RESUMEN
Selenoprotein I (selenoi) is a unique selenocysteine (Sec)-containing protein widely expressed throughout the body. Selenoi belongs to two different protein families: the selenoproteins that are characterized by a redox reactive Sec residue and the lipid phosphotransferases that contain the highly conserved cytidine diphosphate (CDP)-alcohol phosphotransferase motif. Selenoi catalyzes the third reaction of the CDP-ethanolamine branch of the Kennedy pathway within the endoplasmic reticulum membrane. This is not a redox reaction and does not directly involve the Sec residue, making selenoi quite distinct among selenoproteins. Selenoi is also unique among lipid phosphotransferases as the only family member containing a Sec residue near its C-terminus that serves an unknown function. The reaction catalyzed by selenoi involves the transfer of the ethanolamine phosphate group from CDP-ethanolamine to one of two lipid donors, 1,2-diacylglycerol (DAG) or 1-alkyl-2-acylglycerol (AAG), to produce PE or plasmanyl PE, respectively. Plasmanyl PE is subsequently converted to plasmenyl PE by plasmanylethanolamine desaturase. Both PE and plasmenyl PE are critical phospholipids in the central nervous system (CNS), as demonstrated through clinical studies involving SELENOI mutations as well as studies in cell lines and mice. Deletion of SELENOI in mice is embryonic lethal, while loss-of-function mutations in the human SELENOI gene have been found in rare cases leading to a form of hereditary spastic paraplegia (HSP). HSP is an upper motor disease characterized by spasticity of the lower limbs, which is often manifested with other symptoms such as impaired vision/hearing, ataxia, cognitive/intellectual impairment, and seizures. This article will summarize the current understanding of selenoi as a metabolic enzyme and discuss its role in the CNS physiology and pathophysiology.
Asunto(s)
Fosfolípidos , Selenocisteína , Animales , Sistema Nervioso Central/metabolismo , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Etanolaminas/metabolismo , Humanos , Ratones , Fosfolípidos/metabolismo , Fosfotransferasas , Selenoproteínas/metabolismoRESUMEN
The major glycerophospholipid phosphatidylethanolamine (PE) in the nervous system is essential for neural development and function. There are two major PE synthesis pathways, the CDP-ethanolamine pathway in the endoplasmic reticulum (ER) and the phosphatidylserine decarboxylase (PSD) pathway in mitochondria. However, the role played by mitochondrial PE synthesis in maintaining cellular PE homeostasis is unknown. Here, we show that Drosophila pect (phosphoethanolamine cytidylyltransferase) mutants lacking the CDP-ethanolamine pathway, exhibited alterations in phospholipid composition, defective phototransduction, and retinal degeneration. Induction of the PSD pathway fully restored levels and composition of cellular PE, thus rescued the retinal degeneration and defective visual responses in pect mutants. Disrupting lipid exchange between mitochondria and ER blocked the ability of PSD to rescue pect mutant phenotypes. These findings provide direct evidence that the synthesis of PE in mitochondria contributes to cellular PE homeostasis, and suggest the induction of mitochondrial PE synthesis as a promising therapeutic approach for disorders associated with PE deficiency.
Asunto(s)
Carboxiliasas/genética , Citidina Difosfato/análogos & derivados , Retículo Endoplásmico/genética , Degeneración Retiniana/genética , Animales , Carboxiliasas/metabolismo , Citidina Difosfato/deficiencia , Citidina Difosfato/genética , Citidina Difosfato/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Retículo Endoplásmico/metabolismo , Etanolaminas/metabolismo , Homeostasis/genética , Humanos , Metabolismo de los Lípidos/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Fosfatidilserinas/genética , Fosfatidilserinas/metabolismo , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patología , Transducción de Señal/genéticaRESUMEN
Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05â Å resolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.
Asunto(s)
Liasas de Carbono-Oxígeno/química , Dominio Catalítico , Cristalografía por Rayos X , Citidina Difosfato/análogos & derivados , Citidina Difosfato/química , Etanolaminas/química , Humanos , Modelos Moleculares , Estructura Cuaternaria de Proteína , Fosfato de Piridoxal/químicaRESUMEN
The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.
