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Gregory A. Grant - One of the best experts on this subject based on the ideXlab platform.
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D-3-Phosphoglycerate Dehydrogenase.
2018Co-Authors: Gregory A. GrantAbstract:l-Serine is the immediate precursor of d-serine, a major agonist of the N-methyl-d-aspartate (NMDA) receptor. l-Serine is a pivotal amino acid since it serves as a precursor to a large number of essential metabolites besides d-serine. In all non-photosynthetic organisms, including mammals, a major source of l-serine is the phosphorylated pathway of l-serine biosynthesis. The pathway consists of three enzymes, d-3-Phosphoglycerate Dehydrogenase (PGDH), phosphoserine amino transferase (PSAT), and l-phosphoserine phosphatase (PSP). PGDH catalyzes the first step in the pathway by converting d-3-Phosphoglycerate (PGA), an intermediate in glycolysis, to phosphohydroxypyruvate (PHP) concomitant with the reduction of NAD+. In some, but not all organisms, the catalytic activity of PGDH can be regulated by feedback inhibition by l-serine. Three types of PGDH can be distinguished based on their domain structure. Type III PGDHs contain only a nucleotide binding and substrate binding domain. Type II PGDHs contain an additional regulatory domain (ACT domain), and Type I PGDHs contain a fourth domain, termed the ASB domain. There is no consistent pattern of domain content that correlates with organism type, and even when additional domains are present, they are not always functional. PGDH deficiency results in metabolic defects of the nervous system whose systems range from microcephaly at birth, seizures, and psychomotor retardation. Although deficiency of any of the pathway enzymes have similar outcomes, PGDH deficiency is predominant. Dietary or intravenous supplementation with l-serine is effective in controlling seizures but has little effect on psychomotor development. An increase in PGDH levels, due to overexpression, is also associated with a wide array of cancers. In culture, PGDH is required for tumor cell proliferation, but extracellular l-serine is not able to support cell proliferation. This has led to the hypothesis that the pathway is performing some function related to tumor growth other than supplying l-serine. The most well-studied PGDHs are bacterial, primarily from Escherichia coli and Mycobacterium tuberculosis, perhaps because they have been of most interest mechanistically. However, the relatively recent association of PGDH with neuronal defects and human cancers has provoked renewed interest in human PGDH.
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transient kinetic analysis of the interaction of l serine with escherichia coli d 3 Phosphoglycerate Dehydrogenase reveals the mechanism of v type regulation and the order of effector binding
2009Co-Authors: Rodney L Burton, Shawei Chen, Gregory A. GrantAbstract:Pre-steady state stopped-flow analysis of Escherichia coli d-3-Phosphoglycerate Dehydrogenase (PGDH) reveals that the physiological inhibitor, l-serine, exerts its effect on at least two steps in the kinetic mechanism, but to very different degrees. First, there is a small but significant effect on the dissociation constant of NADH, the first substrate to bind in the ordered mechanism. The effect of serine is mainly on the binding off rate, increasing the Kd to 5 and 23 μM from 0.6 and 9 μM, respectively, for the two sets of sites in the enzyme. A more profound effect is seen after the second substrate is added. Serine reduces the amplitude of the signal without a significant effect on the observed rate constants for binding. The serine concentration that reduces the amplitude by 50% is equal to the K0.5 for serine inhibition. The data are consistent with the conclusion that serine binding eliminates a conformational change subsequent to substrate binding by formation of a dead-end quaternary complex cons...
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role of the anion binding site in catalysis and regulation of mycobacterium tuberculosis d 3 Phosphoglycerate Dehydrogenase
2009Co-Authors: Rodney L Burton, Shawei Chen, Gregory A. GrantAbstract:D-3-Phosphoglycerate Dehydrogenase from M. tuberculosis displays substantial substrate inhibition by its physiological substrate in the direction of NADH oxidation, hydroxypyruvic acid phosphate (HPAP). Previous investigations showed that plots of substrate concentration versus activity derived from steady state assays could be fit with the equation for complete uncompetitive inhibition and that the mechanism may be allosteric. This investigation uses a simulation of transient kinetic data to demonstrate that the mechanism is consistent with the interaction of substrate at a second site called the anion-binding site. While substrate addition at the active site is ordered, with HPAP binding before NADH, NADH can compete with the substrate for binding to the allosteric site and thereby eliminate the substrate inhibition. Fluorescence resonance energy transfer analysis of mutants with specific tryptophan residues converted to phenylalanine residues demonstrates that the main interaction of NADH with the enzyme, in the absence of substrate, is at the allosteric anion-binding site. This is further confirmed by mutations of basic residues at the anion-binding site which also demonstrates that these residues are necessary for inhibition by L-serine when it binds to the regulatory domain. This may indicate that a ligand must be bound to the anion-binding site for L-serine inhibition, providing a potential mechanism for low levels of activity in the presence of high levels of inhibitor.
