Dissertations / Theses on the topic 'UDP-glucose pyrophosphorylase'

UDP-glucose pyrophosphorylase (UGPase) is a key component of carbohydrate production in plants, especially with respect to sucrose synthesis/ metabolism, by producing UDP-glucose, a key precursor to sucrose and to many polysaccharides in cell walls. UDP-glucose is also utilized in the synthesis of carbohydrate moiety of glycolipids, glycoproteins and a variety of secondary metabolites, among other functions. The UGPase enzyme may have a rate-limiting function in sugar biosynthesis, and its activity is now known to increase upon variety of abiotic stresses, with possible effects on an overall carbohydrate budget in stressed plants. The enzyme has been proposed to be regulated by (de)oligomerization and it has been estabished that only monomeric form of the enzyme is active. Based on mutant studies, the deoligomerization step (formation of monomers) was found as rate-limiting. A structural model of barley UGPase was recently suggested, based on homology to a human Antigen-X (AGX) protein that has a 40% protein sequence similarity to eukaryotic UGPase. The 3D model shows a bowl-shaped protein with three different domains: (a) N-terminal, (b) central part which includes the nucleotide binding loop (NB-loop) at the active centre and (c) C-terminal which includes an insertion loop (I-loop) that is possibly involved in dimer formation and stabilization. In this study, the model was used as a testable blueprint to verify details of the barley enzyme catalysis and substrate binding, as well as oligomerization process.  In order to test the model, site-directed mutagenesis approaches and heterologous (E. coli) expression system were used to produce several UGPase mutants: Del-NB, lacking 4 amino acids (aa) at the NB region; Del-I-4 and Del-I-8, lacking respectively 4 and 8 aa of the I-loop; and Y192A, by replacing an active-site tyrosine into alanine. The Y192A mutant had about half the apparent activity of the wild-type (wt), whereas Del-I-8 and Del-I-4 had only 0.5 and 0.2 % activity, respectively, of the wt, and Del-NB showed no activity at all. Based on native-PAGE, both Y192A and Del-NB mutants had similar oligomerization status as the wt, i.e. existing as monomer only or a mixture of monomer, dimer and higher order oligomers, depending on incubation conditions. Both Del-I-8 and Del-I-4 were present in all conditions as higher order oligomers. Whereas Y192A mutant had similar Kms with both substrates as the wt protein, significant difference between the Del-I-4 and Del-I-8 mutants and wt could be detected. Both mutants had approximately 16-fold higher Kms for UDP-glucose, and the Kms with PPi were 735- and 1500-fold higher for Del-I-4 and Del-I-8, respectively, when compared to wt.The conclusion of those results: (A) Tyr-192 is not essential for activity and is not involved in substrate binding and/ or oligomerization of the enzyme. (B) The NB-loop is essential for catalysis, as evidenced by a complete lack of activity of the Del-NB mutant, and is not involved in oligomerization. On the other hand, (C) the region corresponding to central part of I-loop is located in the model far from active center, but deletion in this region does affect very strongly both catalysis and substrate binding parameters. This can be explained by the involvement of I-loop in formation of dimers (inactive) from monomers (active), as earlier proposed. Apparently, the Del-I-4 and Del-I-8 mutations lead to an enzyme form with a very high oligomerization ability. This affects both Kms and Vmaxs of the Del-I mutants. Taken together the results verify the essentiality of NB-loop for catalysis support the involvement of I-loop region in oligomerization and, overall, the importance of oligomerization status for enzymatic performance of UGPase.