Asunto(s)
Citidina Difosfato Colina/metabolismo , Citidina Difosfato/análogos & derivados , Etanolaminas/metabolismo , Animales , Colina Quinasa/química , Colina Quinasa/genética , Citidililtransferasa de Colina-Fosfato/química , Citidililtransferasa de Colina-Fosfato/genética , Citidina Difosfato/metabolismo , Estudios de Asociación Genética , Humanos , Distrofias Musculares/congénito , Distrofias Musculares/genética , Distrofias Musculares/patología , Osteocondrodisplasias/congénito , Osteocondrodisplasias/genética , Osteocondrodisplasias/patología , Polimorfismo de Nucleótido SimpleRESUMEN
Metabolic reprogramming has emerged as a key regulator of cell fate decisions. Roles of glucose and amino acid metabolism have been extensively documented, whereas lipid metabolism in pluripotency remains largely unexplored. Using a high-coverage lipidomics approach, we reveal dynamic changes in phospholipids occurring during reprogramming and show that the CDP-ethanolamine (CDP-Etn) pathway for phosphatidylethanolamine (PE) synthesis is required at the early stage of reprogramming. Mechanistically, the CDP-Etn pathway inhibits NF-κB signaling and mesenchymal genes in a Pebp1-dependent manner, without affecting autophagy, resulting in accelerated mesenchymal-to-epithelial transition (MET) and enhanced reprogramming. Furthermore, PE binding to Pebp1 enhances the interaction of Pebp1 with IKKα/ß and reduces the phosphorylation of IKKα/ß. The CDP-Etn-Pebp1 axis is associated with EMT/MET in hepatocyte differentiation, indicating that Etn/PE is a broad-spectrum MET/EMT-regulating metabolite. Collectively, our study reveals an unforeseen connection between phospholipids, cell migration, and pluripotency and highlights the importance of phospholipids in cell fate transitions.
Asunto(s)
Diferenciación Celular , Transición Epitelial-Mesenquimal , Hepatocitos/metabolismo , Fosfatidiletanolaminas/metabolismo , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Animales , Línea Celular , Movimiento Celular , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Etanolaminas/metabolismo , Hepatocitos/citología , Quinasa I-kappa B/metabolismo , Ratones , FN-kappa B/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Probing ligand-target protein interactions provides essential information for deep understanding of biochemical machinery and design of drug screening assays. Native electrospray ionization-mass spectrometry (ESI-MS) is promising for direct analysis of ligand-protein complexes. However, it lacks the ability to distinguish between specific and non-specific ligand-protein interactions, and to further recognize the specifically bound proteins as drug target candidates, which remains as a major challenge in the field of drug developments by far. Herein we report a native-denatured exchange (NDX) mass spectrometry (MS) acquisition approach using a liquid sample-desorption electrospray ionization (LS-DESI) setup, and demonstrate its capability in enabling a change from native detection of noncovalent ligand-protein complexes to denatured analysis using three model ligand-protein complexes including myoglobin, CDP-ribonuclease and N,N',Nâ³-triacetylchitotriose (NAG3)-lysozyme. Notably, we found the NDX-MS approach can readily discriminate specific ligand-protein interactions from nonspecific ones, as revealed by their distinct dynamic profiles of Kd as a function of the DESI spraying flow rate. Consequently, this NDX-MS approach holds promise for future applications to discovering specific protein targets for ligands of interest, and to screening compounds with high specificity to drug targets and thus eliminates off-target effects.
Asunto(s)
Citidina Difosfato/química , Muramidasa/química , Ribonucleasas/química , Trisacáridos/química , Citidina Difosfato/análogos & derivados , Ligandos , Muramidasa/metabolismo , Ribonucleasas/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki =(3.0±0.1)â µm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.