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the relationship between effector binding and inhibition of activity in d 3 Phosphoglycerate Dehydrogenase
2008Co-Authors: Gregory A. GrantAbstract:The binding of L-serine to Phosphoglycerate Dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, approximately 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of approximately 50% inhibition when only one serine is bound and approximately 80-90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 A apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.
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a stopped flow transient kinetic analysis of substrate binding and catalysis in escherichia coli d 3 Phosphoglycerate Dehydrogenase
2008Co-Authors: Rodney L Burton, Jeremiah Hanes, Gregory A. GrantAbstract:Pre-steady state, stopped flow analysis of Escherichia coli d-3-Phosphoglycerate Dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, α-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD+ reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD+ reduction (3.8 s–1) is similar to the kcat of the enzyme (2–3 s–1) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a VH/VD = 1.07 ± 0.06. None of the other rates determined by stopped flow analysis could account for the kcat of the enzyme in either direction (forward kcat = 0.01 s–1, reverse kcat = 2–3 s–1), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.
Shigeki Furuya - One of the best experts on this subject based on the ideXlab platform.
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d 3 Phosphoglycerate Dehydrogenase from the silkworm bombyx mori identification functional characterization and expression
2021Co-Authors: Kohji Yamamoto, Shinya Mohri, Shigeki FuruyaAbstract:D-3-Phosphoglycerate Dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l-serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin-deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l-serine in the larva of B. mori.
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d-Serine in Glia and Neurons Derives from 3-Phosphoglycerate Dehydrogenase
2013Co-Authors: Jeffrey T. Ehmsen, Ting Martin, Hagit Sason, Dina Rosenberg, Tadashi Ogo, Shigeki Furuya, Solomon H. Snyder, Herman WoloskerAbstract:d-Serine is an endogenous ligand for NMDARs generated from l-serine by the enzyme serine racemase (Srr). Both neuronal and glial localizations have been reported for d-serine and Srr. 3-Phosphoglycerate Dehydrogenase is an exclusively astrocytic enzyme that catalyzes the first committed step of l-serine biosynthesis. Using transgenic mice expressing enhanced green fluorescent protein under the Srr promoter and mice with targeted deletion of Srr or 3-Phosphoglycerate Dehydrogenase, we demonstrate predominantly neuronal sources of d-serine dependent on astrocytic supply of l-serine. These findings clarify the cellular basis for the regulation of NMDAR neurotransmission by d-serine.
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selective upregulation of 3 Phosphoglycerate Dehydrogenase phgdh expression in adult subventricular zone neurogenic niche
2009Co-Authors: Masami O Kinoshita, Yoko Shinoda, Masahiko Watanabe, Kazuhisa Sakai, Tsutomu Hashikawa, Takeo Machida, Yoshio Hirabayashi, Shigeki FuruyaAbstract:In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-Phosphoglycerate Dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.
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Inactivation of the 3-Phosphoglycerate Dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency
2008Co-Authors: Shigeki Furuya, Kazuyuki Yoshida, Norihiro Azuma, Hideyuki Tanaka, Tomoko Sayano, Yuriko Kawakami, Jyung Hoon Yang, Satoru Kuhara, Yoshio HirabayashiAbstract:d -3-Phosphoglycerate Dehydrogenase (Phgdh) is a necessary enzyme for de novo l -serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks.
