RESUMO
Starch is the dominant reserve polysaccharide accumulated in the seed of grasses (like wheat). It is the most common carbohydrate in the human diet and a material applied to the bioplastics and biofuels industry. Hence, the complete understanding of starch metabolism is critical to design rational strategies to improve its allocation in plant reserve tissues. ADP-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the key (regulated) step in the synthetic starch pathway. The enzyme comprises a small (S) and a large (L) subunit forming an S2L2 heterotetramer, which is allosterically regulated by orthophosphate, fructose-6P, and 3P-glycerate. ADP-Glc PPase was found in a phosphorylated state in extracts from wheat seeds. The amount of the phosphorylated protein increased along with the development of the seed and correlated with relative increases of the enzyme activity and starch content. Conversely, this post-translational modification was absent in seeds from Ricinus communis. In vitro, the recombinant ADP-Glc PPase from wheat endosperm was phosphorylated by wheat seed extracts as well as by recombinant Ca2+-dependent plant protein kinases. Further analysis showed that the preferential phosphorylation takes place on the L subunit. Results suggest that the ADP-Glc PPase is a phosphorylation target in seeds from grasses but not from oleaginous plants. Accompanying seed maturation and starch accumulation, a combined regulation of ADP-Glc PPase by metabolites and phosphorylation may provide an enzyme with stable levels of activity. Such concerted modulation would drive carbon skeletons to the synthesis of starch for its long-term storage, which later support seed germination.
RESUMO
Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in almost all cases of amyotrophic lateral sclerosis (ALS), and 20% of familial ALS cases are due to mutations in superoxide dismutase 1 (SOD1). Redox regulation is critical to maintain cellular homeostasis, although how this relates to ALS is unclear. Here, we demonstrate that the redox function of protein disulfide isomerase (PDI) is protective against protein misfolding, cytoplasmic mislocalization of TDP-43, ER stress, ER-Golgi transport dysfunction, and apoptosis in neuronal cells expressing mutant TDP-43 or SOD1, and motor impairment in zebrafish expressing mutant SOD1. Moreover, previously described PDI mutants present in patients with ALS (D292N, R300H) lack redox activity and were not protective against ALS phenotypes. Hence, these findings implicate the redox activity of PDI centrally in ALS, linking it to multiple cellular processes. They also imply that therapeutics based on PDI's redox activity will be beneficial in ALS.
RESUMO
This work reports the molecular cloning and heterologous expression of the genes coding for α and ß subunits of pyrophosphate-dependent phosphofructokinase (PPi-PFK) from orange. When expressed individually, both recombinant subunits were produced as highly purified monomeric proteins able to phosphorylate fructose-6-phosphate at the expenses of PPi (specific activity of 0.075 and 0.017 units. mg-1 for α and ß subunits, respectively). On the other hand, co-expression rendered a α3ß3 hexamer with specific activity three orders of magnitude higher than the single subunits. All the conformations of the enzyme were characterized with respect to its kinetic properties and sensitivity to the regulator fructose-2,6-bisphosphate. A thorough review of current knowledge on the matter indicates that this is the first report of the recombinant production of active plant PPi-PFK and the characterization of its different conformations. This is a main contribution for future studies focused to better understand the enzyme properties and how it accomplishes its relevant role in plant metabolism.
Assuntos
Citrus sinensis/enzimologia , Fosfofrutoquinases/metabolismo , Fosfotransferases/metabolismo , Citrus sinensis/genética , Clonagem Molecular , Difosfatos/metabolismo , Frutosedifosfatos/metabolismo , Frutosefosfatos/metabolismo , Expressão Gênica , Cinética , Complexos Multiproteicos , Fosfofrutoquinases/genética , Fosforilação , Fosfotransferases/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas RecombinantesRESUMO
The ADP-glucose pyrophosphorylase from wheat endosperm controls starch synthesis in seeds and has unique regulatory properties compared to others from this family. It comprises two types of subunits, but despite its importance little is known about their roles. Here, we synthesized de novo the wheat endosperm ADP-glucose pyrophosphorylase small (S) and large (L) subunit genes, heterologously expressed them in Escherichia coli, and kinetically characterized the recombinant proteins. To understand their distinct roles, we co-expressed them with well characterized subunits from the potato tuber enzyme to obtain hybrids with one S subunit from one source and an L subunit from the other. After kinetic analyses of these hybrids, we concluded that the unusual insensitivity to activation of the wheat endosperm enzyme is caused by a pre-activation of the L subunit. In addition, the heat stability and sensitivity to phosphate are given by the S subunit.
