKD), HIV-associated nephropathy, focal segmental glomerulosclerosis (FSGS), and lupus nephritis. Sphingomyelin metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate have also gained tremendous interest. We recently described that sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b) is expressed in podocytes where it modulates acid sphingomyelinase activity and acts as a master modulator of danger signaling. Decreased SMPDL3b expression in post-reperfusion kidney biopsies from transplant recipients with idiopathic FSGS correlates with the recurrence of proteinuria in patients and in experimental models of xenotransplantation. Increased SMPDL3b expression is associated with DKD. The consequences of differential SMPDL3b expression in podocytes in these diseases with respect to their pathogenesis will be discussed. Finally, the role of sphingolipids in the formation of lipid rafts in podocytes and their contribution to the maintenance of a functional slit diaphragm in the glomerulus will be discussed. PMID:25126087

  9. Sphingolipid metabolism enzymes as targets for anticancer therapy.

    PubMed

    Kok, J W; Sietsma, H

    2004-05-01

    Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells. Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ceramide. the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis. Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism. Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide. Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells. In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy. In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance. PMID:15134220

  10. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans.

    PubMed

    Tafesse, Fikadu G; Rashidfarrokhi, Ali; Schmidt, Florian I; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L

    2015-10-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  11. Plant sphingolipids: Their importance in cellular organization and adaption.

    PubMed

    Michaelson, Louise V; Napier, Johnathan A; Molino, Diana; Faure, Jean-Denis

    2016-09-01

    Sphingolipids and their phosphorylated derivatives are ubiquitous bio-active components of cells. They are structural elements in the lipid bilayer and contribute to the dynamic nature of the membrane. They have been implicated in many cellular processes in yeast and animal cells, including aspects of signaling, apoptosis, and senescence. Although sphingolipids have a better defined role in animal systems, they have been shown to be central to many essential processes in plants including but not limited to, pollen development, signal transduction and in the response to biotic and abiotic stress. A fuller understanding of the roles of sphingolipids within plants has been facilitated by classical biochemical studies and the identification of mutants of model species. Recently the development of powerful mass spectrometry techniques hailed the advent of the emerging field of lipidomics enabling more accurate sphingolipid detection and quantitation. This review will consider plant sphingolipid biosynthesis and function in the context of these new developments. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:27086144

  12. Sphingolipids in Obesity, Type 2 Diabetes, and Metabolic Disease

    PubMed Central

    Russo, S.B.; Ross, J.S.; Cowart, L.A.

    2014-01-01

    Metabolic disease, including obesity and type 2 diabetes, constitutes a major emerging health crisis in Western nations. Although the symptoms and clinical pathology and physiology of these conditions are well understood, the molecular mechanisms underlying the disease process have largely remained obscure. Sphingolipids, a lipid class with both signaling and structural properties, have recently emerged as key players in most major tissues affected by diabetes and are required components in the molecular etiology of this disease. Indeed, sphingolipids have been shown to mediate loss of insulin sensitivity, to promote the characteristic diabetic pro-inflammatory state, and to induce cell death and dysfunction in important organs such as the pancreas and heart. Furthermore, plasma sphingolipid levels are emerging as potential biomarkers for the decompensation of insulin resistance to frank type 2 diabetes. Despite these discoveries, the roles of specific sphingolipid species and sphingolipid metabolic pathways remain obscure, and newly developed experimental approaches must be employed to elucidate the detailed molecular mechanisms necessary for rational drug development and other clinical applications. PMID:23563667

  13. Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

    PubMed Central

    Schmidt, Florian I.; Freinkman, Elizaveta; Dougan, Stephanie; Dougan, Michael; Esteban, Alexandre; Maruyama, Takeshi; Strijbis, Karin; Ploegh, Hidde L.

    2015-01-01

    The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans. PMID:26431038

  14. [The first steps of chlorophyll synthesis: RNA involvement and regulation

    SciTech Connect

    Soell, D.

    1992-01-01

    Glu-tRNA[sup Glu] is synthesized from glutamate and tRNA[sup Glu] by glutamyl-tRNA synthetase (GluRS). Recent work has demonstrated that Glu-tRNA[sup Glu] has dual functions and is a precursor for protein and 5-aminolevulinate (ALA) synthesis. Current data does not provide compelling evidence for the notion that GluRS is regulated by chlorophyll precursors or in concert with the other enzymes of ALA synthesis. We have redefined the C5-pathway as a two-step route to ALA starting with Glu-tRNA[sup Glu]. Only two enzymes, Glu-tRNA reductase (GluTR) and GSA-2,1-amino-mutase (GSA-AM), are specifically involved in ALA synthesis. We have purified these enzymatic activities from Chlamydomonas and demonstrated that the two purified proteins in the presence of their cofactors NADPH and pyridoxal phosphate are sufficient for the in vitro Glu-tRNA [yields] ALA conversion. We have cloned the genes encoding GluTR. The sequences of the GluTR proteins deduced from these genes share highly conserved regions with those of bacterial origin. We havealso cloned and analyzed the gene encoding GSA-AM from Arabidopsis. As in Salmonella typhimurium, there are indications of the existence of an additional pathway for ALA formation in E. coli. To shed light on the recognition of the single tRNA[sup Glu] by the chloroplast enzymes GluTR, GluRS we characterized a chlorophyll-deficient mutant of Euglena having tRNA[sup Glu] with a point mutation in the T[Psi]C-loop. The altered tRNA supports protein but not ALA synthesis.

  15. Rapid intra-adrenal feedback regulation of glucocorticoid synthesis.

    PubMed

    Walker, J J; Spiga, F; Gupta, R; Zhao, Z; Lightman, S L; Terry, J R

    2015-01-01

    The hypothalamic-pituitary-adrenal axis is a vital neuroendocrine system that regulates the secretion of glucocorticoid hormones from the adrenal glands. This system is characterized by a dynamic ultradian hormonal oscillation, and in addition is highly responsive to stressful stimuli. We have recently shown that a primary mechanism generating this ultradian rhythm is a systems-level interaction where adrenocorticotrophin hormone (ACTH) released from the pituitary stimulates the secretion of adrenal glucocorticoids, which in turn feedback at the level of the pituitary to rapidly inhibit ACTH secretion. In this study, we combine experimental physiology and mathematical modelling to investigate intra-adrenal mechanisms regulating glucocorticoid synthesis. Our modelling results suggest that glucocorticoids can inhibit their own synthesis through a very rapid (within minutes), presumably non-genomic, intra-adrenal pathway. We present further evidence for the existence of a short time delay in this intra-adrenal inhibition, and also that at the initiation of each ACTH stimulus, this local feedback mechanism is rapidly antagonized, presumably via activation of the specific ACTH receptor (MC2R) signalling pathway. This mechanism of intra-adrenal inhibition enables the gland to rapidly release glucocorticoids while at the same time preventing uncontrolled release of glucocorticoids in response to large surges in ACTH associated with stress. PMID:25392395

  16. Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug.

    PubMed

    Guillermet-Guibert, Julie; Davenne, Lise; Pchejetski, Dimitri; Saint-Laurent, Nathalie; Brizuela, Leyre; Guilbeau-Frugier, Céline; Delisle, Marie-Bernadette; Cuvillier, Olivier; Susini, Christiane; Bousquet, Corinne

    2009-04-01

    Defeating pancreatic cancer resistance to the chemotherapeutic drug gemcitabine remains a challenge to treat this deadly cancer. Targeting the sphingolipid metabolism for improving tumor chemosensitivity has recently emerged as a promising strategy. The fine balance between intracellular levels of the prosurvival sphingosine-1-phosphate (S1P) and the proapoptotic ceramide sphingolipids determines cell fate. Among enzymes that control this metabolism, sphingosine kinase-1 (SphK1), a tumor-associated protein overexpressed in many cancers, favors survival through S1P production, and inhibitors of SphK1 are used in ongoing clinical trials to sensitize epithelial ovarian and prostate cancer cells to various chemotherapeutic drugs. We here report that the cellular ceramide/S1P ratio is a critical biosensor for predicting pancreatic cancer cell sensitivity to gemcitabine. A low level of the ceramide/S1P ratio, associated with a high SphK1 activity, correlates with a robust intrinsic pancreatic cancer cell chemoresistance toward gemcitabine. Strikingly, increasing the ceramide/S1P ratio, by using pharmacologic (SphK1 inhibitor or ceramide analogue) or small interfering RNA-based approaches to up-regulate intracellular ceramide levels or reduce SphK1 activity, sensitized pancreatic cancer cells to gemcitabine. Conversely, decreasing the ceramide/S1P ratio, by up-regulating SphK1 activity, promoted gemcitabine resistance in these cells. Development of novel pharmacologic strategies targeting the sphingolipid metabolism might therefore represent an interesting promising approach, when combined with gemcitabine, to defeat pancreatic cancer chemoresistance to this drug. PMID:19372554

  17. Cyclin B synthesis and rapamycin-sensitive regulation of protein synthesis during starfish oocyte meiotic divisions.

    PubMed

    Lapasset, Laure; Pradet-Balade, Bérengère; Vergé, Valérie; Lozano, Jean-Claude; Oulhen, Nathalie; Cormier, Patrick; Peaucellier, Gérard

    2008-11-01

    Translation of cyclin mRNAs represents an important event for proper meiotic maturation and post-fertilization mitoses in many species. Translational control of cyclin B mRNA has been described to be achieved through two separate but related mechanisms: translational repression and polyadenylation. In this paper, we evaluated the contribution of global translational regulation by the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-binding protein) on the cyclin B protein synthesis during meiotic maturation of the starfish oocytes. We used the immunosupressant drug rapamycin, a strong inhibitor of cap-dependent translation, to check for the involvement of this protein synthesis during this physiological process. Rapamycin was found to prevent dissociation of 4E-BP from the initiation factor eIF4E and to suppress correlatively a burst of global protein synthesis occurring at the G2/M transition. The drug had no effect on first meiotic division but defects in meiotic spindle formation prevented second polar body emission, demonstrating that a rapamycin-sensitive pathway is involved in this mechanism. While rapamycin affected the global protein synthesis, the drug altered neither the specific translation of cyclin B mRNA nor the expression of the Mos protein. The expression of these two proteins was correlated with the phosphorylation and the dissociation of the cytoplasmic polyadenylation element-binding protein from eIF4E. PMID:18361417

  18. Genetic Determinants of Circulating Sphingolipid Concentrations in European Populations

    PubMed Central

    Pramstaller, Peter P.; Rudan, Igor; Franklin, Christopher S.; Liebisch, Gerhard; Erdmann, Jeanette; Jonasson, Inger; Zorkoltseva, Irina V.; Pattaro, Cristian; Hayward, Caroline; Isaacs, Aaron; Hengstenberg, Christian; Campbell, Susan; Gnewuch, Carsten; Janssens, A. CecileJ.W.; Kirichenko, Anatoly V.; König, Inke R.; Marroni, Fabio; Polasek, Ozren; Demirkan, Ayse; Kolcic, Ivana; Schwienbacher, Christine; Igl, Wilmar; Biloglav, Zrinka; Witteman, Jacqueline C. M.; Pichler, Irene; Zaboli, Ghazal; Axenovich, Tatiana I.; Peters, Annette; Schreiber, Stefan; Wichmann, H.-Erich; Schunkert, Heribert; Hastie, Nick; Oostra, Ben A.; Wild, Sarah H.; Meitinger, Thomas; Gyllensten, Ulf; van Duijn, Cornelia M.; Wilson, James F.; Wright, Alan; Schmitz, Gerd; Campbell, Harry

    2009-01-01

    Sphingolipids have essential roles as structural components of cell membranes and in cell signalling, and disruption of their metabolism causes several diseases, with diverse neurological, psychiatric, and metabolic consequences. Increasingly, variants within a few of the genes that encode enzymes involved in sphingolipid metabolism are being associated with complex disease phenotypes. Direct experimental evidence supports a role of specific sphingolipid species in several common complex chronic disease processes including atherosclerotic plaque formation, myocardial infarction (MI), cardiomyopathy, pancreatic β-cell failure, insulin resistance, and type 2 diabetes mellitus. Therefore, sphingolipids represent novel and important intermediate phenotypes for genetic analysis, yet little is known about the major genetic variants that influence their circulating levels in the general population. We performed a genome-wide association study (GWAS) between 318,237 single-nucleotide polymorphisms (SNPs) and levels of circulating sphingomyelin (SM), dihydrosphingomyelin (Dih-SM), ceramide (Cer), and glucosylceramide (GluCer) single lipid species (33 traits); and 43 matched metabolite ratios measured in 4,400 subjects from five diverse European populations. Associated variants (32) in five genomic regions were identified with genome-wide significant corrected p-values ranging down to 9.08×10−66. The strongest associations were observed in or near 7 genes functionally involved in ceramide biosynthesis and trafficking: SPTLC3, LASS4, SGPP1, ATP10D, and FADS1–3. Variants in 3 loci (ATP10D, FADS3, and SPTLC3) associate with MI in a series of three German MI studies. An additional 70 variants across 23 candidate genes involved in sphingolipid-metabolizing pathways also demonstrate association (p = 10−4 or less). Circulating concentrations of several key components in sphingolipid metabolism are thus under strong genetic control, and variants in these loci can be

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

    PubMed

    Hajj, Carla; Becker-Flegler, Katrin Anne; Haimovitz-Friedman, Adriana

    2015-06-01

    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. PMID:25719313

  20. Viral serine palmitoyltransferase induces metabolic switch in sphingolipid biosynthesis and is required for infection of a marine alga.