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mouse 3 Phosphoglycerate Dehydrogenase gene genomic organization chromosomal localization and promoter analysis
2004Co-Authors: Junya Mitoma, Shigeki Furuya, Motohiro Shimizu, Yoko Shinoda, Kazuyuki Yoshida, Norihiro Azuma, Hideyuki TanakaAbstract:Abstract d -3-Phosphoglycerate Dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of l -serine biosynthesis in the phosphorylated pathway. We have recently demonstrated that, in developing and mature brain, expression of Phgdh is highly regulated in a cell lineage-specific manner, mainly in neuroepithelial stem cells, radial glia, and astrocytes (J. Neurosci. 21 (2001) 7691; Arch. Histol. Cytol. 66 (2003) 109). To gain insight into the regulatory mechanism of Phgdh expression, we have isolated a mouse genomic clone that contains the entire mouse Phgdh gene. Structural analysis demonstrated that the Phgdh gene spans approximately 27 kilobases (kb) in length and comprises 12 exons with 11 intervening introns. Using fluorescent in situ hybridization (FISH), we mapped the gene to mouse chromosome 3, region F2–F3. Analysis of a 1.8 kb fragment of the 5′-flanking region showed that the classical TATA-box motif near transcription initiation sites was absent. Instead, a GC-rich proximal region containing a potential Sp1 recognition sequence was present; this region is conserved in mouse, rat, and human counterparts. Transient transfection analysis revealed that the cis -acting elements necessary for basal transcription of Phgdh are contained within the −196/+4 proximal sequence of the promoter, in which the conserved Sp1 recognition sites play an important role for basal promoter activity.
Hideyuki Tanaka - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the 3-Phosphoglycerate Dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency
2008Co-Authors: Shigeki Furuya, Kazuyuki Yoshida, Norihiro Azuma, Hideyuki Tanaka, Tomoko Sayano, Yuriko Kawakami, Jyung Hoon Yang, Satoru Kuhara, Yoshio HirabayashiAbstract:d -3-Phosphoglycerate Dehydrogenase (Phgdh) is a necessary enzyme for de novo l -serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks.
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mouse 3 Phosphoglycerate Dehydrogenase gene genomic organization chromosomal localization and promoter analysis
2004Co-Authors: Junya Mitoma, Shigeki Furuya, Motohiro Shimizu, Yoko Shinoda, Kazuyuki Yoshida, Norihiro Azuma, Hideyuki TanakaAbstract:Abstract d -3-Phosphoglycerate Dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of l -serine biosynthesis in the phosphorylated pathway. We have recently demonstrated that, in developing and mature brain, expression of Phgdh is highly regulated in a cell lineage-specific manner, mainly in neuroepithelial stem cells, radial glia, and astrocytes (J. Neurosci. 21 (2001) 7691; Arch. Histol. Cytol. 66 (2003) 109). To gain insight into the regulatory mechanism of Phgdh expression, we have isolated a mouse genomic clone that contains the entire mouse Phgdh gene. Structural analysis demonstrated that the Phgdh gene spans approximately 27 kilobases (kb) in length and comprises 12 exons with 11 intervening introns. Using fluorescent in situ hybridization (FISH), we mapped the gene to mouse chromosome 3, region F2–F3. Analysis of a 1.8 kb fragment of the 5′-flanking region showed that the classical TATA-box motif near transcription initiation sites was absent. Instead, a GC-rich proximal region containing a potential Sp1 recognition sequence was present; this region is conserved in mouse, rat, and human counterparts. Transient transfection analysis revealed that the cis -acting elements necessary for basal transcription of Phgdh are contained within the −196/+4 proximal sequence of the promoter, in which the conserved Sp1 recognition sites play an important role for basal promoter activity.
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targeted disruption of the mouse 3 Phosphoglycerate Dehydrogenase gene causes severe neurodevelopmental defects and results in embryonic lethality
2004Co-Authors: Kazuyuki Yoshida, Shigeki Furuya, Junya Mitoma, Yoko Shinoda, Norihiro Azuma, Hideyuki Tanaka, Soh Osuka, Masahiko WatanabeAbstract:D-3-Phosphoglycerate Dehydrogenase (Phgdh; EC 1.1.1.95) is the first committed enzyme of L-serine biosynthesis in the phosphorylated pathway. To determine the physiological importance of Phgdh-dependent L-serine biosynthesis in vivo, we generated Phgdh-deficient mice using targeted gene disruption in embryonic stem cells. The absence of Phgdh led to a drastic reduction of L-serine metabolites such as phosphatidyl-L-serine and sphingolipids. Phgdh null embryos have small bodies with abnormalities in selected tissues and died after days post-coitum 13.5. Striking abnormalities were evident in the central nervous system in which the Phgdh null mutation culminated in hypoplasia of the telencephalon, diencephalon, and mesencephalon; in particular, the olfactory bulbs, ganglionic eminence, and cerebellum appeared as indistinct structures. These observations demonstrate that the Phgdh-dependent phosphorylated pathway is essential for normal embryonic development, especially for brain morphogenesis.
