249 research outputs found
Molecular Identification of D-Ribulokinase in Budding Yeast and Mammals
No full textpeer reviewedProteomes of even well characterized organisms still contain a high percentage of proteins with unknown or uncertain molecular and/or biological function. A significant fraction of those proteins are predicted to have catalytic properties. Here we aimed at identifying the function of the Saccharomyces cerevisiae Ydr109c protein and of its human homolog FGGY, both of which belong to the broadly conserved FGGY family of carbohydrate kinases. Functionally identified members of this family phosphorylate 3- to 7-carbon sugars or sugar derivatives, but the endogenous substrate of S. cerevisiae Ydr109c and human FGGY has remained unknown. Untargeted metabolomics analysis of an S. cerevisiae deletion mutant of YDR109C revealed ribulose as one of the metabolites with the most significantly changed intracellular concentration as compared to a wild-type strain. In human HEK293 cells, ribulose could only be detected when ribitol was added to the cultivation medium and under this condition, FGGY silencing led to ribulose accumulation. Biochemical characterization of the recombinant purified Ydr109c and FGGY proteins showed a clear substrate preference of both kinases for D-ribulose over a range of other sugars and sugar derivatives tested, including L-ribulose. Detailed sequence and structural analyses of Ydr109c and FGGY as well as homologs thereof furthermore allowed the definition of a 5-residue D-ribulokinase signature motif (TCSLV). The physiological role of the herein identified eukaryotic D-ribulokinase remains unclear, but we speculate that S. cerevisiae Ydr109c and human FGGY could act as metabolite repair enzymes, serving to re-phosphorylate free D-ribulose generated by promiscuous phosphatases from D-ribulose-5-phosphate. In human cells, FGGY can additionally participate in ribitol metabolism.R-STR-4006-00 > Enzymology & Metabolism (Linster) > 01/01/2013 - 19/01/2048 > LINSTER Carol
Metabolite damage and its repair or pre-emption
No full textpeer reviewedIt is increasingly evident that metabolites suffer various kinds of damage, that such damage happens in all organisms, and that cells have dedicated systems for damage repair and containment. Firstly, chemical biology is demonstrating that diverse metabolites are damaged by side-reactions of ‘promiscuous’ enzymes or by spontaneous chemical reactions, that the products are useless or toxic, and that the unchecked buildup of these products can be devastating. Secondly, genetic and genomic evidence from pro- and eukaryotes is implicating a network of novel, conserved enzymes that repair damaged metabolites or somehow pre-empt damage. Metabolite (i.e. small molecule) repair is analogous to macromolecule (DNA and protein) repair and appears from comparative genomic evidence to be equally widespread. Comparative genomics also implies that metabolite repair could be the function of many conserved protein families lacking known activities. How – and how well – cells deal with metabolite damage impacts fields ranging from medical genetics to metabolic engineering
Enzyme complexity in intermediary metabolism.
No full textpeer reviewedA good appraisal of the function of enzymes is essential for the understanding of inborn errors of metabolism. However, it is clear now that the 'one gene, one enzyme, one catalytic function' rule oversimplifies the actual situation. Genes often encode several related proteins, which may differ in their subcellular localisation, regulation or function. Furthermore, enzymes often show several catalytic activities. In some cases, this is because they are multifunctional, possessing two or more different active sites that catalyse different, physiologically related reactions. In enzymes with broad specificity or in multispecificity enzymes, a single type of catalytic site performs the same reaction on different physiological substrates at similar rates. Enzymes that act physiologically in only one reaction often show nonetheless substrate promiscuity: they act at low rates on compounds that resemble their physiological substrate(s), thus forming non-classical metabolites, which are in some cases eliminated by metabolite repair. In addition to their catalytic role, enzymes may have moonlighting functions, i.e. non-catalytic functions that are most often not related with their catalytic activity. Deficiency in such functions may participate in the phenotype of inborn errors of metabolism. Evolution has also made that some enzymes have lost their catalytic activity to become allosteric proteins
L-Ascorbate biosynthesis in higher plants: the role of VTC2
No full textpeer reviewedIn the past year, the last missing enzyme of the L-galactose pathway, the linear form of which appears to represent the major biosynthetic route to L-ascorbate (vitamin C) in higher plants, has been identified as a GDP-L-galactos phosphorylase. This enzyme catalyzes the first committed step in the synthesis of that vital antioxidant and enzyme cofactor. Here, we discuss how GDP-L-galactose phosphorylase enzymes, encoded in Arabidopsis by the paralogous VTC2 and VTC5 genes, function in concert with the other enzymes of the L-galactose pathway to provide plants with the appropriate levels of L-ascorbate. We hypothesize that regulation of L-ascorbate biosynthesis might occur at more than one step and warrants further investigation to allow for the manipulation of vitamin C levels in plants
Vitamin C. Biosynthesis, recycling and degradation in mammals.