    PubMed

    Ziv, Carmit; Malitsky, Sergey; Othman, Alaa; Ben-Dor, Shifra; Wei, Yu; Zheng, Shuning; Aharoni, Asaph; Hornemann, Thorsten; Vardi, Assaf

    2016-03-29

    Marine viruses are the most abundant biological entities in the oceans shaping community structure and nutrient cycling. The interaction between the bloom-forming alga Emiliania huxleyi and its specific large dsDNA virus (EhV) is a major factor determining the fate of carbon in the ocean, thus serving as a key host-pathogen model system. The EhV genome encodes for a set of genes involved in the de novo sphingolipid biosynthesis, not reported in any viral genome to date. We combined detailed lipidomic and biochemical analyses to characterize the functional role of this virus-encoded pathway during lytic viral infection. We identified a major metabolic shift, mediated by differential substrate specificity of virus-encoded serine palmitoyltransferase, a key enzyme of sphingolipid biosynthesis. Consequently, unique viral glycosphingolipids, composed of unusual hydroxylated C17 sphingoid bases (t17:0) were highly enriched in the infected cells, and their synthesis was found to be essential for viral assembly. These findings uncover the biochemical bases of the virus-induced metabolic rewiring of the host sphingolipid biosynthesis during the chemical "arms race" in the ocean. PMID:26984500

  1. Biochemistry and molecular biology of regulation of starch synthesis

    SciTech Connect

    Bloom, M.; Morell, M.; Preiss, J.

    1987-05-01

    Regulation of plant starch synthesis occurs at the level of ADPglc synthetase. 3-P-glycerate (3PGA) activates and P/sub i/ inhibits ADPG synthesis. 3PGA at high concentrations reverses P/sub i/ inhibition. Pyridoxal-P (PLP) activates the spinach leaf enzyme about 5- to 6-fold, but is not as effective as 3PGA which stimulates ADPglc synthesis 20-fold. PLP competes with 3PGA as an activator and reverses P/sub i/ inhibition thus suggesting that PLP binds at or close to the activator site. Reductive phosphopyridoxylation of the spinach leaf ADPglc synthetase with NaBH4 yields an enzyme with 5- to 6-fold higher activity in the absence of activator than the untreated enzyme when about 0.5 mol of (TH)-PLP is bound per mole of native enzyme. This modified enzyme is highly resistant to P/sub i/ inhibition in contrast to the treated enzyme. (TH)-PLP is incorporated into both 54 KD and 51 KD subunits of the enzyme. Incorporation into both peptides is inhibited by both 3PGA and P/sub i/. After incorporation of (TH)-PLP, the 51 KD subunit has been separated from the 54 KD subunit and subjected to tryptic digestion. A major radioactive peptide has been purified by HPLC and sequenced. The determined sequence was Ser-Gly-Ile-Val-Thr-Val-Ile-Lys-Asp-Ala-Leu-Ile-Pro-(Ser)- and is very similar to the deduced amino acid sequence obtained from a cDNA clone of the rice endosperm ADPglc synthetase gene. The amino acid sequence, the putative activator binding region, is situated near the C-terminus.

  2. Hormonal regulation of mannan-binding lectin synthesis in hepatocytes

    PubMed Central

    Sørensen, C M; Hansen, T K; Steffensen, R; Jensenius, J C; Thiel, S

    2006-01-01

    Activation of the complement system via the plasma protein mannan-binding lectin (MBL) provides a first line of defence against infections. The plasma level of MBL is, in part, determined genetically, but may also be influenced by different hormones in vivo. Here we study the hormonal regulation of MBL synthesis from the human hepatocyte cell line HuH-7. Cells were exposed to medium with growth hormone (GH), hydrocortisone, insulin-like growth factor (IGF)-1, insulin, interleukin (IL)-6 or thyroid hormones (T3 or T4). After 3 days the concentration of MBL in the culture supernatants was determined and the amount of mRNA for MBL was measured, relative to mRNA for β2 microglobulin. GH, IL-6, T3 and T4 significantly increased MBL synthesis in a dose-dependent manner, while hydrocortisone, insulin and IGF-1 had no effect. T3 caused a fourfold increase at 1 nM of T3 (P < 0·001) and at 100 nM of T3 the production was increased more than eightfold. The effect of T4 was less potent, reaching an eightfold increase at 1 µM of T4 (P < 0·001). GH augmented the production of MBL threefold at a concentration of 100 ng/ml (P = 0·018) with no further effect up to 10 µg/ml, whereas IL-6 caused only a very weak increase in MBL production. MBL mRNA levels were stable during the first 24 h of T3 stimulation but increased significantly between 24 and 48 h. The results suggest that MBL synthesis in humans may be increased by thyroid hormone and GH, whereas it does not exhibit a classical IL-6-dependent response. PMID:16792688

  3. Genetic regulation of sporopollenin synthesis and pollen exine development.

    PubMed

    Ariizumi, Tohru; Toriyama, Kinya

    2011-01-01

    Pollen acts as a biological protector of male sperm and is covered by an outer cell wall polymer called the exine, which consists of durable sporopollenin. Despite the astonishingly divergent structure of the exine across taxa, the developmental processes of its formation surprisingly do not vary, which suggests the preservation of a common molecular mechanism. The precise molecular mechanisms underlying pollen exine patterning remain highly elusive, but they appear to be dependent on at least three major developmental processes: primexine formation, callose wall formation, and sporopollenin synthesis. Several lines of evidence suggest that the sporopollenin is built up via catalytic enzyme reactions in the tapetum, and both the primexine and callose wall provide an efficient substructure for sporopollenin deposition. Herein, we review the currently accepted understanding of the molecular regulation of sporopollenin biosynthesis and examine unanswered questions regarding the requirements underpinning proper exine pattern formation, as based on genetic evidence. PMID:21275644

  4. Regulation of isopropylmalate isomerase synthesis in Neurospora crassa.

    PubMed Central

    Reichenbecher, V E; Fischer, M; Gross, S R

    1978-01-01

    The capacity to synthetize isopropylmalate isomerase (EC 4.2.1.33) by Neurospora crassa increased during induction in the presence of cycloheximide but was inhibited by proflavine and other inhibitors of RNA synthesis. Turnover of the enzyme once formed appeared negligible, but the message (measured as enzyme-forming capacity) had a half-life of 4 to 8 min. A comparison of the kinetics of induction in the wild type and a newly isolated alpha-isopropylmalate-permeable strain suggested strongly that feedback control by leucine of alpha-isopropylmalate production can adequately serve as the primary physiological regulator of endogenous inducer concentration. Genetic data are presented which implicate the involvement of two unlinked genes, ipm-1 and ipm-2, in determining permeation of alpha-isopropylmalate. PMID:146702

  5. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    DOE PAGESBeta

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

  6. Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol*

    PubMed Central

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-01-01

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton. PMID:23609440

  7. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    SciTech Connect

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

  8. Biotin and Lipoic Acid: Synthesis, Attachment and Regulation

    PubMed Central

    Cronan, John E.

    2014-01-01

    Summary Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well-described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses

  9. The regulation of phosphoenolpyruvate synthesis in pigeon liver

    PubMed Central

    Gevers, W.

    1967-01-01

    -oxoglutarate, and the synthesis of phosphoenolpyruvate. 5. The results suggest that phosphoenolpyruvate formation in pigeon-liver mitochondria is regulated partly by the phosphorylation state of the adenine and guanine nucleotides, and partly by variations in the oxaloacetate concentration, all in the mitochondrial matrix. 6. Phosphoenolpyruvate is assumed to be the metabolite transported from the mitochondria to the cytoplasm during gluconeogenesis from oxaloacetate in pigeon liver. PMID:4962163

  10. Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation.

    PubMed

    Cronan, John E

    2014-05-01

    Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein

  11. Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation.

    PubMed

    Cronan, John E

    2008-09-01

    Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid was discovered 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway, in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin, were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase, followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and thus there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system exerted through BirA, a dual-function protein that both represses

  12. Sphingolipids are potential heat stress signals in Saccharomyces.

    PubMed

    Dickson, R C; Nagiec, E E; Skrzypek, M; Tillman, P; Wells, G B; Lester, R L

    1997-11-28

    The ability of organisms to quickly respond to stresses requires the activation of many intracellular signal transduction pathways. The sphingolipid intermediate ceramide is thought to be particularly important for activating and coordinating signaling pathways during mammalian stress responses. Here we present the first evidence that ceramide and other sphingolipid intermediates are signaling molecules in the Saccharomyces cerevisiae heat stress response. Our data show a 2-3-fold transient increase in the concentration of C18-dihydrosphingosine and C18-phytosphingosine, more than a 100-fold transient increase in C20-dihydrosphingosine and C20-phytosphingosine, and a more stable 2-fold increase in ceramide containing C18-phytosphingosine and a 5-fold increase in ceramide containing C20-phytosphingosine following heat stress. Treatment of cells with dihydrosphingosine activates transcription of the TPS2 gene encoding a subunit of trehalose synthase and causes trehalose, a known thermoprotectant, to accumulate. Dihydrosphingosine induces expression of a STRE-LacZ reporter gene, showing that the global stress response element, STRE, found in many yeast promoter sequences can be activated by sphingolipid signals. The TPS2 promoter contains four STREs that may mediate dihydrosphingosine responsiveness. Using genetic and other approaches it should be possible to identify sphingolipid signaling pathways in S. cerevisiae and quantify the importance of each during heat stress. PMID:9374502

  13. Sphingolipid Therapy in Myocardial Ischemia-Reperfusion Injury

    PubMed Central

    Gundewar, Susheel; Lefer, David J.

    2009-01-01

    Sphingolipids are known to play a significant physiological role in cell growth, cell differentiation, and critical signal transduction pathways. Recent studies have demonstrated a significant role of sphingolipids and their metabolites in the pathogenesis of myocardial ischemia-reperfusion injury. Our laboratory has investigated the cytoprotective effects of N,N,N-Trimethylsphingosine chloride (TMS), a stable N-methylated synthetic sphingolipid analogue on myocardial and hepatic ischemia reperfusion injury in clinically relevant in vivo murine models of ischemia-reperfusion injury. TMS administered intravenously at the onset of ischemia reduced myocardial infarct size in the wild-type and obese (ob/ob) mice. Following myocardial I/R, there was an improvement in cardiac function in the wild-type mice. Additionally, TMS also decreased serum liver enzymes following hepatic I/R in wild-type mice. The cytoprotective effects did not extend to the ob/ob mice following hepatic I/R or to the db/db mice following both myocardial and hepatic I/R. Our data suggests that although TMS is cytoprotective following I/R in normal animals, the cytoprotective actions of TMS are largely attenuated in obese and diabetic animals which may be due to altered signaling mechanisms in these animal models. Here we review the therapeutic role of TMS and other sphingolipids in the pathogenesis of myocardial ischemia reperfusion injury and their possible mechanisms of cardioprotection. PMID:17928150

  14. Sphingosine-1-phosphate lyase in development and disease: Sphingolipid metabolism takes flight

    PubMed Central

    Fyrst, Henrik

    2009-01-01

    Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that catalyses the final step of sphingolipid degradation, namely the irreversible cleavage of the carbon chain at position 2-3 of a long chain base phosphate (LCBP), thereby yielding a long-chain aldehyde and phosphoethanolamine. LCBPs are potent signaling molecules involved in cell proliferation, survival, migration, cell-cell interactions and cell stress responses. Therefore, tight regulation of LCBP signaling is required for proper cell function, and perturbations of this system can lead to alterations in biological processes including development, reproduction and physiology. SPL is a key enzyme in regulating the intracellular and circulating levels of LCBPs and is, therefore, gaining attention as a putative target for pharmacological intervention. This review provides an overview of our current understanding of SPL structure and function, mechanisms involved in SPL regulation and the role of SPL in development and disease. PMID:18558101

  15. Lil3 Assembles with Proteins Regulating Chlorophyll Synthesis in Barley

    PubMed Central

    Gargano, Daniela; Furnes, Clemens; Reisinger, Veronika; Arnold, Janine; Kmiec, Karol; Eichacker, Lutz Andreas

    2015-01-01

    The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.). PMID:26172838

  16. Polyamines function in stress tolerance: from synthesis to regulation

    PubMed Central

    Liu, Ji-Hong; Wang, Wei; Wu, Hao; Gong, Xiaoqing; Moriguchi, Takaya

    2015-01-01

    Plants are challenged by a variety of biotic or abiotic stresses, which can affect their growth and development, productivity, and geographic distribution. In order to survive adverse environmental conditions, plants have evolved various adaptive strategies, among which is the accumulation of metabolites that play protective roles. A well-established example of the metabolites that are involved in stress responses, or stress tolerance, is the low-molecular-weight aliphatic polyamines, including putrescine, spermidine, and spermine. The critical role of polyamines in stress tolerance is suggested by several lines of evidence: firstly, the transcript levels of polyamine biosynthetic genes, as well as the activities of the corresponding enzymes, are induced by stresses; secondly, elevation of endogenous polyamine levels by exogenous supply of polyamines, or overexpression of polyamine biosynthetic genes, results in enhanced stress tolerance; and thirdly, a reduction of endogenous polyamines is accompanied by compromised stress tolerance. A number of studies have demonstrated that polyamines function in stress tolerance largely by modulating the homeostasis of reactive oxygen species (ROS) due to their direct, or indirect, roles in regulating antioxidant systems or suppressing ROS production. The transcriptional regulation of polyamine synthesis by transcription factors is also reviewed here. Meanwhile, future perspectives on polyamine research are also suggested. PMID:26528300

  17. Probing sphingolipid function in plants by the analysis of Arabidopsis mutants with altered sphingolipid content and composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sphingolipids are major components of the plasma membrane and tonoplasts of plant cells. These lipids are enriched in detergent-resistant membrane fractions or lipid-rafts prepared from plasma membrane and have been linked to signaling pathways in plants. Serine palmitoyltransferase (SPT), which c...