Avena sativa L. (2n = 6x = 42, AACCDD genome composition) or common oat is the cereal grain possessing the highest levels of water-soluble seed (1-3,1-4)-β-D-glucan (β-glucan), a hemicellulose important to human health due to its ability to lower serum LDL cholesterol levels. Understanding the mechanisms of β-glucan accumulation in oat endosperm is, consequently, of great interest. We report a genome-wide association study (GWAS) to identify quantitative trait loci (QTLs) controlling β-glucan production in oat, identifying 58 significantly associated markers. Synteny with the barley (Hordeum vulgare L.) genome identified four major regions of interest, the CslF and CslH gene families along with UGPase and AGPase as candidate genes. Subgenome-specific expression of the A, C, and D homoeologs of major β-glucan synthase AsCslF6 revealed that AsCslF6_C is the least expressed in all tissue types and time points, with low-β-glucan varieties recording the highest proportion of AsCslF6_C expression. In order to further investigate the candidate genes identified in our GWAS study and gain a greater understanding of the other cell wall polysaccharides that comprise the total fiber content in oat we sought to characterize five additional genes. Accordingly, we cloned and sequenced the three homoeologs of AsUGP and AsAGPS1. AsAGPS1 is the small subunit 1 gene of the enzyme ADP-glucose pyrophosphorylase (AGPase), which is responsible for catalyzing the first committed step in the starch biosynthesis pathway through the production of ADP-glucose. AsUGP is the gene the codes for UDP-glucose pyrophosphorylase (UGPase) an enzyme responsible for the reversible production of UDP-glucose (UDPG). UDPG is used directly or indirectly as a precursor for the biosynthesis of cell wall polysaccharides. In high β-glucan mutant line ‘OT3044’ we observed increased expression of AsUGP with a corresponding reduction of AsAGPS1 expression. Similarly, we observed an inverse expression pattern in low-fiber mutant line ‘OT3018’, wherein AsUGP expression was decreased in favor of AsAGPS1 expression. Further, we also found evidence that these changes in both AsUGP and AsAGPS1 expression are due primarily to up- or down-regulation in the A-genome homoeoalleles. Additionally, we characterized genes in the CslC family (CslC4, CslC9) and CslA family (CslA7) responsible for xyloglucan and glucomannan synthesis, respectively. High-fiber line ‘HiFi’ showed the least amount of overall expression of these three genes, raising the possibility that the increased β-glucan is due to a reduction in other hemicelluloses. After analyzing homoeolog-specific expression in multiple genes we observed that the A genome consistently had the most highly expressed homoeoallele, hinting at a universal preference for expression of this subgenome. We present hypotheses regarding multiple points in carbohydrate metabolism having the potential to alter β-glucan content in oat.

Photosynthesis captures light from the sun and converts it into carbohydrates, which are utilised by almost all living organisms. The conversion between the different forms of carbohydrates is the basis to form almost all biological molecules.

The main intention of this thesis has been to study the role of UDP-glucose in carbohydrate synthesis and metabolism, and in particular the genes that encode UDP-glucose pyrophosphorylase (UGPase) and UDP-glucose dehydrogenase (UGDH) in plants and their regulation. UGPase converts glucose-1-phosphate to UDP-glucose, which can be utilised for sucrose synthesis, or cell wall polysaccharides among others. UGDH converts UDP-glucose to UDP-glucuronate, which is a precursor for hemicellulose and pectin. As model species I have been working with both Arabidopsis thaliana and poplar.

Sequences for two full-length EST clones of Ugp were obtained from both Arabidopsis and poplar, the cDNAs in Arabidopsis correlate with two genes in the Arabidopsis genomic database.

The derived protein sequences are 90-93% identical within each plants species and 80-83% identical between the two species.

Studies on Ugp showed that the expression is up-regulated by Pi-deficiency, sucrose-feeding and by light exposure in Arabidopsis. Studies with Arabidopsis plants with mutations in sugar/ starch- and Pi-content suggested that the Ugp expression is modulated by an interaction of signals derived from Pi-deficiency, sugar content and light/ dark conditions, where the signals act independently or inhibiting each other, depending on conditions. Okadaic acid, a known inhibitor of certain classes of protein phosphatases, prevented the up-regulation of Ugp by Pi-deficiency and sucrose-feeding. In poplar, sucrose also up-regulated the expression of Ugp. When poplar and Arabidopsis were exposed to cold, an increase of Ugp transcript content was detected as well as an increase in UGPase protein and activity. In poplar, Ugp was found to be expressed in all tissues that were examined (differentiating xylem, phloem, apical leaves and young and mature leaves).

