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Kentaro Hanada - One of the best experts on this subject based on the ideXlab platform.
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Characterization of mutant Serine Palmitoyltransferase 1 in LY-B cells.
Lipids, 2009Co-Authors: Amin Momin, Alfred H. Merrill, Hyejung Park, Jeremy C. Allegood, Martina Leipelt, Samuel Kelly, Kentaro HanadaAbstract:CHO-LY-B cells have been useful in studies of sphingolipid metabolism and function because they lack Serine Palmitoyltransferase (SPT) activity. Cloning and sequencing of the SPT1 transcript of LY-B cells identified the mutation as a guanine to adenine change at nucleotide 738, causing a G246R transformation. Western blots revealed low expression of the mutant SPT1 peptide, but activity was not detectable by mass spectrometric analysis of [13C]-palmitate incorporation into sphinganine, sphingosine, 1-deoxysphinganine, or 1-desoxymethylsphinganine. Treatment of LY-B cells with chemical chaperones (DMSO or glycerol) increased the amounts of mutant SPT1 as well as SPT2, but SPT activity was not restored. This study has established that G246R mutation in hamster SPT1 results in the loss of SPT activity.
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Serine Palmitoyltransferase a key enzyme of sphingolipid metabolism
Biochimica et Biophysica Acta, 2003Co-Authors: Kentaro HanadaAbstract:Abstract The first step in the biosynthesis of sphingolipids is the condensation of Serine and palmitoyl CoA, a reaction catalyzed by Serine Palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5′-phosphate (PLP)-dependent α-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity.
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Serine Palmitoyltransferase a key enzyme of sphingolipid metabolism
Biochimica et Biophysica Acta, 2003Co-Authors: Kentaro HanadaAbstract:The first step in the biosynthesis of sphingolipids is the condensation of Serine and palmitoyl CoA, a reaction catalyzed by Serine Palmitoyltransferase (SPT) to produce 3-ketodihydrosphingosine (KDS). This review focuses on recent advances in the biochemistry and molecular biology of SPT. SPT belongs to a family of pyridoxal 5V (PLP)-dependent a-oxoamine synthases (POAS). Mammalian SPT is a heterodimer of 53-kDa LCB1 and 63-kDa LCB2 subunits, both of which are bound to the endoplasmic reticulum (ER) most likely with the type I topology, whereas other members of the POAS family are soluble homodimer enzymes. LCB2 appears to be unstable unless it is associated with LCB1. Potent inhibitors of SPT structurally resemble an intermediate in a probable multistep reaction mechanism for SPT. Although SPT is a housekeeping enzyme, its activity is regulated transcriptionally and post-transcriptionally, and its up-regulation is suggested to play a role in apoptosis induced by certain types of stress. Specific missense mutations in the human LCB1 gene cause hereditary sensory neuropathy type I, an autosomal dominantly inherited disease, and these mutations confer dominant-negative effects on SPT activity. D 2003 Elsevier Science B.V. All rights reserved.
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Localization, Topology, and Function of the LCB1 Subunit of Serine Palmitoyltransferase in Mammalian Cells
Journal of Biological Chemistry, 2002Co-Authors: Satoshi Yasuda, Masahiro Nishijima, Kentaro HanadaAbstract:Serine Palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.
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d Serine inhibits Serine Palmitoyltransferase the enzyme catalyzing the initial step of sphingolipid biosynthesis
FEBS Letters, 2000Co-Authors: Kentaro Hanada, Tomoko Hara, Masahiro NishijimaAbstract:Abstract Serine Palmitoyltransferase (SPT), responsible for the initial step of sphingolipid biosynthesis, catalyzes condensation of palmitoyl coenzyme A and L -Serine to produce 3-ketodihydrosphingosine (KDS). For determination of the stereochemical specificity of the amino acid substrate, a competition analysis of the production of [ 3 H]KDS from L -[ 3 H]Serine was performed using purified SPT. D -Serine inhibited [ 3 H]KDS production as effectively as non-radioactive L -Serine, whereas neither D -alanine nor D -threonine showed any significant effect. Incubation of purified SPT with [palmitoyl 1- 14 C]palmitoyl coenzyme A and D -Serine did not produce [ 14 C]KDS, while the control incubation with L -Serine did. These results suggest that D -Serine competes with L -Serine for the amino acid recognition site of SPT, but that D -Serine is not utilized by this enzyme to produce KDS.