  18. Biological Effects of Naturally Occurring Sphingolipids, Uncommon Variants, and Their Analogs.

    PubMed

    Lai, Mitchell K P; Chew, Wee Siong; Torta, Federico; Rao, Angad; Harris, Greg L; Chun, Jerold; Herr, Deron R

    2016-09-01

    Sphingolipids (SPs) comprise a highly diverse class of lipids that serve biological roles both as structural components of cell membranes and as mediators of cell signaling. Pharmacologic and genetic manipulation of SPs and their signaling systems have underscored their importance in most biological processes, including central nervous system development and function. Likewise, perturbations of SP accumulation or signaling have been associated with a number of disease states, such as neural tube defects, neuroinflammation, stroke, and dementia. SPs can be endogenously synthesized de novo, and their metabolism is a well-regulated process, so their value as nutraceuticals has not been scrutinized. However, there is evidence that sphingolipid-rich diets can affect lipid homeostasis, and several mycotoxins are SP analogs that are known to cause profound derangement of SP metabolism or signaling. Furthermore, plants and invertebrates have SP species that are not present in mammals. Several of these have been shown to induce biological responses in mammalian cells. These findings suggest that dietary intake of SPs or SP analogs may have significant effects on human health or disease outcome. This manuscript provides an overview of SP metabolism and signaling, their perturbations in neurological diseases, as well as potential impacts of modulating this system in the brain. PMID:27393119

  19. Structure and mechanism of calmodulin binding to a signaling sphingolipid reveal new aspects of lipid-protein interactions.

    PubMed

    Kovacs, Erika; Harmat, Veronika; Tóth, Judit; Vértessy, Beáta G; Módos, Károly; Kardos, József; Liliom, Károly

    2010-10-01

    Lipid-protein interactions are rarely characterized at a structural molecular level due to technical difficulties; however, the biological significance of understanding the mechanism of these interactions is outstanding. In this report, we provide mechanistic insight into the inhibitory complex formation of the lipid mediator sphingosylphosphorylcholine with calmodulin, the most central and ubiquitous regulator protein in calcium signaling. We applied crystallographic, thermodynamic, kinetic, and spectroscopic approaches using purified bovine calmodulin and bovine cerebral microsomal fraction to arrive at our conclusions. Here we present 1) a 1.6-Å resolution crystal structure of their complex, in which the sphingolipid occupies the conventional hydrophobic binding site on calmodulin; 2) a peculiar stoichiometry-dependent binding process: at low or high protein-to-lipid ratio calmodulin binds lipid micelles or a few lipid molecules in a compact globular conformation, respectively, and 3) evidence that the sphingolipid displaces calmodulin from its targets on cerebral microsomes. We have ascertained the specificity of the interaction using structurally related lipids as controls. Our observations reveal the structural basis of selective calmodulin inhibition by the sphingolipid. On the basis of the crystallographic and biophysical characterization of the calmodulin-sphingosylphosphorylcholine interaction, we propose a novel lipid-protein binding model, which might be applicable to other interactions as well. PMID:20522785

  20. Palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB, p38, and ERK in human dermal fibroblasts.

    PubMed

    Oh, Eunhye; Yun, Mihee; Kim, Seong Keun; Seo, Gimoon; Bae, Joon Sung; Joo, Kwon; Chae, Gue Tae; Lee, Seong-Beom

    2014-05-01

    It has been suggested that free fatty acids (FFA) such as palmitate, which are secreted from enlarged adipocytes in the subcutaneous fat of obese subjects, serve as a link between obesity and altered skin functions. Cyclooxygenease-2 (COX-2) and prostanoids participate in the induction of impaired dermal function. In the current study, we investigated the issue of whether palmitate induces COX-2 expression via the sphingolipid pathway-mediated activation of NF-κB or mitogen-activated protein kinase (MAPK) pathways in human dermal fibroblasts. Palmitate treatment significantly induced COX-2 expression and prostaglandin E2 (PGE2) release in human dermal fibroblasts. In addition, pre-treatment with triacsin C, an inhibitor of acyl-CoA synthetase in de novo ceramide synthesis, was found to reduce palmitate-induced COX-2 expression and PGE2 release in human dermal fibroblast. The findings also show that palmitate-induced COX-2 expression and PGE2 release are mediated by the NF-κB, p38, and extracellular signal-regulated kinase (ERK) MAPK pathways. These findings point to a new mechanism for explaining the link between increased FFAs in obesity and impaired dermal function. PMID:24337700

  1. Synthesis and characterization of TEP-EDTA-regulated bioactive hydroxyapatite

    NASA Astrophysics Data System (ADS)

    Haders, Daniel Joseph, II

    Ca2+ concentration enabled the HA crystallization process to be growth dominated, producing films composed of high crystallinity, hexagonal grains on multiple metallic substrates. TEP regulation of HA crystallization enabled the deposition of an adhesive CaTiO3 intermediate layer, and then HA in a continuous, phase sequenced process on Ti6Al4V substrates, the first such process reported in the hydrothermal HA literature. The HA film was found to be deposited by a passivating competitive growth mechanism that enabled the [0001] crystallographic orientation of hexagonal single crystals to be engineered with synthesis time. Bioactivity analysis demonstrated that films were bioactive and bone bonding. Together, these results suggest that these HA films are candidates for use on metallic orthopedic implants, namely Ti6Al4V.

  2. Oxidant-specific regulation of protein synthesis in Candida albicans.

    PubMed

    Sundaram, Arunkumar; Grant, Chris M

    2014-06-01

    Eukaryotic cells typically respond to stress conditions by inhibiting global protein synthesis. The initiation phase is the main target of regulation and represents a key control point for eukaryotic gene expression. In Saccharomyces cerevisiae and mammalian cells this is achieved by phosphorylation of eukaryotic initiation factor 2 (eIF2α). We have examined how the fungal pathogen Candida albicans responds to oxidative stress conditions and show that oxidants including hydrogen peroxide, the heavy metal cadmium and the thiol oxidant diamide inhibit translation initiation. The inhibition in response to hydrogen peroxide and cadmium largely depends on phosphorylation of eIF2α since minimal inhibition is observed in a gcn2 mutant. In contrast, translation initiation is inhibited in a Gcn2-independent manner in response to diamide. Our data indicate that all three oxidants inhibit growth of C. albicans in a dose-dependent manner, however, loss of GCN2 does not improve growth in the presence of hydrogen peroxide or cadmium. Examination of translational activity indicates that these oxidants inhibit translation at a post-initiation phase which may account for the growth inhibition in a gcn2 mutant. As well as inhibiting global translation initiation, phosphorylation of eIF2α also enhances expression of the GCN4 mRNA in yeast via a well-known translational control mechanism. We show that C. albicans GCN4 is similarly induced in response to oxidative stress conditions and Gcn4 is specifically required for hydrogen peroxide tolerance. Thus, the response of C. albicans to oxidative stress is mediated by oxidant-specific regulation of translation initiation and we discuss our findings in comparison to other eukaryotes including the yeast S. cerevisiae. PMID:24699161

  3. Adaptive Control Model Reveals Systematic Feedback and Key Molecules in Metabolic Pathway Regulation

    PubMed Central

    Moffitt, Richard A.; Merrill, Alfred H.; Wang, May D.

    2011-01-01

    Abstract Robust behavior in metabolic pathways resembles stabilized performance in systems under autonomous control. This suggests we can apply control theory to study existing regulation in these cellular networks. Here, we use model-reference adaptive control (MRAC) to investigate the dynamics of de novo sphingolipid synthesis regulation in a combined theoretical and experimental case study. The effects of serine palmitoyltransferase over-expression on this pathway are studied in vitro using human embryonic kidney cells. We report two key results from comparing numerical simulations with observed data. First, MRAC simulations of pathway dynamics are comparable to simulations from a standard model using mass action kinetics. The root-sum-square (RSS) between data and simulations in both cases differ by less than 5%. Second, MRAC simulations suggest systematic pathway regulation in terms of adaptive feedback from individual molecules. In response to increased metabolite levels available for de novo sphingolipid synthesis, feedback from molecules along the main artery of the pathway is regulated more frequently and with greater amplitude than from other molecules along the branches. These biological insights are consistent with current knowledge while being new that they may guide future research in sphingolipid biology. In summary, we report a novel approach to study regulation in cellular networks by applying control theory in the context of robust metabolic pathways. We do this to uncover potential insight into the dynamics of regulation and the reverse engineering of cellular networks for systems biology. This new modeling approach and the implementation routines designed for this case study may be extended to other systems. Supplementary Material is available at www.liebertonline.com/cmb. PMID:21314456

  4. Sphingolipids contribute to acetic acid resistance in Zygosaccharomyces bailii.

    PubMed

    Lindahl, Lina; Genheden, Samuel; Eriksson, Leif A; Olsson, Lisbeth; Bettiga, Maurizio

    2016-04-01

    Lignocellulosic raw material plays a crucial role in the development of sustainable processes for the production of fuels and chemicals. Weak acids such as acetic acid and formic acid are troublesome inhibitors restricting efficient microbial conversion of the biomass to desired products. To improve our understanding of weak acid inhibition and to identify engineering strategies to reduce acetic acid toxicity, the highly acetic-acid-tolerant yeast Zygosaccharomyces bailii was studied. The impact of acetic acid membrane permeability on acetic acid tolerance in Z. bailii was investigated with particular focus on how the previously demonstrated high sphingolipid content in the plasma membrane influences acetic acid tolerance and membrane permeability. Through molecular dynamics simulations, we concluded that membranes with a high content of sphingolipids are thicker and more dense, increasing the free energy barrier for the permeation of acetic acid through the membrane. Z. bailii cultured with the drug myriocin, known to decrease cellular sphingo-lipid levels, exhibited significant growth inhibition in the presence of acetic acid, while growth in medium without acetic acid was unaffected by the myriocin addition. Furthermore, following an acetic acid pulse, the intracellular pH decreased more in myriocin-treated cells than in control cells. This indicates a higher inflow rate of acetic acid and confirms that the reduction in growth of cells cultured with myriocin in the medium with acetic acid was due to an increase in membrane permeability, thereby demonstrating the importance of a high fraction of sphingolipids in the membrane of Z. bailii to facilitate acetic acid resistance; a property potentially transferable to desired production organisms suffering from weak acid stress. PMID:26416641

  5. New norterpenoids and a sphingolipid from Carissa opaca.

    PubMed

    Parveen, Shehla; Saleem, Muhammad; Riaz, Naheed; Ashraf, Muhammad; Qurat-Ul-Ain; Nisar, Muhammad Farrukh; Jabbar, Abdul

    2016-01-01

    Chemical investigations on the aerial parts of Carissa opaca resulted in the isolation and characterization of two new nor-triterpenoids (compounds 1 and 2) and a new sphingolipid (compound 3) together with six known compounds. The structures of all the isolates were established using spectral data. All the isolated compounds showed DPPH radical scavenging and enzyme inhibitory activities against enzymes acetylcholinesterase, butyrylcholinesterase, and lipoxygenase. PMID:27010529

  6. The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine

    PubMed Central

    Mueller, Matthias; Cima, Igor; Noti, Mario; Fuhrer, Andrea; Jakob, Sabine; Dubuquoy, Laurent; Schoonjans, Kristina; Brunner, Thomas

    2006-01-01

    The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis. PMID:16923850

  7. The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine.

    PubMed

    Mueller, Matthias; Cima, Igor; Noti, Mario; Fuhrer, Andrea; Jakob, Sabine; Dubuquoy, Laurent; Schoonjans, Kristina; Brunner, Thomas

    2006-09-01

    The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis. PMID:16923850

  8. Induction of cell wall thickening by the antifungal compound dihydromaltophilin disrupts fungal growth and is mediated by sphingolipid biosynthesis.