Asunto(s)
Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Citidina Trifosfato/análogos & derivados , Inhibidores Enzimáticos/farmacología , Ligasas de Carbono-Nitrógeno/metabolismo , Citidina Difosfato/análogos & derivados , Citidina Trifosfato/química , Citidina Trifosfato/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Phosphatidylethanolamine (PE) is the second most abundant glycerophospholipid in eukaryotic cells. The existence of four only partially redundant biochemical pathways that produce PE, highlights the importance of this essential phospholipid. The CDP-ethanolamine and phosphatidylserine decarboxylase pathways occur in different subcellular compartments and are the main sources of PE in cells. Mammalian development fails upon ablation of either pathway. Once made, PE has diverse cellular functions that include serving as a precursor for phosphatidylcholine and a substrate for important posttranslational modifications, influencing membrane topology, and promoting cell and organelle membrane fusion, oxidative phosphorylation, mitochondrial biogenesis, and autophagy. The importance of PE metabolism in mammalian health has recently emerged following its association with Alzheimer's disease, Parkinson's disease, nonalcoholic liver disease, and the virulence of certain pathogenic organisms.
Asunto(s)
Fosfatidiletanolaminas/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Autofagia , Candida , Carboxiliasas/metabolismo , Membrana Celular/metabolismo , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Etanolaminas/metabolismo , Humanos , Metabolismo de los Lípidos , Metilación , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosforilación Oxidativa , Enfermedad de Parkinson/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , Priones/metabolismo , Procesamiento Proteico-Postraduccional , VirulenciaRESUMEN
Accumulation of diacylglycerol (DG) in muscle is thought to cause insulin resistance. DG is a precursor for phospholipids, thus phospholipid synthesis could be involved in regulating muscle DG. Little is known about the interaction between phospholipid and DG in muscle; therefore, we examined whether disrupting muscle phospholipid synthesis, specifically phosphatidylethanolamine (PtdEtn), would influence muscle DG content and insulin sensitivity. Muscle PtdEtn synthesis was disrupted by deleting CTP:phosphoethanolamine cytidylyltransferase (ECT), the rate-limiting enzyme in the CDP-ethanolamine pathway, a major route for PtdEtn production. While PtdEtn was reduced in muscle-specific ECT knockout mice, intramyocellular and membrane-associated DG was markedly increased. Importantly, however, this was not associated with insulin resistance. Unexpectedly, mitochondrial biogenesis and muscle oxidative capacity were increased in muscle-specific ECT knockout mice and were accompanied by enhanced exercise performance. These findings highlight the importance of the CDP-ethanolamine pathway in regulating muscle DG content and challenge the DG-induced insulin resistance hypothesis.
Asunto(s)
Citidina Difosfato/análogos & derivados , Diglicéridos/metabolismo , Etanolaminas/metabolismo , Resistencia a la Insulina , Músculo Esquelético/metabolismo , Biogénesis de Organelos , Animales , Citidina Difosfato/metabolismo , Glucosa/metabolismo , Metabolismo de los Lípidos , Ratones , Ratones Noqueados , Obesidad/genética , Obesidad/metabolismo , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismoAsunto(s)
Anemia Hemolítica Congénita/diagnóstico , Citidina Difosfato Colina/sangre , Citidina Difosfato/análogos & derivados , Etanolaminas/sangre , Adenosina Desaminasa/sangre , Adenilato Quinasa/sangre , Anciano , Anemia Hemolítica Congénita/sangre , Anemia Hemolítica Congénita/patología , Enfermedad Crónica , Citidina Difosfato/sangre , Femenino , Glucosafosfato Deshidrogenasa/sangre , Glutatión Peroxidasa/sangre , Glutatión Reductasa/sangre , HumanosRESUMEN
Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (â¼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.
Asunto(s)
Carboxiliasas/metabolismo , Mitocondrias/metabolismo , Fosfatidiletanolaminas/biosíntesis , Proteínas Protozoarias/metabolismo , Toxoplasma/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Carboxiliasas/clasificación , Carboxiliasas/genética , Supervivencia Celular , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Diglicéridos/metabolismo , Etanolaminas/metabolismo , Datos de Secuencia Molecular , Filogenia , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4ß4 in Escherichia coli , wherein ß is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and ß2 dimeric motifs within the respective α6 or α4ß4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -ß to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or ß2 associations in the dATP-bound α6 or α4ß4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.