No full textpeer reviewedVitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate
New models to study the cross-talk between the protein repair L-isoaspartyl methyltransferase and cell signalling
No full textIsomerization of L-aspartyl and L-asparaginyl residues to form L-isoaspartyl residues in proteins is one type of protein damage that can occur under physiological conditions and can potentially lead to conformational change, loss of function and enhanced protein degradation. Protein L-isoaspartyl methyltransferase (PCMT or PIMT) is a repair enzyme, which allows the reconversion of L-isoaspartyl residues to L-aspartyl residues in protein. Although the catalytic function of PCMT is known, its physiological roles remain less well understood. Pcmt1 gene knockout in mice leads for example, via molecular mechanisms that remain mostly obscure, to activation of insulin/IGF-1 and MAPK signalling pathways in the brain, and premature death due to massive epileptic seizure events.
In this doctoral research project, we have used both mammalian cells and zebrafish models to investigate the impact of PCMT deficiency on insulin/IGF-1, MAPK and calcium signalling as well as how PCMT may be involved in epilepsy. In mammalian cells we used shRNA and CRISPR/Cas9 technology to reduce or completely silence PCMT expression, with the main objective being to mimic, in cell culture, the activation of the IGF-1 and MAPK signalling pathways observed in Pcmt1 knockout mice in the hope to thereby increase the chances to elucidate the underlying molecular mechanisms. In zebrafish we used an antisense morpholino-based strategy to knock down both PCMT homologs and thereby establish a new whole organism model to further study the physiological functions of PCMT, more particularly in the brain.
Our results indicate that insulin/IGF-1 signalling is not affected by PCMT knockdown or knockout in mammalian cells whereas a time-dependent MAPK pathway activation could be detected in a Pcmt1 knockout mouse hippocampal cell line. In zebrafish, we showed that the two PCMT homologs Pcmt and Pcmtl (Pcmt/l) possess isoaspartyl methyltransferase activity. In pcmt/l knockdown (or morphant) zebrafish larvae we did not detect abnormal electrical activity in the brain, but we identified movement impairment and strongly perturbed brain calcium fluxes. Abnormal calcium responses were also observed in the Pcmt1 knockout mouse hippocampal cell line. We concluded that the interplay between PCMT and growth signalling pathways is highly dependent on experimental model and may not be amenable to investigation in cell culture. Importantly, our results clearly show that PCMT plays a pivotal role in calcium signalling and suggest that PCMT-dependent repair mechanisms may be important to prevent calcium-related neurological disorders
Millifluidic culture improves human midbrain organoid vitality and differentiation
No full textpeer reviewe
Metabolite proofreading, a neglected aspect of intermediary metabolism
No full textpeer reviewedEnzymes of intermediary metabolism are less specific than what is usually assumed: they often act on metabolites that are not their 'true' substrate, making abnormal metabolites that may be deleterious if they accumulate. Some of these abnormal metabolites are reconverted to normal metabolites by repair enzymes, which play therefore a role akin to the proofreading activities of DNA polymerases and aminoacyl-tRNA synthetases. An illustrative example of such repair enzymes is L-2-hydroxyglutarate dehydrogenase, which eliminates a metabolite abnormally made by a Krebs cycle enzyme. Mutations in L-2-hydroxyglutarate dehydrogenase lead to L-2-hydroxyglutaric aciduria, a leukoencephalopathy. Other examples are the epimerase and the ATP-dependent dehydratase that repair hydrated forms of NADH and NADPH; ethylmalonyl-CoA decarboxylase, which eliminates an abnormal metabolite formed by acetyl-CoA carboxylase, an enzyme of fatty acid synthesis; L-pipecolate oxidase, which repairs a metabolite formed by a side activity of an enzyme of L-proline biosynthesis. Metabolite proofreading enzymes are likely quite common, but most of them are still unidentified. A defect in these enzymes may account for new metabolic disorders