    PubMed

    Li, Shaojie; Calvo, Ana M; Yuen, Gary Y; Du, Liangcheng; Harris, Steven D

    2009-01-01

    Dihydromaltophilin (heat-stable antifungal factor [HSAF]) is an antifungal metabolite produced in Lysobacter enzymogenes biocontrol strain C3. This compound induces cell wall thickening in Aspergillus nidulans. Here we show that the cell wall thickening is a general response to HSAF in diverse fungal species. In the A. nidulans model, the thickened cell wall negatively affects hyphal growth. Growth of HSAF-pre-treated hyphae failed to resume at hyphal tips with thick cell wall and the actin cable could not re-polarize at the thickened region of the cell wall, even after the treated hyphae were transferred to drug-free medium. Moreover, HSAF-induced cell wall thickening is mediated by sphingolipid synthesis: HSAF failed to induce cell wall thickening in the absence of ceramide synthase BarA and the sphingolipid synthesis inhibitor myriocin was able to suppress HSAF-induced cell wall thickening. The thickened cell wall could be digested by chitinase suggesting that chitin contributes to the HSAF-induced thickening. Furthermore, HSAF treatment activated the transcription of two chitin synthase encoding genes chsB and chsC. PMID:21462551

  9. Beyond the cherry-red spot: Ocular manifestations of sphingolipid-mediated neurodegenerative and inflammatory disorders.

    PubMed

    Chen, Hui; Chan, Annie Y; Stone, Donald U; Mandal, Nawajes A

    2014-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay-Sachs or Niemann-Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases. PMID:24011710

  10. Beyond the Cherry-Red Spot: Ocular Manifestations of Sphingolipid-mediated Neurodegenerative and Inflammatory Disorders

    PubMed Central

    Stone, Donald U.; Mandal, Nawajes A.

    2013-01-01

    Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay Sachs or Niemann Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases. PMID:24011710

  11. Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

    PubMed

    Grieninger, G; Plant, P W; Liang, T J; Kalb, R G; Amrani, D; Mosesson, M W; Hertzberg, K M; Pindyck, J

    1983-06-27

    Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly. PMID:6307104

  12. Regulation of glucosamine-6-phosphate deaminase synthesis in yeast.

    PubMed

    Singh, B; Datta, A

    1979-02-19

    A basal level of glucosamine-6-phosphate deaminase is detected in yeast cells grown on glucose. However, a burst of enzyme production occurs in the presence of N-acetylglucosamine in pathogenic Candida albicans and non-pathogenic Saccharomyces cervisiae. The enzyme synthesis stops and its concentration in the cells declines rapidly as soon as N-acetylglucosamine is removed from the medium. Experiments with RNA- and protein-synthesis inhibitors indicate that the appearance of new enzyme activity is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibition of DNA synthesis either by hydroxyurea or by mitomycin-C does not impair the synthesis of glucosamine-6-phosphate deaminase. PMID:369615

  13. Glucocorticoids Regulate Tristetraprolin Synthesis and Posttranscriptionally Regulate Tumor Necrosis Factor Alpha Inflammatory Signaling▿

    PubMed Central

    Smoak, Kathleen; Cidlowski, John A.

    2006-01-01

    Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3′ untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment leads to a sustained induction of TTP mRNA expression that is abrogated by RU486. Glucocorticoid induction of TTP mRNA is also blocked by actinomycin D but not by cycloheximide, suggesting a transcriptional mechanism which has been confirmed by transcription run-on experiments. The most widely characterized TTP-regulated gene is the AU-rich tumor necrosis factor alpha (TNF-α) gene. Dexamethasone represses TNF-α mRNA in A549 cells and decreases luciferase expression of a TNF-α 3′ untranslated region reporter plasmid in an orientation-dependent manner. Small interfering RNAs to TTP significantly prevent this effect, and a cell line stably expressing a short-hairpin RNA to TTP conclusively establishes that TTP is critical for dexamethasone inhibition of TNF-α mRNA expression. These studies provide the molecular evidence for glucocorticoid regulation of human TTP and reflect a novel inductive anti-inflammatory signaling pathway for glucocorticoids that acts via posttranscriptional mechanisms. PMID:16982682

  14. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation

    PubMed Central

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-01-01

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders. PMID:26984393

  15. Sphingolipids and Epoxidized Lipid Metabolites in the Control of Gut Immunosurveillance and Allergy

    PubMed Central

    Kunisawa, Jun; Kiyono, Hiroshi

    2016-01-01

    The intestinal immune system ingeniously balances the distinct responses of elimination and tolerance of non-self-substances for the creation and maintenance of homeostatic environments. Accumulating evidence has recently shown that various lipids, including dietary one, are involved in the regulation of intestinal immunity and are associated with biophylaxis and immune disorders. Recent advances in the lipidomics allow the identification of novel pathways of lipid metabolism and lipid metabolites for the control of intestinal immunity. In this paper, we describe the effects and functions of lipids, especially sphingolipids and new lipid metabolites originated from dietary oil on the immunomodulation and on the development and pathogenesis of allergic diseases in the intestine. PMID:26858949

  16. Sphingolipids and Epoxidized Lipid Metabolites in the Control of Gut Immunosurveillance and Allergy.

    PubMed

    Kunisawa, Jun; Kiyono, Hiroshi

    2016-01-01

    The intestinal immune system ingeniously balances the distinct responses of elimination and tolerance of non-self-substances for the creation and maintenance of homeostatic environments. Accumulating evidence has recently shown that various lipids, including dietary one, are involved in the regulation of intestinal immunity and are associated with biophylaxis and immune disorders. Recent advances in the lipidomics allow the identification of novel pathways of lipid metabolism and lipid metabolites for the control of intestinal immunity. In this paper, we describe the effects and functions of lipids, especially sphingolipids and new lipid metabolites originated from dietary oil on the immunomodulation and on the development and pathogenesis of allergic diseases in the intestine. PMID:26858949

  17. Sphingosine-1-phosphate Phosphatase 2 Regulates Pancreatic Islet β-Cell Endoplasmic Reticulum Stress and Proliferation.

    PubMed

    Taguchi, Yoshimitsu; Allende, Maria L; Mizukami, Hiroki; Cook, Emily K; Gavrilova, Oksana; Tuymetova, Galina; Clarke, Benjamin A; Chen, Weiping; Olivera, Ana; Proia, Richard L

    2016-06-01

    Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that regulates basic cell functions through metabolic and signaling pathways. Intracellular metabolism of S1P is controlled, in part, by two homologous S1P phosphatases (SPPases), 1 and 2, which are encoded by the Sgpp1 and Sgpp2 genes, respectively. SPPase activity is needed for efficient recycling of sphingosine into the sphingolipid synthesis pathway. SPPase 1 is important for skin homeostasis, but little is known about the functional role of SPPase 2. To identify the functions of SPPase 2 in vivo, we studied mice with the Sgpp2 gene deleted. In contrast to Sgpp1(-/-) mice, Sgpp2(-/-) mice had normal skin and were viable into adulthood. Unexpectedly, WT mice expressed Sgpp2 mRNA at high levels in pancreatic islets when compared with other tissues. Sgpp2(-/-) mice had normal pancreatic islet size; however, they exhibited defective adaptive β-cell proliferation that was demonstrated after treatment with either a high-fat diet or the β-cell-specific toxin, streptozotocin. Importantly, β-cells from untreated Sgpp2(-/-) mice showed significantly increased expression of proteins characteristic of the endoplasmic reticulum stress response compared with β-cells from WT mice, indicating a basal islet defect. Our results show that Sgpp2 deletion causes β-cell endoplasmic reticulum stress, which is a known cause of β-cell dysfunction, and reveal a juncture in the sphingolipid recycling pathway that could impact the development of diabetes. PMID:27059959

  18. Genes encoding Δ(8)-sphingolipid desaturase from various plants: identification, biochemical functions, and evolution.

    PubMed

    Li, Shu-Fen; Zhang, Guo-Jun; Zhang, Xue-Jin; Yuan, Jin-Hong; Deng, Chuan-Liang; Hu, Zan-Min; Gao, Wu-Jun

    2016-09-01

    ∆(8)-sphingolipid desaturase catalyzes the C8 desaturation of a long chain base, which is the characteristic structure of various complex sphingolipids. The genes of 20 ∆(8)-sphingolipid desaturases from 12 plants were identified and functionally detected by using Saccharomyces cerevisiae system to elucidate the relationship between the biochemical function and evolution of this enzyme. Results showed that the 20 genes all can encode a functional ∆(8)-sphingolipid desaturase, which catalyzes different ratios of two products, namely, 8(Z) and 8(E)-C18-phytosphingenine. The coded enzymes could be divided into two groups on the basis of biochemical functions: ∆(8)-sphingolipid desaturase with a preference for an E-isomer product and ∆(8)-sphingolipid desaturase with a preference for a Z-isomer product. The conversion rate of the latter was generally lower than that of the former. Phylogenetic analysis revealed that the 20 desaturases could also be clustered into two groups, and this grouping is consistent with that of the biochemical functions. Thus, the biochemical function of ∆(8)-sphingolipid desaturase is correlated with its evolution. The two groups of ∆(8)-sphingolipid desaturases could arise from distinct ancestors in higher plants. However, they might have initially evolved from ∆(8)-sphingolipid desaturases in lower organisms, such as yeasts, which can produce E-isomer products only. Furthermore, almost all of the transgenic yeasts harboring ∆(8)-sphingolipid desaturase genes exhibit an improvement in aluminum tolerance. Our study provided new insights into the biochemical function and evolution of ∆(8)-sphingolipid desaturases in plants. PMID:27294968

  19. [Local Protein Synthesis in Dendrites and its Regulation Normally and During Plastic Changes].

    PubMed

    Chesnokova, E A; Kolosov, P M

    2016-01-01

    Specifics and key regulation mechanisms of compartmentalised protein synthesis in dendrites are reviewed. The up-to-date literature data of the subject are analysed. The results of many molecular, cytological and physiological experiments are presented. Also there is some information about a number of neurological diseases connected with dendritic translation regulation malfunction. PMID:27538281

  20. Regulation of troponin C synthesis in primary culture of chicken cardiac muscle cells.

    PubMed

    Malhotra, S B; Bag, J

    1987-01-01

    Cardiac myocyte cell culture from fourteen day old embryonic chicken heart was prepared. This cultured cell system was used to examine the regulation of troponin C (TnC) synthesis in cardiac muscle. To examine the regulation of TnC polypeptide synthesis, cardiac myocyte cells were pulse labelled with 35S-methionine at different days after plating. The synthesis of TnC was measured by determining the amount of radioactivity incorporated into the TnC polypeptide following separation by two dimensional gel electrophoresis. These measurements showed that TnC synthesis was maximum in 36 to 48 h old cultures and reached its lowest level in 4 day old cultures. This was in contrast to the synthesis of actin and tropomyosin. Synthesis of these polypeptides were lowest in 36 to 48 h old cultures and was maximum in 7 day old cultures. To examine whether the synthesis of TnC polypeptide paralleled the levels of TnC mRNA the sequences homologous to quail slow TnC cDNA clone were measured by hybridisation. The results showed that the decrease in the synthesis of troponin C polypeptide cannot be fully explained by the decrease in the steady state level of troponin C mRNA. The possibility of a role of translational control of troponin C mRNA in this process is discussed. PMID:2890096

  1. Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations.

    PubMed

    Monette, Jeffrey S; Gómez, Luis A; Moreau, Régis F; Bemer, Brett A; Taylor, Alan W; Hagen, Tory M

    2010-07-23

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (approximately 10,000 pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (approximately 70 pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  2. Characteristics of the Rat Cardiac Sphingolipid Pool in Two Mitochondrial Subpopulations

    PubMed Central

    Monette, Jeffrey S.; Gómez, Luis A.; Moreau, Régis F.; Bemer, Brett A.; Taylor, Alan W.; Hagen, Tory M.

    2010-01-01

    Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary (IFM) and subsarcolemmal (SSM) mitochondria were isolated, lipids extracted, and sphingolipids quantified by LC-tandem mass spectrometry. Results showed that sphingomyelin (~10,000 pmols/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (~70 pmols/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16 to 24 carbon units in their acyl side-chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45 pmols/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other. PMID:20599536

  3. Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

    PubMed

    Gao, Song; Carson, James A

    2016-01-01

    Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes. PMID:26491045

  4. Regulation of protein synthesis during early limitation of Saccharomyces cerevisiae.

    PubMed Central

    Swedes, J S; Dial, M E; McLaughlin, C S

    1979-01-01

    Arsenate, a competitive inhibitor with phosphate in phosphorylation reactions, has been used to lower adenine and guanine nucleotide levels in Saccharomyces cerevisiae to study nucleotide effects on protein synthesis. By measuring polysome levels, we have shown that initiation of protein synthesis is much more sensitive than elongation or termination to inhibition when the ATP/ADP, GTP/GDP ratios are low. When the arsenate-phosphate molar ratio was 0.27, protein synthesis was inhibited by about 85% and the kinetics of polysome decay was similar to that observed with the initiation inhibitor, verrucarin-76, or with the protein synthesis initiation mutant, ts187, at the restrictive temperature. With this level of arsenate, the adenylate energy charge dropped from 0.9 to 0.7 and the ATP/ADP and GTP/GDP ratios dropped from 6 to 2. The observed correlations between nucleotide ratio changes and inhibition of protein synthesis suggest that the former may be a control signal for the latter. The significance of these in vivo correlations will have to be tested with an in vitro protein synthesizing system. Higher arsenate levels resulted in even lower ATP/ADP, GTP/GDP ratios and in a slower decay of polysomes, implying that, eventually, elongation (in addition to initiation) was being inhibited. PMID:374362

  5. Hemagglutinin clusters in the plasma membrane are not enriched with cholesterol and sphingolipids.

    PubMed

    Wilson, Robert L; Frisz, Jessica F; Klitzing, Haley A; Zimmerberg, Joshua; Weber, Peter K; Kraft, Mary L

    2015-04-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  6. Hemagglutinin Clusters in the Plasma Membrane Are Not Enriched with Cholesterol and Sphingolipids

    PubMed Central

    Wilson, Robert L.; Frisz, Jessica F.; Klitzing, Haley A.; Zimmerberg, Joshua; Weber, Peter K.; Kraft, Mary L.