Asunto(s)
Regulación Alostérica/fisiología , Ribonucleótido Reductasas/metabolismo , Bioingeniería , Dominio Catalítico , Citidina Difosfato/análogos & derivados , Citidina Difosfato/farmacología , Nucleótidos de Desoxiadenina/metabolismo , Desoxirribonucleótidos , Escherichia coli/enzimología , Humanos , Multimerización de Proteína , Proteínas Recombinantes/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidoresRESUMEN
Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.
Asunto(s)
Escherichia coli/enzimología , Mannheimia haemolytica/enzimología , Neisseria meningitidis Serogrupo B/enzimología , Sialiltransferasas/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Electroforesis Capilar , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Fetuínas/metabolismo , Genoma Bacteriano/genética , Concentración de Iones de Hidrógeno , Cinética , Mannheimia haemolytica/genética , Células PC12 , Ratas , Proteínas Recombinantes/metabolismo , Ácidos Siálicos/metabolismo , Solubilidad , Temperatura , Factores de TiempoRESUMEN
Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
Asunto(s)
Membrana Celular/metabolismo , Citidina Difosfato/análogos & derivados , Etanolaminas/metabolismo , Mitocondrias/metabolismo , Fosfatidiletanolaminas/biosíntesis , Fosfatidilserinas/biosíntesis , Animales , Ácidos Araquidónicos/metabolismo , Transporte Biológico , CDPdiacilglicerol-Serina O-Fosfatidiltransferasa/metabolismo , Carboxiliasas/metabolismo , Citidina Difosfato/metabolismo , Descarboxilación , Endocannabinoides/metabolismo , Retículo Endoplásmico/metabolismo , Metilación , Ratones , Ratones Noqueados , Fosfatidilcolinas/metabolismo , Alcamidas Poliinsaturadas/metabolismoRESUMEN
Ribonucleotide reductases (RNRs) provide the precursors for DNA biosynthesis and repair and are successful targets for anticancer drugs such as clofarabine and gemcitabine. Recently, we reported that dATP inhibits E. coli class Ia RNR by driving formation of RNR subunits into α4ß4 rings. Here, we present the first X-ray structure of a gemcitabine-inhibited E. coli RNR and show that the previously described α4ß4 rings can interlock to form an unprecedented (α4ß4)2 megacomplex. This complex is also seen in a higher-resolution dATP-inhibited RNR structure presented here, which employs a distinct crystal lattice from that observed in the gemcitabine-inhibited case. With few reported examples of protein catenanes, we use data from small-angle X-ray scattering and electron microscopy to both understand the solution conditions that contribute to concatenation in RNRs as well as present a mechanism for the formation of these unusual structures.
Asunto(s)
Proteínas de Escherichia coli/química , Exorribonucleasas/química , Cristalografía por Rayos X , Citidina Difosfato/análogos & derivados , Citidina Difosfato/química , Nucleótidos de Desoxiadenina/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/ultraestructura , Exorribonucleasas/antagonistas & inhibidores , Exorribonucleasas/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Sialyloligosaccharides are synthesised by various glycosyltransferases and sugar nucleotides. All of these nucleotides are diphosphate compounds except for cytidine-5'-monophosphosialic acid (CMP-Neu5Ac). To obtain an insight into why cytidine-5'-diphosphosialic acid (CDP-Neu5Ac) has not been used for the sialyltransferase reaction and why it is not found in biological organisms, the compound was synthesised. This synthesis provided the interesting finding that the carboxylic acid moiety of the sialic acid attacks the attached phosphate group. This interaction yields an activated anhydride between carboxylic acid and the phosphate group and leads to hydrolysis of the pyrophosphate linkage. The mechanism was demonstrated by stable isotope-labelling experiments. This finding suggested that CMP-Neu5Ac might also form the corresponding anhydride structure between carboxylic acid and phosphate, and this seems to be the reason why CMP-Neu5Ac is acid labile in relation to other sugar nucleotides. To confirm the role of the carboxylic acid, CMP-Neu5Ac derivatives in which the carboxylic acid moiety in the sialic acid was substituted with amide or ester groups were synthesised. These analogues clearly exhibited resistance to acid hydrolysis. This result indicated that the carboxylic acid of Neu5Ac is associated with its stability in solution. This finding also enabled the development of a novel chemical synthetic method for CMP-Neu5Ac and CMP-sialic acid derivatives.