RESUMO
Cytosolic glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) is involved in a critical energetic step of glycolysis and also has many important functions besides its enzymatic activity. The recombinant wheat NAD-GAPDH was phosphorylated in vitro at Ser205 by a SNF1-Related protein kinase 1 (SnRK1) from wheat heterotrophic (but not from photosynthetic) tissues. The S205D mutant enzyme (mimicking the phosphorylated form) exhibited a significant decrease in activity but similar affinity toward substrates. Immunodetection and activity assays showed that NAD-GAPDH is phosphorylated in vivo, the enzyme depicting different activity, abundance and phosphorylation profiles during development of seeds that mainly accumulate starch (wheat) or lipids (castor oil seed). NAD-GAPDH activity gradually increases along wheat seed development, but protein levels and phosphorylation status exhibited slight changes. Conversely, in castor oil seed, the activity slightly increased and total protein levels do not significantly change in the first half of seed development but both abruptly decreased in the second part of development, when triacylglycerol synthesis and storage begin. Interestingly, phospho-NAD-GAPDH levels reached a maximum when the seed switch their metabolism to mainly support synthesis and accumulation of carbon reserves. After this point the castor oil seed NAD-GAPDH protein levels and activity highly decreased, and the protein stability assays showed that the protein would be degraded by the proteasome. The results presented herein suggest that phosphorylation of NAD-GAPDH during seed development would have impact on the partitioning of triose-phosphate between different metabolic pathways and cell compartments to support the specific carbon, energy and reducing equivalent demands during synthesis of storage products.
RESUMO
HaHB11 is a member of the sunflower homeodomain-leucine zipper I subfamily of transcription factors. The analysis of a sunflower microarray hybridized with RNA from HaHB11-transformed leaf-disks indicated the regulation of many genes encoding enzymes from glycolisis and fermentative pathways. A 1300bp promoter sequence, fused to the GUS reporter gene, was used to transform Arabidopsis plants showing an induction of expression after flooding treatments, concurrently with HaHB11 regulation by submergence in sunflower. Arabidopsis transgenic plants expressing HaHB11 under the control of the CaMV 35S promoter and its own promoter were obtained and these plants exhibited significant increases in rosette and stem biomass. All the lines produced more seeds than controls and particularly, those of high expression level doubled seeds yield. Transgenic plants also showed tolerance to flooding stress, both to submergence and waterlogging. Carbohydrates contents were higher in the transgenics compared to wild type and decreased less after submergence treatments. Finally, transcript levels of selected genes involved in glycolisis and fermentative pathways as well as the corresponding enzymatic activities were assessed both, in sunflower and transgenic Arabidopsis plants, before and after submergence. Altogether, the present work leads us to propose HaHB11 as a biotechnological tool to improve crops yield, biomass and flooding tolerance.
Assuntos
Arabidopsis/fisiologia , Helianthus/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Adaptação Biológica/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Biomassa , Inundações , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.
Assuntos
Galactosefosfatos/metabolismo , Giardia lamblia/enzimologia , Proteínas de Protozoários/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Cisteína/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Giardia lamblia/genética , Glucofosfatos/metabolismo , Cinética , Dados de Sequência Molecular , Oxirredução , Proteínas de Protozoários/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Tiorredoxinas/metabolismo , Fatores de Tempo , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
Sugarcane is a monocot plant that accumulates sucrose to levels of up to 50% of dry weight in the stalk. The mechanisms that are involved in sucrose accumulation in sugarcane are not well understood, and little is known with regard to factors that control the extent of sucrose storage in the stalks. UDP-glucose pyrophosphorylase (UGPase; EC 2.7.7.9) is an enzyme that produces UDP-glucose, a key precursor for sucrose metabolism and cell wall biosynthesis. The objective of this work was to gain insights into the ScUGPase-1 expression pattern and regulatory mechanisms that control protein activity. ScUGPase-1 expression was negatively correlated with the sucrose content in the internodes during development, and only slight differences in the expression patterns were observed between two cultivars that differ in sucrose content. The intracellular localization of ScUGPase-1 indicated partial membrane association of this soluble protein in both the leaves and internodes. Using a phospho-specific antibody, we observed that ScUGPase-1 was phosphorylated in vivo at the Ser-419 site in the soluble and membrane fractions from the leaves but not from the internodes. The purified recombinant enzyme was kinetically characterized in the direction of UDP-glucose formation, and the enzyme activity was affected by redox modification. Preincubation with H2O2 strongly inhibited this activity, which could be reversed by DTT. Small angle x-ray scattering analysis indicated that the dimer interface is located at the C terminus and provided the first structural model of the dimer of sugarcane UGPase in solution.