J Jaeken - One of the best experts on this subject based on the ideXlab platform.
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3-Phosphoglycerate Dehydrogenase deficiency in a patient with West syndrome
2007Co-Authors: M Pineda, M A Vilaseca, R Artuch, S Santos, Mm García González, I Sau, A Aracil, E Schaftingen, J JaekenAbstract:3-Phosphoglycerate Dehydrogenase deficiency is a severe but treatable disorder of serine synthesis, first described in 1996 (Jaeken et al. 1996a). The patient presented with West syndrome, severe psychomotor delay, failure to thrive, microcephaly, atypical ocular movements, and pyramidal signs. Treatment with oral L-serine abolished seizures and improved psychomotor development, hyperexcitability, head growth, cortical and subcortical hypotrophy, and hypomyelination of the brain on MRI scans. 3–Phosphoglycerate Dehydrogenase deficiency is a treatable congential error that probably leads to West syndrome
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congenital microcephaly and seizures due to 3 Phosphoglycerate Dehydrogenase deficiency outcome of treatment with amino acids
2002Co-Authors: T J De Koning, M Pineda, J Jaeken, L Dorland, L Van Maldergem, M Duran, R Gooskens, B T PolltheAbstract:Congenital microcephaly, intractable seizures and severe psychomotor retardation characterize 3-Phosphoglycerate Dehydrogenase (3-PGDH) deficiency, a disorder of L-serine biosynthesis. The enzyme defect results in low concentrations of serine and to a variable degree of glycine in plasma and cerebrospinal fluid. Short-term beneficial effects have been reported of oral treatment with the deficient amino acids. In this paper, we report the first follow-up data of amino acid therapy in five patients treated for 3-7.5 years. Different treatment regimes were used, but a favourable response to amino acids was observed in all patients. A major reduction in seizure frequency occurred in all patients; two patients became free of seizures. Amino acids were well tolerated and no adverse effects were documented. A progress of psychomotor development was only observed in one patient, diagnosed early and treated with a high dosage of L-serine. A favourable outcome of 3-PGDH deficiency depends on early diagnosis and treatment.
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beneficial effects of l serine and glycine in the management of seizures in 3 Phosphoglycerate Dehydrogenase deficiency
1998Co-Authors: T J De Koning, J Jaeken, L Dorland, R Berger, E Van Schaftingen, M Duran, R Gooskens, Nenad Blau, B T PolltheAbstract:3-Phosphoglycerate Dehydrogenase (3-PGDH) deficiency is an inborn error of serine biosynthesis. Patients are affected with congenital microcephaly, psychomotor retardation, and intractable seizures. The effects of oral treatment with amino acids were investigated in 2 siblings. L-Serine up to 500 mg/kg/day was not sufficient for seizure control. Addition of glycine 200 mg/kg/day resulted in complete disappearance of seizures. Electroencephalographic abnormalities gradually resolved after 6 months. We conclude that 3-PGDH can be treated effectively by a combination of L-serine and glycine.
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3 Phosphoglycerate Dehydrogenase deficiency an inborn error of serine biosynthesis
1996Co-Authors: J Jaeken, Michel Detheux, L Van Maldergem, M Foulon, Hubert Carchon, E Van SchaftingenAbstract:Serine concentrations were markedly decreased in the cerebrospinal fluid of two brothers with congenital microcephaly, profound psychomotor retardation, hypertonia, epilepsy, growth retardation, and hypogonadism. The youngest boy also had congenital bilateral cataract. Magnetic resonance imaging of the brain showed evidence of dysmyelination. Plasma serine as well as plasma and cerebrospinal fluid glycine concentrations were also decreased but to a lesser extent. Treatment with oral serine in the youngest patient significantly increased cerebrospinal fluid serine and abolished the convulsions. In fibroblasts of both patients, a decreased activity was demonstrated of 3-Phosphoglycerate Dehydrogenase, the first step of serine biosynthesis (22% and 13% of the mean control value). This is an unusual disorder as the great majority of aminoacidopathies are catabolic defects. It is a severe but potentially treatable inborn error of metabolism that has not been previously reported in man.
Yoshio Hirabayashi - One of the best experts on this subject based on the ideXlab platform.
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selective upregulation of 3 Phosphoglycerate Dehydrogenase phgdh expression in adult subventricular zone neurogenic niche
2009Co-Authors: Masami O Kinoshita, Yoko Shinoda, Masahiko Watanabe, Kazuhisa Sakai, Tsutomu Hashikawa, Takeo Machida, Yoshio Hirabayashi, Shigeki FuruyaAbstract:In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-Phosphoglycerate Dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.