By using antisense strategy, Arabidopsis plants that had a decrease in UGPase activity of up to 30% were obtained. In the antisense plants, the soluble carbohydrate content was reduced in the leaves by at least 50%; in addition the starch content decreased. Despite the changes in carbohydrate content, the growth rate of the antisense plants was not changed compared to wild type plants under normal growth conditions. However, in the antisense lines the UGPase activity and protein content in sliliques and roots increased, perhaps reflecting compensatory up-regulation of second Ugp gene. This correlates with a slightly larger molecular mass of UGPase protein in roots and siliques when compared to that in leaves. Maximal photosynthesis rates were similar for both wild type and antisense plants, but the latter had up to 40% lower dark respiration and slightly lower quantum yield than wild type plants.

Two Ugdh cDNAs from poplar and one from Arabidopsis were sequenced. The highest Ugdh expression was found in xylem and younger leaves. Expression data from sugar and osmoticum feeding experiment in poplar suggested that the Ugdh expression is regulated via an osmoticumdependent pathway.

Trypanosoma brucei, un protiste responsable de la Trypanosomose Humaine Africaine, également connue sous le nom de la maladie du sommeil, est transmis par la mouche tsé-tsé (Glossina sp.). La découverte d'organites de type peroxysome spécialisés dans la glycolyse, appelés glycosomes, a soulevé un certain nombre de questions sur le rôle de cet organite dans la biologie des trypanosomes. Plusieurs voies métaboliques présentes dans le cytosol d'autres eucaryotes, comme la glycolyse et la biosynthèse des sucres nucléotidiques, sont compartimentées dans les glycosomes. Les raisons et les avantages de la présence des enzymes glycolytiques dans l'organite ont été largement discutés, mais la fonctionnalité et le rôle des voies de biosynthèse des sucres nucléotidiques glycosomales ne sont pas connus. Notre étude s'est focalisée sur l'UDP-glucose pyrophosphorylase (UGP), une enzyme impliquée dans la synthèse de l'UDP-glucose (UDP-Glc). Sur la base de la double localisation glycosomale et cytosolique de l'UGP mise en évidence ici à l'aide de plusieurs techniques de localisation subcellulaire, nous avons abordé deux questions en utilisant comme modèle les formes procycliques de T. brucei présentes dans l'insecte vecteur. La première est liée au mécanisme d'import de l'UGP dans les glycosomes, car cette protéine ne possède aucun signal d'adressage aux peroxysomes de type PTS1 ou PTS2. Nous avons montré que l'UGP est importée dans les glycosomes par "piggybacking" en s'associant à la phosphoénolpyruvate décarboxylase (PEPCK) possédant un signal d’adressage PTS1. Les interactions entre l'UGP et la PEPCK ont été montrées in situ et l'identification les régions impliquées dans ces interactions ont été identifiées. Nos résultats suggèrent que le complexe UGP-PEPCK est formé de manière transitoire lors de son import dans les glycosomes nouvellement produits et compétents pour l'import des protéines. La seconde question concerne le rôle de l'UGP dans les glycosomes. Nous avons montré que l'UGP est essentielle à la croissance des trypanosomes et que les voies métaboliques glycosomales et cytosoliques dont l'UGP fait partie sont fonctionnelles. En effet, des mutants viables contenant l'UGP exclusivement dans les glycosomes ou dans le cytosol sont viables et produisent des quantités similaires d'UDP-Glc. La raison d'être de la production glycosomale d'UDP-Glc par l'UGP reste inconnue, mais n'est probablement pas liée aux réactions de glycosylation, étant donné qu'aucune glycosyltransférase n'a été détectée dans l'organite.Un autre aspect de ce travail concerne le rôle des intermédiaires du cycle de l'acide tricarboxylique (TCA) dans le métabolisme mitochondrial des formes procycliques. Dans le tractus digestif de son insecte vecteur, les trypanosomes dépendent de la proline pour alimenter leur métabolisme énergétique. Cependant, la disponibilité d'éventuelles autres sources de carbone pouvant être utilisées par le parasite est actuellement inconnue. Nous avons montré que les intermédiaires du cycle TCA, i.e. succinate, malate et a-cétoglutarate, stimulent la croissance des formes procycliques incubées dans un milieu contenant 2 mM de proline, concentration se situant dans la gamme des quantités mesurées dans l'intestin de la mouche. De plus, le développement de nouvelles approches ont permis d'étudier une branche peu explorée du cycle TCA convertissant le malate en a-cétoglutarate, précédemment décrite comme peu ou pas utilisée par le parasite, quellles que soient les quantités de glucose disponibles. L'activité de cette branche suggère qu'un cycle TCA complet peut être mis en œuvre dans les formes procycliques et probablement dans les autres formes parasitaires de l'insecte. Nos données élargissent le potentiel métabolique des trypanosomes et ouvrent la voie vers une meilleure compréhension du métabolisme de ce parasite dans divers organes de la mouche tsé-tsé, où il évolue
Trypanosoma brucei, a protist responsible for human African trypanosomiasis, also known as sleeping sickness, is transmitted by the tsetse fly (Glossina sp.). The discovery of peroxisome-like organelles specialized in glycolysis called glycosomes, has raised a number of questions about the role of this organelle in the biology of trypanosomes. Several metabolic pathways present in the cytosol of eukaryotes, like glycolysis and sugar nucleotides biosynthesis, are compartmentalized within glycosomes. While the reasons and advantages of having glycolytic enzymes compartmentalized in the organelle have been extensively discussed, little is proposed for sugar nucleotides biosynthetic pathways. This study is focused on the UDP-glucose pyrophosphorylase (UGP), an enzyme involved in the synthesis of UDP-glucose (UDP-Glc). Based on the UGP's dual glycosomal and cytosolic localization evidenced here using several subcellular localization techniques, we addressed two questions using as a model the procyclic forms of T. brucei present in the insect vector. The first one is related to the mechanism of UGP import into glycosomes, since this protein lacks any known peroxisomal targeting signal (PTS1 and PTS2). We demonstrated that UGP is imported into the organelle by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate decarboxylase (PEPCK). Interactions between UGP and PEPCK have been showed in situ and the interacting regions have been identified. Our data suggest that the complex UGP-PEPCK is formed transiently to facilitate the import of UGP and that it is detected in newly formed import-competent glycosomes. The second question concerns the role of UGP in glycosomes. We demonstrated that UGP is essential for the growth of trypanosomes and that mutants containing UGP exclusively in glycosomes or in the cytosol still produce UDP-Glc at similar levels and are viable, which implies that the glycosomal and cytosolic metabolic pathways involving UGP are functional. The glycosomal function of UDP-Glc is currently unknown and probably not related to glycosylation reactions, since no glycosyltransferases have been detected in the organelle.Another aspect of this work concerns the role of tricarboxylic acid (TCA) cycle intermediates in the mitochondrial metabolism of the procyclic trypanosomes. In the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. We showed that TCA cycle intermediates, i.e. succinate, malate and a-ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. In addition, we have implemented new approaches to study cell growth and metabolic pathways in order to investigate mitochondrial metabolism. These new tools have allowed us to study a poorly explored branch of the TCA cycle converting malate to a-ketoglutarate, which was previously described as non-functional or little used in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes and probably in the other trypanosome forms present in the fly. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasite's metabolism in various organ systems of the tsetse fly, where it evolves