    2015-01-01

    The clusters of the influenza envelope protein, hemagglutinin, within the plasma membrane are hypothesized to be enriched with cholesterol and sphingolipids. Here, we directly tested this hypothesis by using high-resolution secondary ion mass spectrometry to image the distributions of antibody-labeled hemagglutinin and isotope-labeled cholesterol and sphingolipids in the plasma membranes of fibroblast cells that stably express hemagglutinin. We found that the hemagglutinin clusters were neither enriched with cholesterol nor colocalized with sphingolipid domains. Thus, hemagglutinin clustering and localization in the plasma membrane is not controlled by cohesive interactions between hemagglutinin and liquid-ordered domains enriched with cholesterol and sphingolipids, or from specific binding interactions between hemagglutinin, cholesterol, and/or the majority of sphingolipid species in the plasma membrane. PMID:25863057

  7. Light-regulated protein and poly(A)+ mRNA synthesis in Neurospora crassa.

    PubMed Central

    Chambers, J A; Hinkelammert, K; Russo, V E

    1985-01-01

    We have examined the effect of illumination upon the patterns of protein synthesis in the filamentous ascomycete Neurospora crassa by pulse labelling and two-dimensional gel electrophoresis. Light did not affect overall rates of protein synthesis but did induce the synthesis of six novel polypeptides whose appearance followed a temporally regulated pattern. When translation products of mRNA from illuminated cultures and dark control cultures were compared it was found that the synthesis of five out of six of the polypeptides specific to illuminated cultures could be seen in vitro. We believe that this is consistent with the hypothesis that light regulates the transcription of some genes in N. crassa, although we cannot exclude effects on mRNA stability or the control of precursor splicing. Images Fig. 2. Fig. 3. PMID:2868891

  8. Sphingosine-1-phosphate synthesis and functions in mast cells

    PubMed Central

    Price, Megan M; Oskeritzian, Carole A; Milstien, Sheldon; Spiegel, Sarah

    2009-01-01

    Sphingolipids are not only major lipid components of all eukaryotic cell membranes, but they also comprise an important family of bioactive signaling molecules that regulate a diverse array of biological responses. The sphingolipid metabolite sphingosine-1-phosphate (S1P), is a key regulator of immune responses. Cellular levels of S1P are determined by the balance between its synthesis, involving two sphingosine kinases (SphK1 and SphK2), and its degradation, involving S1P lyase and S1P phosphatases. S1P mainly signals through its cell-surface receptors and may also have intracellular functions. S1P has important functions in mast cells – the major effectors of allergic responses. Antigen triggering of IgE receptors on mast cells activates both SphKs resulting in the production of S1P that is released and regulates and amplifies mast cell functions, including degranulation as well as cytokine and chemokine release. PMID:19802381

  9. Multiple diguanylate cyclase-coordinated regulation of pyoverdine synthesis in Pseudomonas aeruginosa.

    PubMed

    Chen, Yicai; Yuan, Mingjun; Mohanty, Anee; Yam, Joey Kuok Hoong; Liu, Yang; Chua, Song Lin; Nielsen, Thomas E; Tolker-Nielsen, Tim; Givskov, Michael; Cao, Bin; Yang, Liang

    2015-06-01

    The nucleotide signalling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) plays an essential role in regulating microbial virulence and biofilm formation. C-di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. One intrinsic feature of c-di-GMP signalling is the abundance of DGCs and PDEs encoded by many bacterial species. It is unclear whether the different DGCs or PDEs coordinately establish the c-di-GMP regulation or function independently of each other. Here, we provide evidence that multiple DGCs are involved in regulation of c-di-GMP on synthesis of the major iron siderophore pyoverdine in Pseudomonas aeruginosa. Constitutive expression of the WspG or YedQ DGC in P. aeruginosa is able to induce its pyoverdine synthesis. Induction of pyoverdine synthesis by high intracellular c-di-GMP depends on the synthesis of exopolysaccharides and another two DGCs, SiaD and SadC. SiaD was found to boost the c-di-GMP synthesis together with constitutively expressing YedQ. The exopolysaccharides and the SiaD DGC were found to modulate the expression of the RsmY/RsmZ ncRNAs. Induction of the RsmY/RsmZ ncRNAs might enhance the pyoverdine synthesis through SadC. Our study sheds light on a novel multiple DGC-coordinated c-di-GMP regulatory mechanism of bacteria. PMID:25683454

  10. A Gibberellin-Mediated DELLA-NAC Signaling Cascade Regulates Cellulose Synthesis in Rice[OPEN

    PubMed Central

    Huang, Debao; Wang, Shaogan; Zhang, Baocai; Shang-Guan, Keke; Shi, Yanyun; Zhang, Dongmei; Liu, Xiangling; Wu, Kun; Xu, Zuopeng; Fu, Xiangdong; Zhou, Yihua

    2015-01-01

    Cellulose, which can be converted into numerous industrial products, has important impacts on the global economy. It has long been known that cellulose synthesis in plants is tightly regulated by various phytohormones. However, the underlying mechanism of cellulose synthesis regulation remains elusive. Here, we show that in rice (Oryza sativa), gibberellin (GA) signals promote cellulose synthesis by relieving the interaction between SLENDER RICE1 (SLR1), a DELLA repressor of GA signaling, and NACs, the top-layer transcription factors for secondary wall formation. Mutations in GA-related genes and physiological treatments altered the transcription of CELLULOSE SYNTHASE genes (CESAs) and the cellulose level. Multiple experiments demonstrated that transcription factors NAC29/31 and MYB61 are CESA regulators in rice; NAC29/31 directly regulates MYB61, which in turn activates CESA expression. This hierarchical regulation pathway is blocked by SLR1-NAC29/31 interactions. Based on the results of anatomical analysis and GA content examination in developing rice internodes, this signaling cascade was found to be modulated by varied endogenous GA levels and to be required for internode development. Genetic and gene expression analyses were further performed in Arabidopsis thaliana GA-related mutants. Altogether, our findings reveal a conserved mechanism by which GA regulates secondary wall cellulose synthesis in land plants and provide a strategy for manipulating cellulose production and plant growth. PMID:26002868

  11. Regulation of Escherichia coli aspartate transcarbamylase synthesis by guanosine tetraphosphate and pyrimidine ribonucleoside triphosphates.

    PubMed

    Turnbough, C L

    1983-02-01

    The effects of guanosine tetraphosphate (ppGpp) and pyrimidine ribonucleoside triphosphates on Escherichia coli aspartate transcarbamylase (ATCase) synthesis were examined. To determine the effect of ppGpp, a stringent (relA+) and relaxed (relA) isogenic pair of E. coli K-12 strains was starved for isoleucine, and the residual rate of synthesis of this enzyme was measured. It was necessary to starve the strains for uracil before the isoleucine limitation to maintain similar, low levels of UTP, the putative pyrimidine effector of ATCase synthesis. The isoleucine starvation of the stringent strain caused an immediate 10-fold increase in the intracellular concentration of ppGpp, which was coincident with the cessation of the synthesis of the enzyme. The elevated level of ppGpp then decayed until it reached an intracellular concentration similar to that found in unstarved cells. Enzyme synthesis resumed at this time. In the relaxed strain, the intracellular concentration of ppGpp did not increase upon isoleucine starvation and synthesis of the enzyme was not repressed. These experiments strongly indicated that ppGpp acts as a negative effector of ATCase synthesis. The repression of ATCase synthesis by ppGpp was demonstrated directly by using a Salmonella typhimurium (relA) in vitro coupled transcription-translation system with a lambda specialized transducing phage carrying the E. coli K-12 operon encoding the subunits of this enzyme (pyrBI) as a source of DNA. This in vitro system was also used to measure the effects of UTP and CTP on ATCase synthesis. Increasing the concentration of UTP in the in vitro reaction mixture resulted in strong repression of this synthesis, whereas increasing the CTP concentration did not affect synthesis significantly. Possible mechanisms for the regulation of pyr gene expression, including attenuation control, are discussed. PMID:6337130

  12. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS.

    PubMed

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. PMID:26868933

  13. Quantitative profiling of sphingolipids in wild Cordyceps and its mycelia by using UHPLC-MS

    PubMed Central

    Mi, Jia-Ning; Wang, Jing-Rong; Jiang, Zhi-Hong

    2016-01-01

    In the present study, 101 sphingolipids in wild Cordyceps and its five mycelia were quantitatively profiled by using a fully validated UHPLC-MS method. The results revealed that a general rank order for the abundance of different classes of sphingolipids in wild Cordyceps and its mycelia is sphingoid bases/ceramides > phosphosphingolipids > glycosphingolipids. However, remarkable sphingolipid differences between wild Cordyceps and its mycelia were observed. One is that sphingoid base is the dominant sphingolipid in wild Cordyceps, whereas ceramide is the major sphingolipid in mycelia. Another difference is that the abundance of sphingomyelins in wild Cordyceps is almost 10-folds higher than those in most mycelia. The third one is that mycelia contain more inositol phosphorylceramides and glycosphingolipids than wild Cordyceps. Multivariate analysis was further employed to visualize the difference among wild Cordyceps and different mycelia, leading to the identification of respective sphingolipids as potential chemical markers for the differentiation of wild Cordyceps and its related mycelia. This study represents the first report on the quantitative profiling of sphingolipids in wild Cordyceps and its related mycelia, which provided comprehensive chemical evidence for the quality control and rational utilization of wild Cordyceps and its mycelia. PMID:26868933

  14. Developmental Regulation across the Life Span: Toward a New Synthesis

    ERIC Educational Resources Information Center

    Haase, Claudia M.; Heckhausen, Jutta; Wrosch, Carsten

    2013-01-01

    How can individuals regulate their own development to live happy, healthy, and productive lives? Major theories of developmental regulation across the life span have been proposed (e.g., dual-process model of assimilation and accommodation; motivational theory of life-span development; model of selection, optimization, and compensation), but they…

  15. Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis.

    PubMed

    Magnin-Robert, Maryline; Le Bourse, Doriane; Markham, Jonathan; Dorey, Stéphan; Clément, Christophe; Baillieul, Fabienne; Dhondt-Cordelier, Sandrine

    2015-11-01

    Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids. PMID:26378098

  16. Modifications of Sphingolipid Content Affect Tolerance to Hemibiotrophic and Necrotrophic Pathogens by Modulating Plant Defense Responses in Arabidopsis1[OPEN

    PubMed Central

    Magnin-Robert, Maryline; Le Bourse, Doriane; Markham, Jonathan; Dorey, Stéphan; Clément, Christophe; Baillieul, Fabienne; Dhondt-Cordelier, Sandrine

    2015-01-01

    Sphingolipids are emerging as second messengers in programmed cell death and plant defense mechanisms. However, their role in plant defense is far from being understood, especially against necrotrophic pathogens. Sphingolipidomics and plant defense responses during pathogenic infection were evaluated in the mutant of long-chain base phosphate (LCB-P) lyase, encoded by the dihydrosphingosine-1-phosphate lyase1 (AtDPL1) gene and regulating long-chain base/LCB-P homeostasis. Atdpl1 mutants exhibit tolerance to the necrotrophic fungus Botrytis cinerea but susceptibility to the hemibiotrophic bacterium Pseudomonas syringae pv tomato (Pst). Here, a direct comparison of sphingolipid profiles in Arabidopsis (Arabidopsis thaliana) during infection with pathogens differing in lifestyles is described. In contrast to long-chain bases (dihydrosphingosine [d18:0] and 4,8-sphingadienine [d18:2]), hydroxyceramide and LCB-P (phytosphingosine-1-phosphate [t18:0-P] and 4-hydroxy-8-sphingenine-1-phosphate [t18:1-P]) levels are higher in Atdpl1-1 than in wild-type plants in response to B. cinerea. Following Pst infection, t18:0-P accumulates more strongly in Atdpl1-1 than in wild-type plants. Moreover, d18:0 and t18:0-P appear as key players in Pst- and B. cinerea-induced cell death and reactive oxygen species accumulation. Salicylic acid levels are similar in both types of plants, independent of the pathogen. In addition, salicylic acid-dependent gene expression is similar in both types of B. cinerea-infected plants but is repressed in Atdpl1-1 after treatment with Pst. Infection with both pathogens triggers higher jasmonic acid, jasmonoyl-isoleucine accumulation, and jasmonic acid-dependent gene expression in Atdpl1-1 mutants. Our results demonstrate that sphingolipids play an important role in plant defense, especially toward necrotrophic pathogens, and highlight a novel connection between the jasmonate signaling pathway, cell death, and sphingolipids. PMID:26378098

  17. Regulation of muscle protein synthesis and the effects of catabolic states.

    PubMed

    Gordon, Bradley S; Kelleher, Andrew R; Kimball, Scot R

    2013-10-01

    Protein synthesis and degradation are dynamically regulated processes that act in concert to control the accretion or loss of muscle mass. The present article focuses on the mechanisms involved in the impairment of protein synthesis that are associated with skeletal muscle atrophy. The vast majority of mechanisms known to regulate protein synthesis involve modulation of the initiation phase of mRNA translation, which comprises a series of reactions that result in the binding of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The function of the proteins involved in both events has been shown to be repressed under atrophic conditions such as sepsis, cachexia, chronic kidney disease, sarcopenia, and disuse atrophy. The basis for the inhibition of protein synthesis under such conditions is likely to be multifactorial and includes insulin/insulin-like growth factor 1 resistance, pro-inflammatory cytokine expression, malnutrition, corticosteroids, and/or physical inactivity. The present article provides an overview of the existing literature regarding mechanisms and signaling pathways involved in the regulation of mRNA translation as they apply to skeletal muscle wasting, as well as the efficacy of potential clinical interventions such as nutrition and exercise in the maintenance of skeletal muscle protein synthesis under atrophic conditions. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting. PMID:23769967

  18. Regulation of Muscle Protein Synthesis and the Effects of Catabolic States

    PubMed Central

    Gordon, Bradley S.; Kelleher, Andrew R.; Kimball, Scot R.