Asunto(s)
Citidina Difosfato/análogos & derivados , Ácido N-Acetilneuramínico Citidina Monofosfato/síntesis química , Citidina Monofosfato/análogos & derivados , Ácidos Siálicos/síntesis química , Citidina Difosfato/síntesis química , Citidina Difosfato/química , Citidina Monofosfato/síntesis química , Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Estructura Molecular , Ácidos Siálicos/química , EstereoisomerismoRESUMEN
A novel enzyme, induced by choline, ethanolamine, glycine betaine or dimethylglycine, was released at low temperature and phosphate from Pseudomonas fluorescens (CECT 7229) suspensions at low cell densities. It is a CDP-ethanolamine pyrophosphatase/(dihexanoyl)glycerophosphoethanolamine phosphodiesterase (CGDEase) less active on choline derivatives, and inactive on long-chain phospholipids, CDP-glycerol and other NDP-X compounds. The reaction pattern was typical of phospholipase C (PLC), as either phosphoethanolamine or phosphocholine was produced. Peptide-mass analyses, gene cloning and expression provided a molecular identity for CGDEase. Bioinformatic studies assigned it to the PLC branch of the phospholipase C/acid phosphatase (PLC/APase) superfamily, revealed an irregular phylogenetic distribution of close CGDEase relatives, and suggested their genes are not in operons or conserved contexts. A theoretical CGDEase structure was supported by mutagenesis of two predicted active-site residues, which yielded essentially inactive mutants. Biological relevance is supported by comparisons with CGDEase relatives, induction by osmoprotectants (not by osmotic stress itself) and repression by micromolar phosphate. The low bacterial density requirement was related to phosphate liberation from lysed bacteria in denser populations, rather than to a classical quorum-sensing effect. The results fit better a CGDEase role in phosphate scavenging than in osmoprotection.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Fosfatos/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Pseudomonas fluorescens/enzimología , Pirofosfatasas/metabolismo , Dominio Catalítico , Citidina Difosfato/análogos & derivados , Citidina Difosfato/metabolismo , Etanolaminas/metabolismo , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Familia de Multigenes , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/genética , Pseudomonas fluorescens/química , Pseudomonas fluorescens/genética , Pirofosfatasas/química , Pirofosfatasas/genética , Especificidad por SustratoRESUMEN
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside 5'-diphosphates to the corresponding deoxynucleotides supplying the dNTPs required for DNA replication and DNA repair. Class I RNRs require two subunits, alpha and beta, for activity. Humans possess two beta subunits: one involved in S phase DNA replication (beta) and a second in mitochondrial DNA replication (beta' or p53R2) and potentially DNA repair. Gemcitabine (F(2)C) is used clinically as an anticancer agent, and its phosphorylated metabolites target many enzymes involved in nucleotide metabolism, including RNR. The present investigation with alpha (specific activity of 400 nmol min(-1) mg(-1)) and beta' (0.6 Y./beta'2 and a specific activity of 420 nmol min(-1) mg(-1)) establishes that F(2)CDP is a substoichiometric inactivator of RNR. Incubation of this alpha/beta' with [1'-(3)H]-F(2)CDP or [5-(3)H]-F(2)CDP and reisolation of the protein by Sephadex G-50 chromatography resulted in recovery 0.5 equiv of covalently bound sugar and 0.03 equiv of tightly associated cytosine to alpha2. SDS-PAGE analysis (loaded without boiling) of the inactivated RNR showed that 60% of alpha migrates as a 90 kDa protein and 40% as a 120 kDa protein. Incubation of [1'-(3)H]-F(2)CDP with active site mutants C444S/A, C218S/A, and E431Q/D-alpha and the C-terminal tail C787S/A and C790S/A mutants reveals that no sugar label is bound to the active site mutants of alpha and that, in the case of C218S-alpha, alpha migrates as a 90 kDa protein. Analysis of the inactivated wt-alpha/beta' RNR by size exclusion chromatography indicates a quaternary structure of alpha6beta'6. A mechanism of inactivation common with halpha/beta is presented.