Mechanism and regulation of the formation of D-glucuronate, the precursor of vitamin C, in rat liver
No full textLe L-ascorbate, un antioxydant et cofacteur enzymatique important chez les animaux et les plantes, est synthétisé par une grande majorité de vertébrés et par toutes les espèces de plantes analysées jusqu'à ce jour. L'enzyme catalysant l'étape finale de la voie de biosynthèse de l'ascorbate est toutefois absente chez les primates (y compris les humains) et le cobaye, et pour eux ce composé est donc devenu une vitamine (vitamine C). Une des premières étapes de la voie de biosynthèse consiste en la réduction du D-glucuronate, le précurseur de la vitamine C chez les animaux, en L-gulonate. Ce dernier peut être converti en ascorbate par lactonisation et oxydation, mais peut également entrer dans la voie des pentoses dans laquelle il est successivement oxydé et décarboxylé en L-xylulose. La voie des pentoses est opérationnelle chez tous les mammifères et son fonctionnement chez l'homme est démontré par le fait qu'une déficience en L-xylulose réductase mène à une excrétion urinaire anormalement élevée de L-xylulose (pentosurie). Il est connu depuis longtemps que l'administration d'une série de médicaments tels que l'aminopyrine, le barbital et la chlorétone, augmente la formation de vitamine C chez les animaux et l'excrétion de L-xylulose chez les sujets pentosuriques. L'objectif de cette thèse était d'élucider le mécanisme à la base de ces effets stimulateurs. L'observation que certains xénobiotiques stimulent à la fois l'excrétion de vitamine C et de L-xylulose indique qu'ils augmentent la formation d'un précurseur commun des voies métaboliques impliquées. Ceci nous a amenés à mettre au point une méthode de dosage spectrophotométrique pour le D-glucuronate basée sur l'utilisation de deux enzymes bactériennes, la mannonate déshydrogénase et l'uronate isomérase. Ces enzymes ont été surepxrimées chez E. coli, purifiées à quasi-homogénéité et partiellement caractérisées. Nous avons observé que l'uronate isomérase est inhibée par l'EDTA et stimulée par le Zn2+, le Co2+ et le Mn2+. La méthode de dosage enzymatique développée est spécifique et sensible, et peut également être utilisée pour mesurer les concentrations de ?-glucuronides et de glucuronate 1-phosphate. A l'aide de ce dosage, nous avons pu montrer que certains agents (par exemple l'aminopyrine, l'antipyrine et la chlorétone) connus pour stimuler la formation de vitamine C in vivo, mais également d'autres composés (métyrapone, proadifen, clotrimazole) augmentent de plusieurs fois, et en moins de 10 minutes, la formation de glucuronate dans des hépatocytes isolés de rat. Cet effet était considérablement amplifié en présence de sorbinil, un inhibiteur de la glucuronate réductase. L'augmentation de la formation de glucuronate était accompagnée par une augmentation de la formation de vitamine C et par une diminution d'environ deux fois de la concentration en UDP-glucuronate. Les agents stimulateurs ne donnaient généralement pas lieu à la formation de quantités détectables de ?-glucuronides et n'avaient pas d'effet sur la concentration de glutathion. D'un autre côté, le résorcinol, qui est un bon substrat de la glucuronoconjugaison, et des agents diminuant la concentration de glutathion (diamide, L-buthionine sulfoximine) ne stimulent pas la formation de glucuronate. Ces résultats indiquent que l'effet stimulateur des xénobiotiques sur la formation de vitamine C est médié par une augmentation rapide de la conversion d'UDP-glucuronate en glucuronate libre, n'impliquant apparemment pas un cycle de glucuronoconjugaison-déconjugaison ou une déplétion en glutathion. Nous avons ensuite voulu reproduire les effets de l'aminopyrine, de la métyrapone et de la chlorétone sur la formation de glucuronate dans un système acellulaire. Les trois composés stimulent la formation de glucuronate à partir d'UDP-glucuronate dans des extraits de foie à condition que ceux-ci soient enrichis en ATP-Mg. La formation et