    2013-01-01

    Protein synthesis and degradation are dynamically regulated processes that act in concert to control the accretion or loss of muscle mass. The present article focuses on the mechanisms involved in the impairment of protein synthesis that are associated with skeletal muscle atrophy. The vast majority of mechanisms known to regulate protein synthesis involve modulation of the initiation phase of mRNA translation, which comprises a series of reactions that result in the binding of initiator methionyl-tRNAi and mRNA to the 40S ribosomal subunit. The function of the proteins involved in both events has been shown to be repressed under atrophic conditions such as sepsis, cachexia, chronic kidney disease, sarcopenia, and disuse atrophy. The basis for the inhibition of protein synthesis under such conditions is likely to be multifactorial and includes insulin/insulin-like growth factor 1 resistance, pro-inflammatory cytokine expression, malnutrition, corticosteroids, and/or physical inactivity. The present article provides an overview of the existing literature regarding mechanisms and signaling pathways involved in the regulation of mRNA translation as they apply to skeletal muscle wasting, as well as the efficacy of potential clinical interventions such as nutrition and exercise in the maintenance of skeletal muscle protein synthesis under atrophic conditions. PMID:23769967

  19. Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation

    PubMed Central

    Tcherkezian, Joseph; Brittis, Perry A.; Thomas, Franziska; Roux, Philippe P.; Flanagan, John G.

    2010-01-01

    Summary Extracellular signals regulate protein translation in many cell functions. A key advantage of control at the translational level is the opportunity to regulate protein synthesis within specific cellular subregions. However, little is known about mechanisms that may link extracellular cues to translation with spatial precision. Here we show that a transmembrane receptor, DCC, forms a binding complex containing multiple translation components, including eukaryotic initiation factors, ribosomal large and small subunits, and monosomes. In neuronal axons and dendrites DCC colocalizes in particles with translation machinery, and newly synthesized protein. The extracellular ligand netrin promoted DCC-mediated translation and disassociation of translation components. The functional and physical association of a cell surface receptor with the translation machinery leads to a generalizable model for localization and extracellular regulation of protein synthesis, based on a transmembrane translation regulation complex. PMID:20434207

  20. AMPD2 Regulates GTP Synthesis and is Mutated in a Potentially-Treatable Neurodegenerative Brainstem Disorder

    PubMed Central

    Akizu, Naiara; Cantagrel, Vincent; Schroth, Jana; Cai, Na; Vaux, Keith; McCloskey, Douglas; Naviaux, Robert K.; Vleet, Jeremy Van; Fenstermaker, Ali G.; Silhavy, Jennifer L.; Scheliga, Judith S.; Toyama, Keiko; Morisaki, Hiroko; Sonmez, Fatma Mujgan; Celep, Figen; Oraby, Azza; Zaki, Maha S.; Al-Baradie, Raidah; Faqeih, Eissa; Saleh, Mohammad; Spencer, Emily; Rosti, Rasim Ozgur; Scott, Eric; Nickerson, Elizabeth; Gabriel, Stacey; Morisaki, Takayuki; Holmes, Edward W.; Gleeson, Joseph G.

    2013-01-01

    Purine biosynthesis and metabolism, conserved in all living organisms, is essential for cellular energy homeostasis and nucleic acids synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a new distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH), due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation, which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a new, potentially treatable early-onset neurodegenerative disease. PMID:23911318

  1. Characterization of secretory sphingomyelinase activity, lipoprotein sphingolipid content and LDL aggregation in ldlr−/− mice fed on a high-fat diet

    PubMed Central

    Deevska, Gergana M.; Sunkara, Manjula; Morris, Andrew J.; Nikolova-Karakashian, Mariana N.

    2012-01-01

    The propensity of LDLs (low-density lipoproteins) for aggregation and/or oxidation has been linked to their sphingolipid content, specifically the levels of SM (sphingomyelin) and ceramide. To investigate this association in vivo, ldlr (LDL receptor)-null mice (ldlr−/−) were fed on a modified (atherogenic) diet containing saturated fats and cholesterol. The diet led to significantly elevated SM content in all serum lipoproteins. In contrast, ceramide increased only in the LDL particles. MS-based analyses of the lipid acyl chain composition revealed a marked elevation in C16:0 fatty acid in SM and ceramide, consistent with the prevalence of palmitic acid in the modified diet. The diet also led to increased activity of the S-SMase [secretory SMase (sphingomyelinase)], a protein that is generated by ASMase (acid SMase) and acts on serum LDL. An increased macrophage secretion seemed to be responsible for the elevated S-SMase activity. ASMase-deficient mice (asm−/−/ldlr−/−) lacked S-SMase activity and were protected from diet-induced elevation in LDL ceramide. LDL from asm−/−/ldlr−/− mice fed on the modified diet were less aggregated and oxidized than LDL from asm+/+/ldlr−/− mice. When tested in vitro, the propensity for aggregation was dependent on the SM level: only LDL from animals on modified diet that have high SM content aggregated when treated with recombinant S-SMase. In conclusion, LDL-SM content and S-SMase activity are up-regulated in mice fed on an atherogenic diet. S-SMase mediates diet-induced changes in LDL ceramide content and aggregation. S-SMase effectiveness in inducing aggregation is dependent on diet-induced enrichment of LDL with SM, possibly through increased hepatic synthesis. PMID:22712892

  2. Regulation of plant immunity through modulation of phytoalexin synthesis.

    PubMed

    Zernova, Olga V; Lygin, Anatoli V; Pawlowski, Michelle L; Hill, Curtis B; Hartman, Glen L; Widholm, Jack M; Lozovaya, Vera V

    2014-01-01

    Soybean hairy roots transformed with the resveratrol synthase and resveratrol oxymethyl transferase genes driven by constitutive Arabidopsis actin and CsVMV promoters were characterized. Transformed hairy roots accumulated glycoside conjugates of the stilbenic compound resveratrol and the related compound pterostilbene, which are normally not synthesized by soybean plants. Expression of the non-native stilbenic phytoalexin synthesis in soybean hairy roots increased their resistance to the soybean pathogen Rhizoctonia solani. The expression of the AhRS3 gene resulted in 20% to 50% decreased root necrosis compared to that of untransformed hairy roots. The expression of two genes, the AhRS3 and ROMT, required for pterostilbene synthesis in soybean, resulted in significantly lower root necrosis (ranging from 0% to 7%) in transgenic roots than in untransformed hairy roots that had about 84% necrosis. Overexpression of the soybean prenyltransferase (dimethylallyltransferase) G4DT gene in soybean hairy roots increased accumulation of the native phytoalexin glyceollin resulting in decreased root necrosis. PMID:24914895

  3. Rapid quantitative analysis of sphingolipids in seafood using HPLC with evaporative light-scattering detection: its application in tissue distribution of sphingolipids in fish.

    PubMed

    Duan, Jingjing; Sugawara, Tatsuya; Hirata, Takashi

    2010-01-01

    Sphingolipids are ubiquitous in all eukaryotic organisms and known to be essential constituents of cellular membranes. Recently, various physiological functions of dietary sphingolipids, such as preventing cancer, improving skin barrier and contributing to central nervous system myelination have been demonstrated. To characterize the sphingolipids from fish as food components, tissue distribution of sphingomyelin and glycosylceramide (ceramide monohexoside, CMH) in fish were determined in this study. We established a rapid, accurate and effective method for separation, purification and determination of sphingolipids by using high-performance liquid chromatography with evaporative light-scattering detector (ELSD-HPLC). Sphingolipids were extracted and quantified from pacific saury (Cololabis saira). Sphingomyelin in different tissues of Cololabis saira ranged from 2.5 +/- 0.2 mg/g to 27.6 +/- 2.1 mg/g, the content in brain was the highest, followed by eyes, and CMH contents were less than 23.0 +/- 2.4 mg/g in all tissues. These results revealed that fish contained CMH and sphingomyelin as same levels as most of the terrestrial organisms and suggested marine organisms could be used as a potential source of precious and useful complex lipids. PMID:20720382

  4. The Sphingolipid Receptor S1PR2 Is a Receptor for Nogo-A Repressing Synaptic Plasticity

    PubMed Central

    Arzt, Michael E.; Weinmann, Oliver; Obermair, Franz J.; Pernet, Vincent; Zagrebelsky, Marta; Delekate, Andrea; Iobbi, Cristina; Zemmar, Ajmal; Ristic, Zorica; Gullo, Miriam; Spies, Peter; Dodd, Dana; Gygax, Daniel; Korte, Martin; Schwab, Martin E.

    2014-01-01

    Nogo-A is a membrane protein of the central nervous system (CNS) restricting neurite growth and synaptic plasticity via two extracellular domains: Nogo-66 and Nogo-A-Δ20. Receptors transducing Nogo-A-Δ20 signaling remained elusive so far. Here we identify the G protein-coupled receptor (GPCR) sphingosine 1-phosphate receptor 2 (S1PR2) as a Nogo-A-Δ20-specific receptor. Nogo-A-Δ20 binds S1PR2 on sites distinct from the pocket of the sphingolipid sphingosine 1-phosphate (S1P) and signals via the G protein G13, the Rho GEF LARG, and RhoA. Deleting or blocking S1PR2 counteracts Nogo-A-Δ20- and myelin-mediated inhibition of neurite outgrowth and cell spreading. Blockade of S1PR2 strongly enhances long-term potentiation (LTP) in the hippocampus of wild-type but not Nogo-A−/− mice, indicating a repressor function of the Nogo-A/S1PR2 axis in synaptic plasticity. A similar increase in LTP was also observed in the motor cortex after S1PR2 blockade. We propose a novel signaling model in which a GPCR functions as a receptor for two structurally unrelated ligands, a membrane protein and a sphingolipid. Elucidating Nogo-A/S1PR2 signaling platforms will provide new insights into regulation of synaptic plasticity. PMID:24453941

  5. The Impact of Cholesterol, DHA, and Sphingolipids on Alzheimer's Disease

    PubMed Central

    Grimm, Marcus O. W.; Zimmer, Valerie C.; Lehmann, Johannes; Grimm, Heike S.; Hartmann, Tobias

    2013-01-01

    Alzheimer's disease (AD) is a devastating neurodegenerative disorder currently affecting over 35 million people worldwide. Pathological hallmarks of AD are massive amyloidosis, extracellular senile plaques, and intracellular neurofibrillary tangles accompanied by an excessive loss of synapses. Major constituents of senile plaques are 40–42 amino acid long peptides termed β-amyloid (Aβ). Aβ is produced by sequential proteolytic processing of the amyloid precursor protein (APP). APP processing and Aβ production have been one of the central scopes in AD research in the past. In the last years, lipids and lipid-related issues are more frequently discussed to contribute to the AD pathogenesis. This review summarizes lipid alterations found in AD postmortem brains, AD transgenic mouse models, and the current understanding of how lipids influence the molecular mechanisms leading to AD and Aβ generation, focusing especially on cholesterol, docosahexaenoic acid (DHA), and sphingolipids/glycosphingolipids. PMID:24575399