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and accumulation in sugarcane is poorly understood. The present study was therefore aimed at the development of an in situ hybridisation (ISH) technique to study differential gene expression among the various cell types of the sugarcane culm. This technique in conjunction with northern and western blotting was then used to determine the sites of cellular and tissue specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose metabolism. This study revealed that the determination of the influencing parameters associated with the development of an ISH protocol was essential for the successful detection of the endogenous RNA sequences in sugarcane internodal tissues. The parameters that were investigated included the type of embedding medium, duration of fixation period, pre-treatment procedures and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the analysis of cytological gene expression at all stages of sugarcane development. The second part of this study revealed very localised transcript expression for UDP-Glc DH, PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all expressed in the parenchyma cells that were associated with the vascular bundles and the stem storage compartment, suggesting that the parenchyma cells distributed throughout the stem in the different tissue types complement each other in function for the purposes of phloem loading, unloading and assimilate transport processes. Complimentary northern and western hybridisations demonstrated that internode 7 represents a shift in the sink from utilisation to storage. This is evident by the observed decline in both the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage of development. The relative mRNA and protein abundances for the three enzymes showed a similar trend. Higher levels of the gene transcripts and translated products were observed in the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was expressed exclusively in parenchyma cells that were associated with the vascular bundles and those serving a storage function in the stem pith and UGPase was found to be localised in the phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such findings have demonstrated an increase in resolution with which gene expression can be examined at a cellular level. Hence, the results from this study have demonstrated that the knowledge of metabolic compartmentation between different tissue and cell types is a requisite to understanding the function(s) of individual enzymes within complex structures such as the sugarcane culm.
AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by sukrosemetabolisme, te bepaal. Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes. Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens alle fases van suikkerrietontwikkeling moontlik gemaak het. Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed. RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die suikerrietstingel te bepaal.