Teresa M. Dunn - One of the best experts on this subject based on the ideXlab platform.
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Use of isotopically labeled substrates reveals kinetic differences between human and bacterial Serine Palmitoyltransferase.
Journal of Lipid Research, 2019Co-Authors: Peter J. Harrison, Teresa M. Dunn, David J. Clarke, James H. Naismith, Kenneth Gable, Niranjanakumari Somashekarappa, Van Kelly, Dominic J. CampopianoAbstract:Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-Serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, Serine Palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-Serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Sphingomonas paucimobilis Our data show that S. paucimobilis SPT activity displays a clear isotope effect with [2,3,3-D]l-Serine, whereas the human SPT isoform does not. This suggests that although both human and S. paucimobilis SPT catalyze the same chemical reaction, there may well be underlying subtle differences in their catalytic mechanisms. Our results suggest that it is the activating small subunits of human SPT that play a key role in these mechanistic variations. This study also highlights that it is important to consider the type and location of isotope labels on a substrate when they are to be used in in vitro and in vivo studies.
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The Chemical Basis of Serine Palmitoyltransferase Inhibition by Myriocin
2016Co-Authors: John M. Wadsworth, Teresa M. Dunn, David J. Clarke, Jonathan Lowther, Ashley E. Beattie, Patrick R. R. Langridge-smith, Howard B. Broughton, James H. Naismith, Stephen A. Mcmahon, Dominic J. CampopianoAbstract:Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of l-Serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent enzyme Serine Palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected ‘retro-aldol-like’ cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition
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ormdl Serine Palmitoyltransferase stoichiometry determines effects of ormdl3 expression on sphingolipid biosynthesis
Journal of Lipid Research, 2015Co-Authors: Deanna L. Siow, Teresa M. Dunn, Manjula Sunkara, Andrew J. Morris, Binks W. WattenbergAbstract:The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, Serine Palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.
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ORMDL/Serine Palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis.
Journal of Lipid Research, 2015Co-Authors: Deanna L. Siow, Teresa M. Dunn, Manjula Sunkara, Andrew J. Morris, Binks W. WattenbergAbstract:The ORM1 (Saccharomyces cerevisiae)-like proteins (ORMDLs) and their yeast orthologs, the Orms, are negative homeostatic regulators of the initiating enzyme in sphingolipid biosynthesis, Serine Palmitoyltransferase (SPT). Genome-wide association studies have established a strong correlation between elevated expression of the endoplasmic reticulum protein ORMDL3 and risk for childhood asthma. Here we test the notion that elevated levels of ORMDL3 decrease sphingolipid biosynthesis. This was tested in cultured human bronchial epithelial cells (HBECs) (an immortalized, but untransformed, airway epithelial cell line) and in HeLa cells (a cervical adenocarcinoma cell line). Surprisingly, elevated ORMDL3 expression did not suppress de novo biosynthesis of sphingolipids. We determined that ORMDL is expressed in functional excess relative to SPT at normal levels of expression. ORMDLs and SPT form stable complexes that are not increased by elevated ORMDL3 expression. Although sphingolipid biosynthesis was not decreased by elevated ORMDL3 expression, the steady state mass levels of all major sphingolipids were marginally decreased by low level ORMDL3 over-expression in HBECs. These data indicate that the contribution of ORMDL3 to asthma risk may involve changes in sphingolipid metabolism, but that the connection is complex.