  6. Transcriptional regulation of repressor synthesis in mycobacteriophage L5.

    PubMed

    Nesbit, C E; Levin, M E; Donnelly-Wu, M K; Hatfull, G F

    1995-09-01

    Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. Lysogeny is maintained by the putative repressor, the gene 71 product, which also mediates immunity to superinfection. We show here that there are three promoters located upstream of gene 71 which are active in an L5 lysogen but which do not require any phage-encoded proteins. In early lytic growth, gene 71 is also transcribed from a promoter, Pleft, located at the right end of the genome and which appears to be a target of gp71 regulation. A model is given for the regulation of L5 life cycles. PMID:8594325

  7. Regulation of Synthesis and Roles of Hyaluronan in Peritoneal Dialysis

    PubMed Central

    Bowen, Timothy; Meran, Soma; Williams, Aled P.; Newbury, Lucy J.; Sauter, Matthias; Sitter, Thomas

    2015-01-01

    Hyaluronan (HA) is a ubiquitous extracellular matrix glycosaminoglycan composed of repeated disaccharide units of alternating D-glucuronic acid and D-N-acetylglucosamine residues linked via alternating β-1,4 and β-1,3 glycosidic bonds. HA is synthesized in humans by HA synthase (HAS) enzymes 1, 2, and 3, which are encoded by the corresponding HAS genes. Previous in vitro studies have shown characteristic changes in HAS expression and increased HA synthesis in response to wounding and proinflammatory cytokines in human peritoneal mesothelial cells. In addition, in vivo models and human peritoneal biopsy samples have provided evidence of changes in HA metabolism in the fibrosis that at present accompanies peritoneal dialysis treatment. This review discusses these published observations and how they might contribute to improvement in peritoneal dialysis. PMID:26550568

  8. Trafficking and Functions of Bioactive Sphingolipids: Lessons from Cells and Model Membranes

    PubMed Central

    Zhou, Kecheng; Blom, Tomas

    2015-01-01

    Ceramide and sphingosine and their phosphorylated counterparts are recognized as “bioactive sphingolipids” and modulate membrane integrity, the activity of enzymes, or act as ligands of G protein-coupled receptors. The subcellular distribution of the bioactive sphingolipids is central to their function as the same lipid can mediate diametrically opposite effects depending on its location. To ensure that these lipids are present in the right amount and in the appropriate organelles, cells employ selective lipid transport and compartmentalize sphingolipid-metabolizing enzymes to characteristic subcellular sites. Our knowledge of key mechanisms involved in sphingolipid signaling and trafficking has increased substantially in the past decades—thanks to advances in biochemical and cell biological methods. In this review, we focus on the bioactive sphingolipids and discuss how the combination of studies in cells and in model membranes have contributed to our understanding of how they behave and function in living organisms. PMID:26715852

  9. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters.

    PubMed

    Eich, Christina; Manzo, Carlo; de Keijzer, Sandra; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  10. Inhibition of neutral sphingomyelinase-2 perturbs brain sphingolipid balance and spatial memory in mice

    PubMed Central

    Tabatadze, Nino; Savonenko, Alena; Song, Hongjun; Bandaru, Veera Venkata Ratnam; Chu, Michael; Haughey, Norman J.

    2010-01-01

    The sphingolipid ceramide is a bioactive signaling lipid that is thought to play important roles in modulating synaptic activity, in part by regulating the function of excitatory postsynaptic receptors. However, the molecular mechanisms by which ceramide exerts its effects on synaptic activity remain largely unknown. We recently demonstrated that a rapid generation of ceramide by neutral sphingomyelinase-2 (nSMase2; also known as sphingomyelin phosphodiesterase-3) played a key role in modulating excitatory postsynaptic currents by controlling the insertion and clustering of NMDA receptors (Wheeler et al. 2009). We now demonstrate that nSMase2 plays a role in memory. Inhibition of nSMase2 impaired spatial and episodic-like memory in mice. At the molecular level, inhibition of nSMase2 decreased ceramide, increased PSD-95, increased the number of AMPA receptors and altered the subunit composition of NMDA receptors. Our study identifies nSMase2 as an important component for efficient memory formation and underscores the importance of ceramide in regulating synaptic events related to learning and memory. PMID:20629193

  11. Changes in membrane sphingolipid composition modulate dynamics and adhesion of integrin nanoclusters

    PubMed Central

    Eich, Christina; Manzo, Carlo; Keijzer, Sandra de; Bakker, Gert-Jan; Reinieren-Beeren, Inge; García-Parajo, Maria F.; Cambi, Alessandra

    2016-01-01

    Sphingolipids are essential constituents of the plasma membrane (PM) and play an important role in signal transduction by modulating clustering and dynamics of membrane receptors. Changes in lipid composition are therefore likely to influence receptor organisation and function, but how this precisely occurs is difficult to address given the intricacy of the PM lipid-network. Here, we combined biochemical assays and single molecule dynamic approaches to demonstrate that the local lipid environment regulates adhesion of integrin receptors by impacting on their lateral mobility. Induction of sphingomyelinase (SMase) activity reduced sphingomyelin (SM) levels by conversion to ceramide (Cer), resulting in impaired integrin adhesion and reduced integrin mobility. Dual-colour imaging of cortical actin in combination with single molecule tracking of integrins showed that this reduced mobility results from increased coupling to the actin cytoskeleton brought about by Cer formation. As such, our data emphasizes a critical role for the PM local lipid composition in regulating the lateral mobility of integrins and their ability to dynamically increase receptor density for efficient ligand binding in the process of cell adhesion. PMID:26869100

  12. A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans

    PubMed Central

    Zhu, Huanhu; Shen, Huali; Sewell, Aileen K; Kniazeva, Marina; Han, Min

    2013-01-01

    Regulation of animal development in response to nutritional cues is an intensely studied problem related to disease and aging. While extensive studies indicated roles of the Target of Rapamycin (TOR) in sensing certain nutrients for controlling growth and metabolism, the roles of fatty acids and lipids in TOR-involved nutrient/food responses are obscure. Caenorhabditis elegans halts postembryonic growth and development shortly after hatching in response to monomethyl branched-chain fatty acid (mmBCFA) deficiency. Here, we report that an mmBCFA-derived sphingolipid, d17iso-glucosylceramide, is a critical metabolite in regulating growth and development. Further analysis indicated that this lipid function is mediated by TORC1 and antagonized by the NPRL-2/3 complex in the intestine. Strikingly, the essential lipid function is bypassed by activating TORC1 or inhibiting NPRL-2/3. Our findings uncover a novel lipid-TORC1 signaling pathway that coordinates nutrient and metabolic status with growth and development, advancing our understanding of the physiological roles of mmBCFAs, ceramides, and TOR. DOI: http://dx.doi.org/10.7554/eLife.00429.001 PMID:23705068

  13. Regulation of sphingomyelin metabolism.

    PubMed

    Bienias, Kamil; Fiedorowicz, Anna; Sadowska, Anna; Prokopiuk, Sławomir; Car, Halina

    2016-06-01

    Sphingolipids (SFs) represent a large class of lipids playing diverse functions in a vast number of physiological and pathological processes. Sphingomyelin (SM) is the most abundant SF in the cell, with ubiquitous distribution within mammalian tissues, and particularly high levels in the Central Nervous System (CNS). SM is an essential element of plasma membrane (PM) and its levels are crucial for the cell function. SM content in a cell is strictly regulated by the enzymes of SM metabolic pathways, which activities create a balance between SM synthesis and degradation. The de novo synthesis via SM synthases (SMSs) in the last step of the multi-stage process is the most important pathway of SM formation in a cell. The SM hydrolysis by sphingomyelinases (SMases) increases the concentration of ceramide (Cer), a bioactive molecule, which is involved in cellular proliferation, growth and apoptosis. By controlling the levels of SM and Cer, SMSs and SMases maintain cellular homeostasis. Enzymes of SM cycle exhibit unique properties and diverse tissue distribution. Disturbances in their activities were observed in many CNS pathologies. This review characterizes the physiological roles of SM and enzymes controlling SM levels as well as their involvement in selected pathologies of the Central Nervous System, such as ischemia/hypoxia, Alzheimer disease (AD), Parkinson disease (PD), depression, schizophrenia and Niemann Pick disease (NPD). PMID:26940196

  14. Pharmacogenetics of resistance to cisplatin and other anti-cancer drugs and the role of sphingolipid metabolism

    PubMed Central

    Alexander, Stephen; Swatson, William S.; Alexander, Hannah

    2014-01-01

    Summary Dictyostelium discoideum has proven to be a useful lead genetic system for identifying novel genes and pathways responsible for the regulation of sensitivity to the widely used anti-cancer drug cisplatin. Resistance to cisplatin is a major factor limiting the efficacy of the drug in treating many types of cancer. Studies using unbiased insertional mutagenesis in D. discoideum have identified the pathway of sphingolipid metabolism as a key regulator in controlling sensitivity to cisplatin. Using the genetic tools including directed homologous recombination and ectopic gene expression available with D. discoideum has shown how pharmacological modulation of this pathway can increase sensitivity to cisplatin, and these results have been extensively translated to, and validated in, human cells. Strategies, experimental conditions and methods are presented to enable further study of resistance to cisplatin as well as other important drugs. PMID:23494308

  15. Cell cycle-dependent regulation of extra-adrenal glucocorticoid synthesis in murine intestinal epithelial cells.

    PubMed

    Atanasov, Atanas G; Leiser, Dominic; Roesselet, Corinne; Noti, Mario; Corazza, Nadia; Schoonjans, Kristina; Brunner, Thomas

    2008-12-01

    Glucocorticoids are anti-inflammatory steroids with important applications in the treatment of inflammatory diseases. Endogenous glucocorticoids are mainly produced by the adrenal glands, although there is increasing evidence for extra-adrenal sources. Recent findings show that intestinal crypt cells produce glucocorticoids, which contribute to the maintenance of intestinal immune homeostasis. Intestinal glucocorticoid synthesis is critically regulated by the transcription factor liver receptor homologue-1 (LRH-1). As expression of steroidogenic enzymes and LRH-1 is restricted to the proliferating cells of the crypts, we aimed to investigate the role of the cell cycle in the regulation of LRH-1 activity and intestinal glucocorticoid synthesis. We here show that either pharmacological or molecular modulation of cell cycle progression significantly inhibited expression of steroidogenic enzymes and synthesis of glucocorticoids in intestinal epithelial cells. Synchronization of intestinal epithelial cells in the cell cycle revealed that expression of steroidogenic enzymes is preferentially induced at the G(1)/S stage. Differentiation of immature intestinal epithelial cells to mature nonproliferating cells also resulted in reduced expression of steroidogenic enzymes. This cell cycle-related effect on intestinal steroidogenesis was found to be mediated through the regulation of LRH-1 transcriptional activity. This mechanism may restrict intestinal glucocorticoid synthesis to the proliferating cells of the crypts. PMID:18711026

  16. Simultaneous Quantification of Sphingolipids in Small Quantities of Liver by LC-MS/MS

    PubMed Central

    Saigusa, Daisuke; Okudaira, Michiyo; Wang, Jiao; Kano, Kuniyuki; Kurano, Makoto; Uranbileg, Baasanjav; Ikeda, Hitoshi; Yatomi, Yutaka; Motohashi, Hozumi; Aoki, Junken

    2014-01-01

    Sph, S1P, and Cer, derived from the membrane sphingolipids, act as intracellular and intercellular mediators, involved in various (path) physiological functions. Accordingly, determining the distributions and concentrations of these sphingolipid mediators in body tissues is an important task. Consequently, a method for determination of sphingolipids in small quantities of tissue is required. Sphingolipids analysis has been dependent on improvements in mass spectrometry (MS) technology. Additionally, decomposition of sphingosine-1-phosphate (S1P) in the tissue samples before preparation for MS has hindered analysis. In the present study, a method for stabilization of liver samples before MS preparation was developed using a heat stabilizer (Stabilizor™ T1). Then, a LC-MS/MS method using a triple-quadrupole mass spectrometer with a C8 column was developed for simultaneous determination of sphingolipids in small quantities of liver specimens. This method showed good separation and validation results. Separation was performed with a gradient elution of solvent A (5 mmol L−1 ammonium formate in water, pH 4.0) and solvent B (5 mmol L−1 ammonium formate in 95% acetonitrile, pH 4.0) at 300 μL min−1. The lower limit of quantification was less than 132 pmol L−1, and this method was accurate (∼13.5%) and precise (∼7.13%) for S1P analysis. The method can be used to show the tissue distribution of sphingolipids. PMID:26819890

  17. Abscisic Acid Negatively Regulates Elicitor-Induced Synthesis of Capsidiol in Wild Tobacco1[W

    PubMed Central

    Mialoundama, Alexis Samba; Heintz, Dimitri; Debayle, Delphine; Rahier, Alain; Camara, Bilal; Bouvier, Florence

    2009-01-01

    In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8′-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8′-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants. PMID:19420326

  18. Regulation of macrophage IL-12 synthesis by Leishmania phosphoglycans.

    PubMed

    Piedrafita, D; Proudfoot, L; Nikolaev, A V; Xu, D; Sands, W; Feng, G J; Thomas, E; Brewer, J; Ferguson, M A; Alexander, J; Liew, F Y