L'UDP-N-Acétyl-glucosamine (UDP-GlcNAc) est un précurseur clé de composants de la paroi cellulaire bactérienne et fongique, du glycolipide d'ancrage GPI, ainsi que des N- et O-glycannes des glycoprotéines. Pour comprendre les mécanismes d'acétylation et d'uridylation qui conduisent à la synthèse de l'UDP-GIcNAc chez les eucaryotes, la GlcN6P acétyltransférase de S. Cerevisiae (ScGNA1) et les deux isoformes (AGX1 et AGX2) issues de l'épissage alternatif du gène humain de la GlcNAc1P uridyltransférase ont été exprimées dans des souches d'E. Coli, purifiées et cristallisées. Les méthodes cristallographiques MAD et de remplacement moléculaire ont été utilisées pour résoudre leurs structures sous formes apo et/ou complexées aux produits ou substrats de la réaction, à des résolutions allant de 1. 3 à 2. 4 Angstrom. ScGNA1 adopte un repliement α/ß caractéristique des membres de la superfamille GNAT. Cette protéine s'assemble sous forme d'un dimère stabilisé par l'échange, entre les deux sous-unités, du dernier brin ß. Cet assemblage dimérique est crucial pour la formation du site de liaison du substrat accepteur. AGX1 et AGX2 qui diffèrent par un segment de 17 résidus chez AGX2, adoptent un repliement commun, apparenté à celui des membres de la superfamille SGC, mais possèdent en solution, un assemblage oligomérique différent. Cette différence modifie l'environnement du site actif et suggère un rôle de l'épissage alternatif du gène d'AGX dans la régulation de l'activité GlcNAc1P uridyltransférase. Enfin, nos résultats permettent l'identification des acides aminés potentiellement impliqués dans la catalyse et la reconnaissance des substrats et supportent l'hypothèse d'un mécanisme de type simple déplacement pour les réactions d'acétylation et d'uridylation