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Expression of the ORMDLS, Modulators of Serine Palmitoyltransferase, Is Regulated by Sphingolipids in Mammalian Cells
Journal of Biological Chemistry, 2014Co-Authors: Sita D. Gupta, Teresa M. Dunn, Kenneth Gable, Aikaterini Alexaki, Panagiotis Chandris, Richard L. Proia, Jeffrey M. HarmonAbstract:Abstract The relationship between Serine Palmitoyltransferase (SPT) activity and ORMDL regulation of sphingolipid biosynthesis was investigated in mammalian HEK293 cells. Each of the three human ORMDLs reduced the increase in long-chain base synthesis seen after overexpression of wild-type SPT or SPT containing the C133W mutation in hLCB1, which produces the non-catabolizable sphingoid base, 1-deoxySa. ORMDL-dependent repression of sphingoid base synthesis occurred whether SPT was expressed as individual subunits or as a heterotrimeric single-chain SPT fusion protein. Overexpression of the single-chain SPT fusion protein under the control of a tetracycline-inducible promoter in stably transfected cells resulted in increased endogenous ORMDL expression. This increase was not transcriptional; there was no significant increase in any of the ORMDL mRNAs. Increased ORMDL protein expression required SPT activity since overexpression of a catalytically inactive SPT with a mutation in hLCB2a had little effect. Significantly, increased ORMDL expression was also blocked by myriocin inhibition of SPT as well as fumonisin inhibition of the ceramide synthases, suggesting that increased expression is a response to a metabolic signal. Moreover, blocking ORMDL induction with fumonisin treatment resulted in significantly greater increases in in vivo SPT activity than was seen when ORMDLs were allowed to increase, demonstrating the physiological significance of this response.
Konrad Sandhoff - One of the best experts on this subject based on the ideXlab platform.
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cis-4-Methylsphingosine Decreases Sphingolipid Biosynthesis by Specifically Interfering with Serine Palmitoyltransferase Activity in Primary Cultured Neurons
Journal of Biological Chemistry, 1997Co-Authors: Gerhild Van Echten-deckert, Richard R. Schmidt, Alexandra Zschoche, Thomas Bär, Andrea Raths, Thomas Heinemann, Konrad SandhoffAbstract:The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of Serine Palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on Serine Palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on Serine Palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on Serine Palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.
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subcellular localization and membrane topology of Serine Palmitoyltransferase 3 dehydrosphinganine reductase and sphinganine n acyltransferase in mouse liver
Journal of Biological Chemistry, 1992Co-Authors: Elisabet C. Mandon, Gerhild Van Echten, I Ehses, Jiirgen Rother, Konrad SandhoffAbstract:Abstract Serine Palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of Serine Palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (Serine Palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits Serine Palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.
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sphingolipid biosynthesis in cultured neurons down regulation of Serine Palmitoyltransferase by sphingoid bases
FEBS Journal, 1991Co-Authors: Elisabet C. Mandon, Gerhild Van Echten, Rolf Birk, Richard R. Schmidt, Konrad SandhoffAbstract:Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of Serine Palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 μM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4–8 h with 50 μM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10–50 μM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of Serine Palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of Serine Palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of Serine Palmitoyltransferase is quite specific.
Christina Chan - One of the best experts on this subject based on the ideXlab platform.