l'hydrolyse du glucuronate 1-phosphate ne sont cependant pas affectées par la métyrapone et les autres agents. Ceci indique que, contrairement à ce qui est généralement admis, le glucuronate 1-phosphate n'est pas un intermédiaire dans la formation de glucuronate à partir d'UDP-glucuronate et suggère que cette réaction consiste en une hydrolyse directe de l'UDP-glucuronate en UDP et glucuronate par une "UDP-glucuronidase". Cette activité enzymatique possède plusieurs propriétés similaires à celle catalysée par les UDP-glucuronosyltransférases (association avec la fraction microsomiale, stimulation par l'UDP-N-acétylglucosamine, sensibilité aux détergents) et l'observation qu'elle est inhibée par des substrats de la glucuronoconjugaison ...L-Ascorbate, an important antioxidant and enzyme cofactor in animals and plants, is synthesized by a great majority of vertebrates and by all plant species examined so far. Primates, including humans, and guinea pigs do not form ascorbate because they lack the enzyme catalyzing the final step of the ascorbate biosynthesis pathway, and for them ascorbate has thus become a vitamin (vitamin C). An early step of the biosynthetic pathway consists of the reduction of D-glucuronate, the precursor of vitamin C in animals, to L-gulonate. The latter can be converted to ascorbate through lactonization and oxidation, but can also enter the pentose pathway by which it is successively oxidized and decarboxylated to L-xylulose. The pentose pathway is operational in all mammals and its occurrence in man is demonstrated by the fact that a deficiency in L-xylulose reductase leads to urinary excretion of abnormal amounts of L-xylulose (pentosuria). It has been known for a long time that the administration of a series of drugs including aminopyrine, barbital and chloretone, increases the formation of vitamin C in animals and the excretion of L-xylulose in pentosuric subjects. The aim of this thesis was to elucidate the mechanism underlying the stimulatory effects observed. The finding that some xenobiotics stimulate the excretion of both vitamin C and L-xylulose indicates that they increase the formation of a common precursor of the metabolic pathways involved. This prompted us to set up a spectrophotometric assay for D-glucuronate based on the use of two bacterial enzymes, mannonate dehydrogenase and uronate isomerase. These enzymes have been overexpressed in E. coli, purified to near-homogeneity and partially characterized. Uronate isomerase was found to be inhibited by EDTA and stimulated by Zn2+, Co2+ and Mn2+. The enzymatic assay developed is specific and sensitive, and can also be used to determine the concentration of ?-glucuronides and glucuronate 1-phosphate. Using this assay, we showed that agents (e.g. aminopyrine, antipyrine and chloretone) known to stimulate vitamin C formation in vivo, but also some other compounds (metyrapone, proadifen, clotrimazole) induced in less than 10 minutes a several-fold increase in the formation of glucuronate in isolated rat hepatocytes. This effect was considerably amplified by the presence of sorbinil, an inhibitor of glucuronate reductase. The increase in glucuronate formation was accompanied by an increase in the formation of vitamin C and an approximate 2-fold decrease in the concentration of UDP-glucuronate. The stimulating compounds did not generally give rise to the formation of detectable amounts of ?-glucuronides and exerted no effect on glutathione levels. On the other hand, resorcinol, a good substrate of glucuronidation, and glutathione-depleting agents (diamide, L-buthionine sulfoximine) did not stimulate the formation of glucuronate. These observations indicated that the stimulatory effect of some xenobiotics on vitamin C formation is mediated by a rapid increase in the conversion of UDP-glucuronate to free glucuronate, which does apparently not involve a glucuronidation-deglucuronidation cycle or glutathione depletion. We then wanted to reproduce the effects of aminopyrine, metyrapone and chloretone on the formation of glucuronate in a cell-free system. The three compounds stimulated glucuronate formation