Assuntos
Membrana Celular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/biossíntese , Caules de Planta/enzimologia , Saccharum/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/biossíntese , Membrana Celular/química , Modelos Moleculares , Fosforilação/fisiologia , Proteínas de Plantas/química , Caules de Planta/química , Estrutura Terciária de Proteína , UTP-Glucose-1-Fosfato Uridililtransferase/química , Uridina Difosfato Glucose/biossíntese , Uridina Difosfato Glucose/químicaRESUMO
Glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. Herein we report the molecular cloning, heterologous expression and characterization of Gol dehydrogenase (GolDHase, EC 1.1.1.14) from peach (Prunus persica) fruits. The recombinant enzyme showed kinetic parameters similar to those reported for orthologous enzymes purified from apple and pear fruits. The activity of recombinant GolDHase was strongly inhibited by Cu(2+) and Hg(2+), suggesting that it might have cysteine residues critical for functionality. Oxidizing compounds (such as diamide, hydrogen peroxide and oxidized glutathione) inactivated the enzyme, whereas its activity was restored after incubation with reduced glutathione and thioredoxin from Escherichia coli. Recombinant thioredoxin h from peach fruits also recovered the activity of oxidized GolDHase. Our results suggest that peach fruit GolDHase could be redox regulated in vivo and this would be of relevance to determine carbon assimilation and partitioning in plants accumulating sugar alcohols.
Assuntos
Carbono/metabolismo , Regulação da Expressão Gênica de Plantas , Oxirredutases/genética , Prunus/enzimologia , Sorbitol/metabolismo , Tiorredoxina h/genética , Clonagem Molecular , Cobre/farmacologia , Diamida/farmacologia , Frutas/enzimologia , Frutas/genética , Glutationa/farmacologia , Peróxido de Hidrogênio/farmacologia , Cinética , Mercúrio/farmacologia , Modelos Biológicos , Oxirredução , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prunus/genética , Proteínas Recombinantes , Tiorredoxina h/metabolismoRESUMO
Adaptation to aerobic life leads organisms to sense reactive oxygen species and use the signal for coordination of the entire metabolism. Glycolysis in plants is a particular network where specific steps, like oxidation of glyceraldehydes-3-phosphate (Ga3P), are critical in order for it to function. The triose-phosphate can be converted into 3-phosphoglycerate through the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12) producing ATP and NADH, or via the non-phosphorylating enzyme (np-Ga3PDHase; EC 1.2.1.9) generating NADPH. In this work we found redox regulation to be a posttranslational mechanism allowing the fine-tuning of the triose-phosphate fate. Both enzymes were inactivated after oxidation by reactive oxygen and nitrogen species. Kinetic studies determined that Ga3PDHase is marked (63-fold) more sensitive to oxidants than np-Ga3PDHase. Thioredoxin-h reverted the oxidation of both enzymes (although with differences between them), suggesting a physiological redox regulation. The results support a metabolic scenario where the cytosolic triose-phosphate dehydrogenases are regulated under changeable redox conditions. This would allow coordinate production of NADPH or ATP through glycolysis, with oxidative signals triggering reducing power synthesis in the cytosol. The NADPH increment would favor antioxidant responses to cope with the oxidative situation, while the thioredoxin system would positively feedback NADPH production by maintaining np-Ga3PDHase at its reduced active state.