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Palmitate-activated astrocytes via Serine Palmitoyltransferase increase BACE1 in primary neurons by sphingomyelinases
Neurobiology of Aging, 2013Co-Authors: Li Liu, Rebecca Martin, Christina ChanAbstract:Astrocytes play a critical role in neurodegenerative diseases, including Alzheimer's disease (AD). Previously, we showed that saturated free fatty acid, palmitic acid (PA), upregulates β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) level and amyloidogenesis in primary rat neurons mediated by astrocytes. However, the molecular mechanisms by which conditioned media from PA-treated astrocytes upregulates BACE1 level in neurons are unknown. This study demonstrates that Serine Palmitoyltransferase (SPT) in the astrocytes increases ceramide levels, which enhances the release of cytokines that mediate the activation of neural and acidic sphingomyelinase (SMase) in the neurons, to propagate the deleterious effects of PA (i.e., BACE1 upregulation). In support of the relevance of SPT in AD, our laboratory recently measured and found SPT levels to be significantly upregulated in AD brains as compared with controls. Cytokines, namely tumor necrosis factor-α and interleukin-1β, released into the conditioned media of PA-treated astrocytes activate neural and acidic SMase in the neurons. Neutralizing the cytokines in the PA-treated astrocyte conditioned media reduced BACE1 upregulation. However, inhibiting SPT in the astrocytes decreased the levels of both tumor necrosis factor-α and interleukin-1β in the conditioned media, which in turn reduced the SMase activities and BACE1 level in primary neurons. Thus, our results suggest that the activation of the astrocytes by PA is mediated by SPT, and the activated astrocytes increases BACE1 level in the neurons; the latter is mediate by the SMases.
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MicroRNA-137/181c Regulates Serine Palmitoyltransferase and In Turn Amyloid β, Novel Targets in Sporadic Alzheimer's Disease
Journal of Neuroscience, 2011Co-Authors: Hirosha Geekiyanage, Christina ChanAbstract:The contribution of mutations in amyloid precursor protein (APP) and presenilin (PSEN) to familial Alzheimer's disease (AD) is well established. However, little is known about the molecular mechanisms leading to amyloid β (Aβ) generation in sporadic AD. Increased brain ceramide levels have been associated with sporadic AD, and are a suggested risk factor. Serine Palmitoyltransferase (SPT) is the first rate-limiting enzyme in the de novo ceramide synthesis. However, the regulation of SPT is not yet understood. Evidence suggests that it may be posttranscriptionally regulated. Therefore, we investigated the role of miRNAs in the regulation of SPT and amyloid β (Aβ) generation. We show that SPT is upregulated in a subgroup of sporadic AD patient brains. This is further confirmed in mouse model studies of risk factors associated with AD. We identified that the loss of miR-137, -181c, -9, and 29a/b-1 increases SPT and in turn Aβ levels, and provides a mechanism for the elevated risk of AD associated with age, high-saturated-fat diet, and gender. Finally, these results suggest SPT and the respective miRNAs may be potential therapeutic targets for sporadic AD.
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microrna 137 181c regulates Serine Palmitoyltransferase and in turn amyloid β novel targets in sporadic alzheimer s disease
The Journal of Neuroscience, 2011Co-Authors: Hirosha Geekiyanage, Christina ChanAbstract:The contribution of mutations in amyloid precursor protein (APP) and presenilin (PSEN) to familial Alzheimer's disease (AD) is well established. However, little is known about the molecular mechanisms leading to amyloid β (Aβ) generation in sporadic AD. Increased brain ceramide levels have been associated with sporadic AD, and are a suggested risk factor. Serine Palmitoyltransferase (SPT) is the first rate-limiting enzyme in the de novo ceramide synthesis. However, the regulation of SPT is not yet understood. Evidence suggests that it may be posttranscriptionally regulated. Therefore, we investigated the role of miRNAs in the regulation of SPT and amyloid β (Aβ) generation. We show that SPT is upregulated in a subgroup of sporadic AD patient brains. This is further confirmed in mouse model studies of risk factors associated with AD. We identified that the loss of miR-137, -181c, -9, and 29a/b-1 increases SPT and in turn Aβ levels, and provides a mechanism for the elevated risk of AD associated with age, high-saturated-fat diet, and gender. Finally, these results suggest SPT and the respective miRNAs may be potential therapeutic targets for sporadic AD.
Hiroko Ikushiro - One of the best experts on this subject based on the ideXlab platform.