Orientadores: Marcelo Menossi Teixeira, Ricardo Aparicio
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O agronegócio da cana de açúcar movimenta cerca de R$ 40 bilhões por ano no Brasil. A cadeia produtiva da cana de açúcar como atividade na economia é responsável por 1,5% do produto interno bruto (PIB) nacional e um dos principais componentes econômicos é a quantidade de sacarose acumulada nos colmos. No entanto, a síntese de sacarose e sua acumulação em plantas superiores é o resultado do produto de uma extensa rede de interações. Quando descarregada nas células do parênquima de armazenamento, a sacarose é metabolizada por diferentes enzimas, sendo a UDP-glucose pirofosforilase (UGPase) uma das enzimas responsáveis pela síntese de sacarose em cana de açúcar. O objetivo deste trabalho foi avaliar o padrão de expressão do gene ScUGPase-1 e os mecanismos regulatórios que controlam a atividade da proteína UGPase de cana de açúcar. Análises por RT-qPCR revelaram que a expressão do gene ScUGPase-1 diminui ao longo da maturação dos colmos e o gene é mais expresso nos entrenós em comparação com o tecido de folha. Porém, nenhuma diferença de expressão significativa foi observada entre dois cultivares contrastantes em teor de sacarose. In vivo, a localização subcelular da proteína ScUGPase-1 indicou uma associação à membrana nos tecidos de folha e colmo. Utilizando anticorpo primário fosfo-específico, observamos a fosforilação da proteína ScUGPase-1 apenas na fração solúvel e microssomal do tecido de folha. In vitro, a proteína ScUGPase-1 formou um complexo com a proteína recombinante caseína quinase 1 (CK1) e sua atividade foi afetada por agentes óxido-redutores. Para complementar os dados de óxido-redução, análises de espalhamento de luz a baixo ângulo (SAXS) forneceram o primeiro modelo estrutural do dímero da proteína ScUGPase-1 em solução, destacando que a interface de dimerização está localizada na região C-terminal. Os dados indicam que a fosforilação, interação protéica e oligomerização podem exercem um papel importante na regulação da proteína ScUGPase-1 durante a síntese de sacarose em cana de açúcar.
Abstract:The sugarcane agribusiness generates around R$ 40 billion per year in Brazil, while the entire supply chain of sugarcane is responsible for 1.5% of the gross domestic product (GDP). Sugarcane productivity is mainly determined by the accumulation of sucrose in the culms. However, the synthesis and accumulation of sucrose in plants is the result of an extensive network. When sucrose is unloaded in the storage parenchyma cells, it is metabolized by different enzymes, and UDP-glucose pyrophosphorylase (UGPase) is one of the enzymes responsible for the synthesis of sucrose in sugarcane. The objective of this work was to gain insights on the ScUGPase-1 expression pattern and the regulatory mechanisms that control protein activity. ScUGPase-1 transcript levels were negatively correlated with sucrose content in the internodes and only a slight difference in the expression pattern was observed between two cultivars that differ in their sucrose content. The intracellular localization of ScUGPase-1 indicated association with membranes in both leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo in the soluble and membrane fractions from leaves, but not from internodes. In vitro, the purified recombinant enzyme interacted with recombinant protein casein kinase 1 and its activity was affected by redox modification. To complement the redox data, Small-Angle X-ray Scattering provided the first structural model of the dimer of sugarcane UGPase in solution, highlighting that the dimer interface is located at the C-terminal. The data indicated that phosphorylation, protein interaction and oligomerization may play an important role in the regulation of ScUGPase-1 activity
Doutorado
Genetica de Microorganismos
Mestre em Genética e Biologia Molecular

La reaction de glucoronoconjugaison, catalysee par les udp-glucuronosyltranferases (ugts), est un processus implique dans le metabolisme des xenobiotiques (medicaments, substances toxiques) mais aussi des molecules endognes. L'activite de ces enzymes participe ainsi au controle de la concentration de certains mediateurs de reponses physiologiques comme les hormones et les molecules odorantes. L'objectif de ce travail est l'identification et l'etude de la regulation de l'expression d'ugts chez le rat, par differents facteurs dans des tissus ou leur presence a probablement des consequences fonctionnelles differentes. Dans la cadre d'une etude cinetique de l'influence d'un traitement par le ciprofibrate, nous avons observe que l'augmentation d'activite deglucurono- -conjugaison de la bilirubine dans le foie de rat n'est pas correlee a l'expression des isoformes ugt1a1 (forme majeure), ugt1a2 ou encore ugt1a5 (formes majeures).

Un cycle de substrats ternaire est une structure métabolique composée de trois substrats convertis par trois activités enzymatiques. Pour l'étude de ce système, seuls les flux d'interconversion sont pris en compte. Les modifications des concentrations des substrats d'un cycle ternaire, à l'état stationnaire, sont décrites en fonction des valeurs des paramètres du système (somme des concentrations des substrats et vitesses maximales des enzymes). Des phénomènes de bistabilité sont mis en évidence de façon théorique. Un cycle expérimental avec les substrats UTP, UDPglucose et UDP est étudié en parallèle avec le modèle théorique correspondant. Différentes enzymes (UDPglucose pyrophosphorylase, glycogène synthétase, pyruvate kinase et nucléoside diphosphokinase), de cinétique plus ou moins complexe, sont utilisées. Les vitesses de ces enzymes sont fonction des affinités pour les substrats et les produits, ainsi que des effets dus aux métabolites présents dans le système. Les cycles expérimentaux montrent des évolutions temporelles en accord avec les prédictions de modélisation. Les comportements observés sont une amplification de la réponse à l'état stationnaire, avec cependant un temps de réponse du système entre deux états stationnaires extrêmement long.