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Inactivation of the 3-Phosphoglycerate Dehydrogenase gene in mice: changes in gene expression and associated regulatory networks resulting from serine deficiency
2008Co-Authors: Shigeki Furuya, Kazuyuki Yoshida, Norihiro Azuma, Hideyuki Tanaka, Tomoko Sayano, Yuriko Kawakami, Jyung Hoon Yang, Satoru Kuhara, Yoshio HirabayashiAbstract:d -3-Phosphoglycerate Dehydrogenase (Phgdh) is a necessary enzyme for de novo l -serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks.
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functional analysis of mouse 3 Phosphoglycerate Dehydrogenase phgdh gene promoter in developing brain
2004Co-Authors: Motohiro Shimizu, Shigeki Furuya, Junya Mitoma, Yoko Shinoda, Yoshio Hirabayashi, Tadashi Okamura, Ichiro Miyoshi, Noriyuki Kasai, Yasuo SuzukiAbstract:D-3-Phosphoglycerate Dehydrogenase (Phgdh; EC 1.1.1.95) is a necessary enzyme for de novo L-serine biosynthesis via the phosphorylated pathway. Targeted disruption of the mouse Phgdh gene has been shown to result in embryonic lethality, accompanied by severe abnormalities in brain development. Phgdh is expressed exclusively by neuroepithelium and radial glia in developing brain and later mainly by astrocytes. To elucidate the molecular mechanism that regulates such cell-type-specific expression of Phgdh in developing brain, an upstream 3.5-kilobase-pair (kbp) region of the gene harboring the promoter was characterized in primary cultures and transgenic mice. Analysis of Phgdh 5'-nested deletions in transfected cultures indicated that overall reporter luciferase levels were higher in glial cultures than those in neuronal cultures. Although basal promoter activity of the gene appeared to depend on an Sp1 binding sequence residing between -193 and -184 in both glial and neuronal cultures, an upstream 5'-flanking region between -1,794 and -1,095 contributed to up-regulation of Phgdh transcription in a glial-cell-specific manner. In the cerebral cortex of transgenic mouse embryos, the Phgdh promoter-LacZ transgene DNA containing -1,794/+4 promoter sequences directed beta-galactosidase (beta-Gal) expression mainly to Phgdh-positive neuroepithelium and radial glia. This glial preference diminished when beta-Gal expression was driven solely by the upstream 0.2-kbp minimal promoter. However, glial preference of beta-Gal expression was restored by placing the 700-base-pair 5'-DNA segment upstream of the minimal promoter. These observations suggest the presence of cis-acting elements that confer the cell type specificity of Phgdh transcription in the distal promoter region.
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l serine regulates the activities of microglial cells that express very low level of 3 Phosphoglycerate Dehydrogenase an enzyme for l serine biosynthesis
2001Co-Authors: Hiroki Sugishita, Shigeki Furuya, Junya Mitoma, Yoshio Hirabayashi, Yasuhide Kuwabara, Kazuko Toku, Lisa Doi, Lihua Yang, Nobuji Maeda, Masahiro SakanakaAbstract:Microglia are well known to become activated during various kinds of neuropathological events. The factors that are responsible for the activation, however, are not fully determined. In the present study, L-Ser was shown to enhance production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF alpha) by lipopolysaccharide (LPS)-stimulated cultured rat microglial cells. L-Ser, however, did not enhance the expression of mRNAs encoding inducible NO synthase, IL-6 and TNF alpha. On the other hand, astrocytes did not depend on L-Ser for release of IL-6 and TNF alpha. The expression of an enzyme 3-Phosphoglycerate Dehydrogenase (3PGDH), which is essential for L-Ser biosynthesis from a glycolytic intermediate 3-Phosphoglycerate, was investigated. As revealed by Western blotting and immunocytochemical staining, 3PGDH-protein expression in vitro was the highest in astrocytes, intermediate in neurons and the lowest in microglial cells. Semiquantitative RT-PCR showed that microglial cells expressed 3PGDH-mRNA at a lower level than astrocytes. In frozen sections from rat forebrain, only astrocytes were immunoreactive for 3PGDH. The present study suggested that L-Ser is able to modulate microglial function mainly at the translation level because microglial cells cannot synthesize sufficient amount of L-Ser due to the scarce expression of 3PGDH.