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Role of a conserved arginine residue during catalysis in Serine Palmitoyltransferase.
FEBS Letters, 2011Co-Authors: Jonathan Lowther, Hiroko Ikushiro, Hideyuki Hayashi, Guillaume Charmier, Marine C. C. Raman, Dominic J. CampopianoAbstract:All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent Serine Palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-Serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-Serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.
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Mechanistic enzymology of Serine Palmitoyltransferase
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2011Co-Authors: Hiroko Ikushiro, Hideyuki HayashiAbstract:Serine Palmitoyltransferase, which is one of the α-oxamine synthase family enzymes, catalyzes the condensation reaction of L-Serine and palmitoyl-CoA to form 3-ketodihydrosphingosine, the first and rate-determining step of the sphingolipid biosynthesis. As with other α-oxamine synthase family enzymes, the catalytic reaction is composed of multiple elementary steps, and the mechanism to control these steps to avoid side reactions has been the subject of intensive research in recent years. Combined spectroscopic, kinetic, and structural studies have revealed the finely controlled stereochemical mechanism, in which the His residue conserved among the α-oxamine synthase family enzymes plays a central and critical role. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.
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structural insights into the enzymatic mechanism of Serine Palmitoyltransferase from sphingobacterium multivorum
Journal of Biochemistry, 2009Co-Authors: Hiroko Ikushiro, Mohammad Mainul Islam, Akihiro Okamoto, Jun Hoseki, T Murakawa, Shigeru Fujii, Ikuko Miyahara, Hideyuki HayashiAbstract:: Serine Palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-Serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with l-Serine was determined at 2.3 A resolution. The electron density map showed the Schiff base formation between l-Serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the Calpha-H bond of the PLP-l-Serine aldimine was not perpendicular to the PLP-Schiff base plane. This conformation is unfavourable for the alpha-proton abstraction by Lys244 and the reaction is expected to stop at the PLP-l-Serine aldimine. Structural modelling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of l-Serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the l-Serine Calpha-H bond perpendicular to the PLP-Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the alpha-deprotonation of l-Serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.
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Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase
Journal of Biological Chemistry, 2009Co-Authors: Yuka Shiraiwa, Hiroko Ikushiro, Hideyuki HayashiAbstract:Serine Palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-Serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His159) instead of an aromatic amino acid residue. His159 was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-Serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-Serine. These results indicate that His159 is not only the anchoring site for l-Serine but regulates the α-deprotonation of l-Serine by fixing the conformation of the PLP-l-Serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-Serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His159 promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His159 plays multiple roles by exploiting the stereochemistry of Dunathan's conjecture.
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Reactions of Serine Palmitoyltransferase with Serine and molecular mechanisms of the actions of Serine derivatives as inhibitors.
Biochemistry, 2004Co-Authors: Hiroko Ikushiro, Hideyuki Hayashi, Hiroyuki KagamiyamaAbstract:Serine Palmitoyltransferase (SPT) is a key enzyme in sphingolipid biosynthesis and catalyzes the decarboxylative condensation of l-Serine and palmitoyl coenzyme A to 3-ketodihydrosphingosine. We have succeeded in the overproduction of a water-soluble homodimeric SPT from Sphingomonas paucimobilis EY2395T in Escherichia coli. The recombinant SPT showed the characteristic absorption and circular dichroism spectra derived from its coenzyme pyridoxal 5‘-phosphate. On the basis of the spectral changes of SPT, we have analyzed the reactions of SPT with compounds related to l-Serine and product, and showed the following new aspects: First, we analyzed the binding of l-Serine and 3-hydroxypropionate and found that the spectral change in SPT by the substrate is caused by the formation of an external aldimine intermediate and not by the formation of the Michaelis complex. Second, various Serine analogues were also examined; the data indicated that the α-carboxyl group of l-Serine was quite important for substrate ...