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Citidina Difosfato/análogos & derivados , Inhibidores Enzimáticos/toxicidad , Ribonucleótido Reductasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/aislamiento & purificación , Cromatografía en Gel , Citidina Difosfato/química , Citidina Difosfato/toxicidad , Daño del ADN/genética , Reparación del ADN/genética , Inhibidores Enzimáticos/química , Humanos , Mutagénesis Sitio-Dirigida , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/genética , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/aislamiento & purificación , Ribonucleótido Reductasas/metabolismo , Ribonucleótido Reductasas/fisiologíaRESUMEN
Gemcitabine 5'-diphosphate (F(2)CDP) is a potent inhibitor of ribonucleotide reductases (RNRs), enzymes that convert nucleotides (NDPs) to deoxynucleotides and are essential for DNA replication and repair. The Escherichia coli RNR, an alpha2beta2 complex, when incubated with 1 equiv of F(2)CDP catalyzes the release of two fluorides and cytosine concomitant with enzyme inactivation. In the presence of reductant (thioredoxin/thioredoxin reductase/NADPH or DTT), the enzyme inactivation results from its covalent labeling of alpha with the sugar of F(2)CDP (one label/alpha2beta2). SDS-PAGE analysis of the inactivated RNR without boiling of the sample reveals that alpha migrates as an 87 and 110 kDa protein in a ratio of 0.6:0.4. When the reductant is omitted, RNR is inactivated by loss of the essential tyrosyl radical and formation of a new radical. Inactivation studies with C225S-alpha in the presence or absence of reductants, reveal it behaves like wt-RNR in the absence of reductant. Inactivated C225S-alpha migrates as an 87 kDa protein and is not covalently modified. C225 is one of the cysteines in RNR's active site that supplies reducing equivalents to make dNDPs. To identify the new radical formed, [1'-(2)H]-F(2)CDP was studied with wt- and C225S-RNR by 9 and 140 GHz EPR spectroscopy. These studies revealed that the new radical is a nucleotide derived with g values of g(x) 2.00738, g(y) 2.00592, and g(z) 2.00230 and with altered hyperfine interactions (apparent triplet collapsed to a doublet) relative to [1'-(1)H]-F(2)CDP. The EPR features are very similar to those we recently reported for the nucleotide radical generated with CDP and E441Q-RNR.
Asunto(s)
Citidina Difosfato/análogos & derivados , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Colesterol/fisiología , Citidina Difosfato/toxicidad , Inhibidores Enzimáticos/metabolismo , Oxidación-Reducción , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiología , PorcinosRESUMEN
Phosphatidylethanolamine (PE) is an important inner membrane phospholipid mostly synthesized de novo via the PE-Kennedy pathway and by the decarboxylation of phosphatidylserine. CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the formation of CDP-ethanolamine, which is often the rate regulatory step in the PE-Kennedy pathway. In the current investigation, we show that the reduced CDP-ethanolamine formation in Pcyt2(+/-) mice limits the rate of PE synthesis and increases the availability of diacylglycerol. This results in the increased formation of triglycerides, which is facilitated by stimulated de novo fatty acid synthesis and increased uptake of pre-existing fatty acids. Pcyt2(+/-) mice progressively accumulate more diacylglycerol and triglycerides with age and have modified fatty acid composition, predominantly in PE and triglycerides. Pcyt2(+/-) additionally have an inherent blockage in fatty acid utilization as energy substrate and develop impaired tolerance to glucose and insulin at an older age. Accordingly, gene expression analyses demonstrated the up-regulation of the main lipogenic genes and down-regulation of mitochondrial fatty acid beta-oxidation genes. These data demonstrate for the first time that to preserve membrane PE phospholipids, Pcyt2 deficiency generates compensatory changes in triglyceride and energy substrate metabolism, resulting in a progressive development of liver steatosis, hypertriglyceridemia, obesity, and insulin resistance, the main features of the metabolic syndrome.