    1999-01-01

    It is now generally accepted that IFN-gamma, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL-12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL-12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL-12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL-12 release in a dose-dependent manner. Inhibition was dependent on the galactose(beta1-4)mannose(alpha1)-PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF-alpha or IL-6 in activated macrophages. The inhibition of IL-12(p40) production was at the transcriptional level, but was not mediated through NF kappaB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts. PMID:9933105

  19. Regulation of phosphatidylcholine synthesis in rat liver endoplasmic reticulum.

    PubMed Central

    Sribney, M; Knowles, C L; Lyman, E M

    1976-01-01

    The biosynthesis of phosphatidylcholine in rat liver microsomal preparations catalysed by CDP-choline-1,2-diacylglycerol cholinephosphotransferase (EC 2.7.8.2) was inhibited by a combination of ATP and CoA or ATP and pantetheine. ATP alone at high concentrations (20 mM) inhibits phosphatidylcholine formation to the extent of 70%. In the presence of 0.1 mM-CoA, ATP (2 mM) inhibits to the extent of 80% and in the presence of 1 mM-pantetheine to the extent of 90%. ADP and other nucleotide triphosphates in combination with either CoA or pantetheine are only 10-30% as effective in inhibiting phosphatidylcholine synthesis. AMP(CH2)PP [adenosine 5'-(alphabeta-methylene)triphosphate] together with CoA inhibits to the extent of 59% and with pantetheine by 48%. AMP-P(CH2)P [adenosine 5'-(betagamma-methylene)triphosphate] together with either CoA or pantetheine had no significant effect on phosphatidylcholine formation. Other closely related derivatives of pantothenic acid were without effect either alone or in the presence of ATP, as were thiol compounds such as cysteine, homocysteine, cysteamine, dithiothreitol and glutathione. Several mechanisms by which this inhibition might take place were ruled out and it is concluded that ATP together with either CoA or pantetheine interacts reversibly with phosphatidylcholine synthetase to cause temporarily the inhibition of phosphatidylcholine formation. PMID:182154

  20. A key developmental regulator controls the synthesis of the antibiotic erythromycin in Saccharopolyspora erythraea.

    PubMed

    Chng, Chinping; Lum, Amy M; Vroom, Jonathan A; Kao, Camilla M

    2008-08-12

    Saccharopolyspora erythraea makes erythromycin, an antibiotic commonly used in human medicine. Unusually, the erythromycin biosynthetic (ery) cluster lacks a pathway-specific regulatory gene. We isolated a transcriptional regulator of the ery biosynthetic genes from S. erythraea and found that this protein appears to directly link morphological changes caused by impending starvation to the synthesis of a molecule that kills other bacteria, i.e., erythromycin. DNA binding assays, liquid and affinity chromatography, MALDI-MS analysis, and de novo sequencing identified this protein (M(r) = 18 kDa) as the S. erythraea ortholog of BldD, a key regulator of development in Streptomyces coelicolor. Recombinant S. erythraea BldD bound to all five regions containing promoters in the ery cluster as well as to its own promoter, the latter with an order-of-magnitude stronger than to the ery promoters. Deletion of bldD in S. erythraea decreased the erythromycin titer in a liquid culture 7-fold and blocked differentiation on a solid medium. Moreover, an industrial strain of S. erythraea with a higher titer of erythromycin expressed more BldD than a wild-type strain during erythromycin synthesis. Together, these results suggest that BldD concurrently regulates the synthesis of erythromycin and morphological differentiation. The ery genes are the first direct targets of a BldD ortholog to be identified that are positively regulated. PMID:18685110

  1. A key developmental regulator controls the synthesis of the antibiotic erythromycin in Saccharopolyspora erythraea

    PubMed Central

    Chng, Chinping; Lum, Amy M.; Vroom, Jonathan A.; Kao, Camilla M.

    2008-01-01

    Saccharopolyspora erythraea makes erythromycin, an antibiotic commonly used in human medicine. Unusually, the erythromycin biosynthetic (ery) cluster lacks a pathway-specific regulatory gene. We isolated a transcriptional regulator of the ery biosynthetic genes from S. erythraea and found that this protein appears to directly link morphological changes caused by impending starvation to the synthesis of a molecule that kills other bacteria, i.e., erythromycin. DNA binding assays, liquid and affinity chromatography, MALDI-MS analysis, and de novo sequencing identified this protein (Mr = 18 kDa) as the S. erythraea ortholog of BldD, a key regulator of development in Streptomyces coelicolor. Recombinant S. erythraea BldD bound to all five regions containing promoters in the ery cluster as well as to its own promoter, the latter with an order-of-magnitude stronger than to the ery promoters. Deletion of bldD in S. erythraea decreased the erythromycin titer in a liquid culture 7-fold and blocked differentiation on a solid medium. Moreover, an industrial strain of S. erythraea with a higher titer of erythromycin expressed more BldD than a wild-type strain during erythromycin synthesis. Together, these results suggest that BldD concurrently regulates the synthesis of erythromycin and morphological differentiation. The ery genes are the first direct targets of a BldD ortholog to be identified that are positively regulated. PMID:18685110

  2. PERK Regulates Working Memory and Protein Synthesis-Dependent Memory Flexibility.

    PubMed

    Zhu, Siying; Henninger, Keely; McGrath, Barbara C; Cavener, Douglas R

    2016-01-01

    PERK (EIF2AK3) is an ER-resident eIF2α kinase required for memory flexibility and metabotropic glutamate receptor-dependent long-term depression, processes known to be dependent on new protein synthesis. Here we investigated PERK's role in working memory, a cognitive ability that is independent of new protein synthesis, but instead is dependent on cellular Ca2+ dynamics. We found that working memory is impaired in forebrain-specific Perk knockout and pharmacologically PERK-inhibited mice. Moreover, inhibition of PERK in wild-type mice mimics the fear extinction impairment observed in forebrain-specific Perk knockout mice. Our findings reveal a novel role of PERK in cognitive functions and suggest that PERK regulates both Ca2+ -dependent working memory and protein synthesis-dependent memory flexibility. PMID:27627766

  3. Regulation of Brome Mosaic Virus Gene Expression by Restriction of Initiation of Protein Synthesis

    PubMed Central

    Chroboczek, Jadwiga; Puchkova, Ludmiła; Zagórski, Włodzimierz

    1980-01-01

    The translation of total and individual brome mosaic virus (BMV) RNAs was examined in a wheat germ cell-free system in the presence of various inhibitors. Inhibitors of the initiation of polypeptide synthesis, e.g., potassium ions, 7-methylguanosine 5′ -monophosphate, and aurintricarboxylic acid, were shown not only to inhibit overall BMV protein synthesis but also to change the ratio of BMV polypeptides synthesized. Under conditions restrictive for initiation, the translation of nonstructural BMV genes was suppressed, but coat protein synthesis proceeded at a high rate. A similar discrimination among BMV messengers was exerted by a regulatory protein kinase isolated from wheat germ. These results suggest that the regulation of the expression of BMV genes is based on a difference in the mechanism of formation of initiation complexes for individual BMV messages. Images PMID:16789194

  4. Extra-adrenal glucocorticoid synthesis: immune regulation and aspects on local organ homeostasis.

    PubMed

    Talabér, Gergely; Jondal, Mikael; Okret, Sam

    2013-11-01

    Systemic glucocorticoids (GCs) mainly originate from de novo synthesis in the adrenal cortex under the control of the hypothalamus-pituitary-adrenal (HPA)-axis. However, research during the last 1-2 decades has revealed that additional organs express the necessary enzymes and have the capacity for de novo synthesis of biologically active GCs. This includes the thymus, intestine, skin and the brain. Recent research has also revealed that locally synthesized GCs most likely act in a paracrine or autocrine manner and have significant physiological roles in local homeostasis, cell development and immune cell activation. In this review, we summarize the nature, regulation and known physiological roles of extra-adrenal GC synthesis. We specifically focus on the thymus in which GC production (by both developing thymocytes and epithelial cells) has a role in the maintenance of proper immunological function. PMID:23707789

  5. Regulation of peptidoglycan synthesis by outer membrane proteins

    PubMed Central

    Typas, Athanasios; Banzhaf, Manuel; van den Berg van Saparoea, Bart; Verheul, Jolanda; Biboy, Jacob; Nichols, Robert J.; Zietek, Matylda; Beilharz, Katrin; Kannenberg, Kai; von Rechenberg, Moritz; Breukink, Eefjan; den Blaauwen, Tanneke; Gross, Carol A.; Vollmer, Waldemar

    2011-01-01

    Summary Growth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth. PMID:21183073

  6. Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis

    PubMed Central

    Aitchison, Adam J.; Arsenault, Daniel J.; Ridgway, Neale D.

    2015-01-01

    The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs. PMID:26108622

  7. Synthesis of multiple Pseudomonas aeruginosa biofilm matrix exopolysaccharides is post-transcriptionally regulated

    PubMed Central

    Ma, Luyan; Wang, Juan; Wang, Shiwei; Anderson, Erin M.; Lam, Joseph S.; Parsek, Matthew R.; Wozniak, Daniel J.

    2015-01-01

    Summary Exopolysaccharide is a critical biofilm matrix component, yet little is known about how the synthesis of multiple exopolysaccharides is regulated. Pseudomonas aeruginosa can produce several biofilm matrix exopolysaccharides that include alginate, Psl and Pel. Here we demonstrated that AlgC, a key enzyme that provides sugar precursors for the synthesis of alginate and lipopolysaccharides (LPS) is also required for both Psl and Pel production. We showed that forced-synthesis of Psl in alginate-producing mucoid bacteria reduced alginate production but this was not due to transcription of the alginate biosynthesis-operon. Likewise, when either alginate or Psl were overproduced, levels of B-band LPS decreased. Induction of Pel resulted in a reduction of Psl levels. Because the effects of reduced exopolysaccharide synthesis when another is overproduced didn’t appear to be regulated at the transcriptional level, this suggests that the biosynthesis pathways of Psl, Pel, alginate, and LPS compete for common sugar precursors. As AlgC is the only enzyme that provides precursors for each of these exopolysaccharides, we propose that AlgC is a key checkpoint enzyme that coordinates the total amount of exopolysaccharide biosynthesis by controlling sugar precursor pool. Our data also provide a plausible strategy that P. aeruginosa utilizes to modulate its biofilm matrix exopolysaccharides. PMID:22513190

  8. Revisiting Human Cholesterol Synthesis and Absorption: The Reciprocity Paradigm and its Key Regulators.

    PubMed

    Alphonse, Peter A S; Jones, Peter J H

    2016-05-01

    Hypercholesterolemia is a major risk factor for cardiovascular disease. Cholesterol homeostasis in the body is governed by the interplay between absorption, synthesis, and excretion or conversion of cholesterol into bile acids. A reciprocal relationship between cholesterol synthesis and absorption is known to regulate circulating cholesterol in response to dietary or therapeutic interventions. However, the degree to which these factors affect synthesis and absorption and the extent to which one vector shifts in response to the other are not thoroughly understood. Also, huge inter-individual variability exists in the manner in which the two systems act in response to any cholesterol-lowering treatment. Various factors are known to account for this variability and in light of recent experimental advances new players such as gene-gene interactions, gene-environmental effects, and gut microbiome hold immense potential in offering an explanation to the complex traits of inter-individual variability in human cholesterol metabolism. In this context, the objective of the present review is to provide an overview on cholesterol metabolism and discuss the role of potential factors such as genetics, epigenetics, epistasis, and gut microbiome, as well as other regulators in modulating cholesterol metabolism, especially emphasizing the reciprocal relationship between cholesterol synthesis and absorption. Furthermore, an evaluation of the implications of this push-pull mechanism on cholesterol-lowering strategies is presented. PMID:26620375

  9. Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

    PubMed Central

    Yakunin, A F; Hallenbeck, P C

    1997-01-01

    The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis. PMID:9045800

  10. Gonadotropin-releasing hormone regulates spine density via its regulatory role in hippocampal estrogen synthesis

    PubMed Central

    Prange-Kiel, Janine; Jarry, Hubertus; Schoen, Michael; Kohlmann, Patrick; Lohse, Christina; Zhou, Lepu; Rune, Gabriele M.

    2008-01-01

    Spine density in the hippocampus changes during the estrus cycle and is dependent on the activity of local aromatase, the final enzyme in estrogen synthesis. In view of the abundant gonadotropin-releasing hormone receptor (GnRH-R) messenger RNA expression in the hippocampus and the direct effect of GnRH on estradiol (E2) synthesis in gonadal cells, we asked whether GnRH serves as a regulator of hippocampal E2 synthesis. In hippocampal cultures, E2 synthesis, spine synapse density, and immunoreactivity of spinophilin, a reliable spine marker, are consistently up-regulated in a dose-dependent manner at low doses of GnRH but decrease at higher doses. GnRH is ineffective in the presence of GnRH antagonists or aromatase inhibitors. Conversely, GnRH-R expression increases after inhibition of hippocampal aromatase. As we found estrus cyclicity of spine density in the hippocampus but not in the neocortex and GnRH-R expression to be fivefold higher in the hippocampus compared with the neocortex, our data strongly suggest that estrus cycle–dependent synaptogenesis in the female hippocampus results from cyclic release of GnRH. PMID:18227283