Le rétrovirus HTLV-1 (Human T-cell Leukemia Virus type 1) est l'agent responsable de la leucémie T de l'adulte (ATL) et de la paraparésie spastique tropicale (TSP/HAM). Le HTLV est présent de manière endémique au Japon, en Afrique centrale, en Océanie, en Amérique du sud et aux Caraïbes. Chez les patients, le HTLV est détecté principalement dans les lymphocytes T CD4+. Le récepteur du HTLV est présent chez tous les vertébrés, et toutes les lignées cellulaires de vertébrés semblent l'exprimer in vitro. L'organisation de la glycoprotéine d'enveloppe (Env) du HTLV est similaire à celle des MLV (Murine Leukemia Virus), avec un domaine de liaison au récepteur situé dans la partie N-terminale.
Au cours de cette thèse, nous avons dérivés des produits tronqués de l'Env, sur la base de cette organisation, nous permettant de mesurer l'expression de surface du récepteur HTLV. Nous avons ainsi pu mettre en évidence que le récepteur HTLV n'est pas détectable à la surface des lymphocytes T CD4+, mais que son expression est induite par l'activation lymphocytaire. Nous avons également montré qu'un traitement à l'IL-7 des lymphocytes T de sang de cordon, qui provoque la prolifération, induit l'expression du récepteur HTLV. Ces observations nous ont permis de conclure que le récepteur HTLV est un marqueur d'activation des lymphocytes associé à l'état prolifératif.
Par la suite, nous avons montré que l'expression de l'Env HTLV induit un blocage de la production de lactate. Nous avons pu établir que ce blocage est dû à l'inhibition par l'Env HTLV du transport de glucose dans la cellule, induisant un blocage de la glycolyse. Ces observations nous ont conduit à évaluer si le principal transporteur du glucose GLUT1 pouvait être un récepteur pour le virus HTLV. Effectivement, nous avons pu démontrer que GLUT1 permet à la fois la liaison à l'Env HTLV et l'entrée de particules virales.
Afin de comprendre cette interaction, nous avons construit des molécules chimériques entre GLUT1 et GLUT3. Nous avons ainsi observé que 7 acides aminés de la 6ème boucle extracellulaire de GLUT1 confèrent l'activité de liaison à l'Env. Cependant cette propriété n'est pas suffisante pour permettre l'entrée virale, et la présence concomitante de la 1ère, la 5ème et la 6ème boucle extracellulaire est requise pour permettre l'entrée virale, suggérant l'existence d'une nouvelle activité associée à la notion de récepteur rétroviral.
L'ensemble de ces données nous a enfin conduit à proposer de nouveaux modèles de pathogenèse. La prise en compte de l'inhibition de l'activité de transport de GLUT1 par l'Env autorise en effet de nouvelles hypothèses pour expliquer l'émergence de clones leucémiques et la démyélinisation associée à la TSP/HAM.

碩士
國立臺灣大學
化學研究所
100
Glycans and glycoconjugates are important biomolecules, which participate in various cellular processes, e.g. cancer growth, infiltration, and metastasis. Although many chemical synthetic methods have been developed during last decade, the synthesis of glycoconjugates, especially the stereoselective glycosylation, remains to be problematic. In contrast, enzymatic synthesis using glycosyltransferases is advantageous in terms of not only chemical selectivities but also green chemistry. In this study, we looked into arabidopsis thaliana UDP-sugar pyrophosphorylase (AtUSP), an enzyme that catalyses the conversion of monosaccharide-1-phosphate to the respective UDP-sugars with broad specificity, and its application for the synthesis of glycoconjugate containing non-natural sugar. We attempted to use kinase to transfer non-nature monosacharides to monosacharide-1-phosphate followed by AtUSP-catalyzed transfermation of these monosacharides-1-phosphates to the respective UDP-sugars. To examine the substrate tolerance of AtUSP, we synthesized glucose and galactose analogues containing azido, fluorous, methyl, vinyl, and alkynyl groups at the C-6 position. The azido and fluorous ananlogues were prepared form C-6 OH free derivatives by SN2 reaction and fluorination, respectively, using DAST. Alkenyl and alkynyl mimics were synthesized from the corresponding aldehydes via Wittig olefination and Bestman-ohira reaction. Finally, methyl analogues were obtained by hydrogenation of corresponding alkenyl derivatives. We also synthesized GlcNAc and GalNAc analogues with different azidoalkylcarbonyl groups and pentynoyl groups. We applied amide bond formation to synthesize these compounds. This methodology is straightforward and may be applicable for the preparation of other sugar analogues. The evaluation of the synthesized analogues as substrates is under way. However, when we used kinase to catalyze the formation of monosaccharide-1-phosphates, the reaction rate is too slow to obtain any kinetic data. The problem may be solved by using more kinases or chemical method to synthesize these monosaccharide-1-phosphates to test AtUSP more quickly.