Assuntos
Citosol/metabolismo , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/metabolismo , Gliceraldeído 3-Fosfato/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Gliceraldeído 3-Fosfato Desidrogenase (NADP+)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Glicólise , Cinética , Redes e Vias Metabólicas , Oxirredução , Proteínas de Plantas/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Triticum/citologia , Triticum/genética , Triticum/metabolismoRESUMO
Amoebiasis is an intestinal infection caused by the human pathogen Entamoeba histolytica and representing the third leading cause of death by parasites in the world. Host-parasite interactions mainly involve anchored glycoconjugates localized in the surface of the parasitic cell. In protozoa, synthesis of structural oligo- and polysaccharides occurs via UDP-glucose, generated in a reaction catalyzed by UDP-glucose pyrophosphorylase. We report the molecular cloning of the gene coding for this enzyme from genomic DNA of E. histolytica and its recombinant expression in Escherichia coli cells. The purified enzyme was kinetically characterized, catalyzing UDP-glucose synthesis and pyrophosphorolysis with V(max) values of 95 U/mg and 3 U/mg, respectively, and affinity for substrates comparable to those found for the enzyme from other sources. Enzyme activity was affected by redox modification of thiol groups. Different oxidants, including diamide, hydrogen peroxide and sodium nitroprusside inactivated the enzyme. The process was completely reverted by reducing agents, mainly cysteine, dithiothreitol, and thioredoxin. Characterization of the enzyme mutants C94S, C108S, C191S, C354S, C378S, C108/378S, M106S and M106C supported a molecular mechanism for the redox regulation. Molecular modeling confirmed the role of specific cysteine and methionine residues as targets for redox modification in the entamoebic enzyme. Our results suggest that UDP-glucose pyrophosphorylase is a regulated enzyme in E. histolytica. Interestingly, results strongly agree with the occurrence of a physiological redox mechanism modulating enzyme activity, which would critically affect carbohydrate metabolism in the protozoon.
Assuntos
Entamoeba histolytica/enzimologia , UTP-Glucose-1-Fosfato Uridililtransferase/metabolismo , Clonagem Molecular , Espaço Intracelular/enzimologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oxirredução , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , UTP-Glucose-1-Fosfato Uridililtransferase/química , UTP-Glucose-1-Fosfato Uridililtransferase/genéticaRESUMO
Non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) plays a key metabolic role in higher plants. Purification to homogeneity of enzymes found in relatively low abundance in plants represents a major technical challenge that can be solved by molecular gene cloning and heterologous expression. To apply this strategy to np-Ga3PDHase we performed the cloning of the gapN gene from Arabidopsis thaliana and Triticum aestivum, followed by the heterologous expression in Escherichia coli by two different strategies. Soluble expression of the Arabidopsis enzyme in the pET32c+ vector required a chaperone co-expression system (pGro7). The system using E. coli BL21-CodonPlus cells and the pRSETB vector was successful for expression of a soluble His(6)-taged recombinant wheat enzyme producing 2.5 mg of electrophoretically pure protein per liter of cell culture after a single chromatographic purification step. Both systems were effective for the expression of functional plant np-Ga3PDHases, however the expression of the Arabidopsis enzyme in pRSETB was affordable but not as optimal as for the wheat protein. This would be associated with a different codon usage preference between this specific plant and E. coli. Considering the relevant role played by np-Ga3PDHase in plant metabolism, it is experimentally valuable the development of a procedure to obtain adequate amounts of highly purified enzyme, which envisages the viability to perform studies of structure-to-function relationships to better understand the enzyme kinetics and regulation, as well as carbon and energy metabolism in higher plants.
Assuntos
Arabidopsis/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/isolamento & purificação , Triticum/enzimologia , Arabidopsis/genética , Western Blotting , Clonagem Molecular , Eletroforese , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Cinética , Dados de Sequência Molecular , Fosforilação , Triticum/genéticaRESUMO
Glutaredoxins play an important role in cellular functionality. A putative dithiol glutaredoxin is encoded in the genome of Trypanosoma cruzi. We cloned the gene and obtained the recombinant protein, which behaved as a typical thioltransferase. Activity was variable and dependent on the nature of reducer or oxidant agent used, or both. Epimastigote extracts exhibited similar activity, suggesting the occurrence of the protein in the parasite. Results support a redox scenario in T. cruzi, with glutaredoxin being involved mainly in reduction of glutathione disulfide as well as in deglutathionylation of target proteins.
Assuntos
Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Clonagem Molecular , Dados de Sequência Molecular , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Trypanosoma cruzi/genéticaRESUMO
The full coding sequence for thioredoxin from Trypanosoma cruzi (TcTRX) strain Tulahuen O has been cloned into the pRSETA vector. The protein was expressed in Escherichia coli with an N-terminal extension of six histidine residues for purification through metal ion chromatography. The biological activity of recombinant TcTRX was proved utilizing the insulin reduction assay. Amino acid sequence alignment indicates a high identity of TcTRX with thioredoxins from different sources. Immunocytochemistry assays showed that TcTRX is present in epimastigote forms of T. cruzi, thus, indicating that the gene is expressed in vivo, rather than being a pseudogene. The in vivo occurrence of TcTRX points out the necessity of considering this protein as a molecular component of the redox metabolism in trypanosomatids.