from UDP-glucuronate in liver extracts provided these were enriched with ATP-Mg. The formation and hydrolysis of glucuronate 1-phosphate were, however, unaffected by metyrapone and the other agents. This indicated that, unlike as generally believed, glucuronate 1-phosphate is not an intermediate in the formation of glucuronate from UDP-glucuronate and suggested that this reaction consists of direct hydrolysis of UDP-glucuronate to UDP and glucuronate by a UDP-glucuronidase. This enzymatic activity shared several properties with that catalyzed by UDP-glucuronosyltransferases (association with the microsomal fraction, stimulation by UDP-N-acetylglucosamine, sensitivity to detergents) and the observation that it was inhibited by substrates of glucuronidation indicated that it could in fact be contributed by one or several isoform(s) of the UDP-glucuronosyltransferase superfamily. Inhibitors of ?-glucuronidase and esterases had no effect on glucuronate formation, further arguing against involvement of a glucuronidation-deglucuronidation cycle. The stimulatory effect of metyrapone and other agents was lost in washed microsomes, but could be restored by adding a heated liver high-speed supernatant or CoASH. In conclusion, our results indicate that the increase in vitamin C formation in animals and of L-xylulose excretion in pentosuric subjects induced by several xenobiotics is mediated by a rapid stimulatory effect of these agents on UDP-glucuronidase, an enzyme closely related to UDP-glucuronosyltransferases. The presence of ATP-Mg and CoASH, which together inhibit UDP-glucuronidase activity, is necessary to observe the stimulatory effect of xenobiotics in liver microsomes.Thèse de doctorat en sciences biomédicales (biochimie et biologie cellulaire) (BCHM)-- UCL, 200
A tradução da comédia teatral em The Importance of Being Earnest: tradução comentada e anotada
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Comunicação e Expressão. Programa de Pós-graduação em Estudos da TraduçãoEsta dissertação consiste em uma discussão sobre a tradução teatral de textos de humor seguida da tradução comentada e anotada de The Importance of Being Earnest, de Oscar Wilde. No primeiro capítulo, são discutidas questões a respeito da tradução teatral, partindo-se do conceito de concretizações textuais de Patrice Pavis, como as noções de speakability e performance. No segundo capítulo, é feito um estudo sobre duas outras traduções da peça para o português brasileiro, analisando-se as escolhas feitas pelos tradutores bem como a evolução da tradução dessa peça ao longo da segunda metade do século vinte. O trabalho culmina com a discussão de questões pertinentes a minha tradução da peça, onde são tecidos os comentários pertinentes às escolhas ou mudanças realizadas na tradução apresentada: as questões dos nomes próprios, dos pronomes de tratamento e do humor. A nova tradução, que apresenta uma cena inédita ao público de língua portuguesa, e suas notas, encontra-se no apêndice deste trabalho. This dissertation consists of a discussion on theatrical translation of humoristic texts followed by a noted and commented translation of Oscar Wilde#s The Importance of Being Earnest. In the first chapter, questions related to theatrical translation are discussed starting from Patrice Pavis#s concepts of textual concretizations, such as the notions of speakability and performance. In the second chapter, there are analyses of two other translations of the play into Brazilian Portuguese focusing upon the translator#s choices as well as the evolution of the play#s translation through the second half of the twentieth century. This dissertation ends with a discussion on questions related to my translation of the play. In this final chapter, we can find commentaries concerning the choices and changes made in the translation presented, such as the question of the names of the characters, the personal pronouns, addresses and humour. The retranslation, which presents a new scene to Brazilian audiences, and the notes are in the appendix of this dissertation
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