博士
國立清華大學
分子醫學研究所
100
This thesis consists of 3 chapters. The first 2 chapters describe the molecular evolution of prokaryotic exceptionally large-sized genes (ELSGs) and nucleotide-sugar pyrophosphorylases/phosphorylases (NDP-sugar PPases/Pases). The third chapter describes a computer-aided drug design for searching bacterial UDP-glucose pyrophosphorylase (UGPase) inhibitors. In chapter 1, we have noted that, in contrast to the average gene size of approximately 1 kb in bacteria, 3 genes > 13 kb were present in Vibrio vulnificus. The finding prompted us to investigate the prevalence, possible function, and origin of ELSGs (>10 kb) in prokaryotes. Forty-two ELSGs (0.03%) were identified after searching more than 170,000 genes in 46 bacterial and 11 archaeal species. Homology analysis of these ELSGs indicated that, in addition to encoding non-ribosomal peptide synthetic enzymes, many ELSGs likely encode membrane-anchored proteins. Dot-matrix plot analysis of these ELSGs indicated that domain-duplication contributed significantly to size expansion. Other size expansion mechanisms were direct gene fusion, recombination of different genes, and horizontal gene transfer. In summary, ELSGs are commonly present in prokaryotes, and the evolutionary processes that have contributed to the formation of ELSGs are relatively heterogeneous. NDP-sugar PPases/Pases play a central role in providing sugar donor for the formation of glycoconjugates. Despite each of the enzymes has unique substrate specificity, they share homologous protein sequences and are therefore difficult to annotate. Chapter 2 describes our effort to better categorize these enzymes. More than 2,500 NDP-sugar PPases/Pases sequences were collected from the KEGG in this study and 86 representative sequences were selected. Phylogenetic and domain analyses revealed that members of GDP-Man PPase had the most diverse protein sequences implying that this enzyme is evolutionally closer to the common ancestor of the NDP-sugar PPases/Pases than other members of the family. Most NDP-sugar PPases/Pases were apparently derived by gene duplication although horizontal gene transfer, as in the case of eukaryotic UDP-Glc PPase, also contributed to the gene diversification. An evolutionary model for this group of enzymes was established by combining phylogenetic analysis and domain profiling. The core domains of each of the enzymes, trend of domain gain and loss, and evolutionary transition were demonstrated. These non-redundant 86 representative sequences may be used as the reference sequences for NDP-sugar PPases/Pases categorization. The low levels of sequence homology between prokaryotic and eukaryotic UGPase makes the enzyme a good target for antimicrobial drug development. Chapter 3 describes 22 UDP-Glc analogs and 97 candidate compounds, respectively selected from the ZINC and NCI compound databases using the protein docking program, LigandFit, that have potential to inhibit the bacterial UGPase activity. Five of the 97 candidate compounds were also identified by another docking program, Libdock. These 27 compounds represent potential bacterial UGPase inhibitors. The research interpreted the evolution of prokaryotic ELSGs, the categorization and evolutionary history of NDP-sugar PPases/Pases, and obtained the potential bacterial UGPases candidate inhibitors. The next step, we hope to obtain or synthesis these candidate compounds, or to screen the approved drugs in drug product database (new uses for old drugs) to perform the antimicrobial assay for the research and development of novel antimicrobial agents.