Uridyltransferase Research Articles

Loss of UGP2 in brain leads to a severe epileptic encephalopathy, emphasizing that bi-allelic isoform-specific start-loss mutations of essential genes can cause genetic diseases

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders, resulting in early-onset, therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy, severe developmental delay, progressive microcephaly, visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent, homozygous variant (chr2:64083454A > G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform, which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells, leading to altered glycogen metabolism, upregulated unfolded protein response and premature neuronal differentiation, as modeled during pluripotent stem cell differentiation in vitro. In contrast, the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly, it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease, even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies.

Differential expression of genes involved in the response of Prunus spp. rootstocks under soil flooding

The State of Rio Grande do Sul is used to produce the largest volume of peaches in Brazil. However, productivity in the state is lower than elsewhere in the country. One of the challenges faced by producers in the area is the occurrence soil drainage problems. These problems are observed mainly in Pelotas and its surroundings, and depending on the time of year, may produce flooding issues that hamper both the development and productivity of cultures. In situations in which soil is flooded, oxygen levels are insufficient for root growth, can hinder cellular respiration and may lead to a cellular energy crisis. In addition, global climate change is affecting the climate, and such changes are expected to increase the frequency and severity of flood events in many the agricultural regions. In order to design studies that aim to enhance the selection of flood tolerant genotypes, it is essential to understand the physiological and molecular changes that occur in plants under conditions of oxygen deprivation. Since the production of peach trees in the region is accomplished using rootstocks, the aim of this study was to evaluate gas exchange parameters and gene expression changes involved in glycolytic, fermentative and ethylene metabolism in the peach cultivar NR0170401 and plum cultivars Marianna 2624 and Myrobalan 29C under flooded soil conditions. To this end, gas exchange parameters [net photosynthetic rate (A), and stomatal conductance (gs)] and the expression of nine genes [ACO1 (1-aminocyclopropane-1-carboxylic acid oxidase), ACS5 (1-aminoacyclopropane 1-carboxylic acid synthase), ADH1 (alcohol dehydrogenase 1), G6P (glucose-6-phosphate plastidic transporter), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), LDH1 (lactate dehydrogenase 1), PDC (Pyruvate decarboxylase), SAMs (SAM synthase), and UDPg (UDP-glucose pyrophosphorylase] were evaluated under conditions of flooding relative to controls. An evaluation of gas exchange showed that the plum cultivars are more tolerant to flooding than the peach cultivar studied. RT-qPCR results showed that the three evaluated rootstocks activated fermentative metabolism in roots in the first six hours post-flooding period, and root tissues were more highly affected than leaves. 'Marianna 2426' appears to have a more efficient gene response system compared to the other cultivars studied under conditions of oxygen deprivation. Although more studies are needed, such information has the potential to facilitate the selection of rootstocks used for peach production, for orchard management and for plant breeding programs aiming to produce new cultivars with increased flood tolerance.

Two Homologous Enzymes of the GalU Family in Rhodococcus opacus 1CP-RoGalU1 and RoGalU2.

Uridine-5’-diphosphate (UDP)-glucose is reported as one of the most versatile building blocks within the metabolism of pro- and eukaryotes. The activated sugar moiety is formed by the enzyme UDP-glucose pyrophosphorylase (GalU). Two homologous enzymes (designated as RoGalU1 and RoGalU2) are encoded by most Rhodococcus strains, known for their capability to degrade numerous compounds, but also to synthesize natural products such as trehalose comprising biosurfactants. To evaluate their functionality respective genes of a trehalose biosurfactant producing model organism—Rhodococcus opacus 1CP—were cloned and expressed, proteins produced (yield up to 47 mg per L broth) and initially biochemically characterized. In the case of RoGalU2, the Vmax was determined to be 177 U mg−1 (uridine-5’-triphosphate (UTP)) and Km to be 0.51 mM (UTP), respectively. Like other GalUs this enzyme seems to be rather specific for the substrates UTP and glucose 1-phosphate, as it accepts only dTTP and galactose 1-phoshate in addition, but both with solely 2% residual activity. In comparison to other bacterial GalU enzymes the RoGalU2 was found to be somewhat higher in activity (factor 1.8) even at elevated temperatures. However, RoGalU1 was not obtained in an active form thus it remains enigmatic if this enzyme participates in metabolism.

Optimization of UDP-glucose supply module and production of ginsenoside F1 in Saccharomyces cerevisiae

Ginsenoside F1 is a rare ginsenoside in medicinal plants such as Panax ginseng,P. notogingseng and P. quinquefolius. It has strong pharmacological activities of anti-tumor,anti-oxidation and anti-aging. In order to directly produce ginsenoside F1 by using inexpensive raw materials such as glucose,we integrated the codon-optimized P.ginseng dammarenediol-Ⅱ synthase(Syn Pg DDS),P.ginseng protopanaxadiol synthase(Syn Pg PPDS),P. ginseng protopanaxatriol synthase(Syn Pg PPTS) genes and Arabidopsis thaliana cytochrome P450 reductase(At CPR1) gene into triterpene chassis strain BY-T3. The transformant BY-PPT can produce protopanaxatriol. Then we integrated the Sacchromyces cerevisiae phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2) and UDP-glucose pyrophosphorylase 1(UGP1) genes into chassis strain BY-PPT. The UDP-glucose supply module increased UDP-glucose production by 8. 65 times and eventually reached to 44. 30 mg·L-1 while confirmed in the transformant BY-PPT-GM. Next,we integrated the UDPglucosyltransferase Pg3-29 gene which can catalyze protopanaxatriol to produce ginsenoside F1 into chassis strain BY-PPT-GM. The transformant BY-F1 produced a small amount of ginsenoside F1 which was measured as 0. 5 mg·L-1. After the fermentation process was optimized,the titer of ginsenoside F1 could be increased by 900 times to 450. 5 mg·L-1. The high-efficiency UDP-glucose supply module in this study can provide reference for the construction of cell factories for production of saponin,and provide an important basis for further obtaining high-yield ginsenoside yeast cells.

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

Comparative transcriptome analysis reveals the genetic basis underlying the biosynthesis of polysaccharides in Hericium erinaceus

BackgroundHericium erinaceus, also known as lion’s mane mushroom, is a widely distributed edible and medicinal fungus in Asian countries. H. erinaceus harbors diverse bioactive metabolites with anticancer, immunomodulating, anti-inflammatory, antimicrobial, antihypertensive, antidiabetic and neuroprotective properties. Although the chemical synthesis processes of these bioactive metabolites are known, the biosynthetic processes remain unknown.ResultsIn this study, we obtained the transcriptomes of six H. erinaceus strains using next-generation RNA sequencing and investigated the characteristics of the transcriptomes and biosynthesis of bioactive compounds, especially polysaccharides. The transcriptomes ranged in size from 46.58 to 58.14 Mb, with the number of unigenes ranging from 20,902 to 37,259 across the six H. erinaceus strains. Approximately 60% of the unigenes were successfully annotated by comparing sequences against different databases, including the nonredundant (NR), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), clusters of orthologous groups for eukaryotic complete genomes (KOG) and Swiss-Prot databases. Most of the transcripts were putatively involved in signal transduction, carbohydrate metabolism, translation, transport and catabolism, and amino acid metabolism. Genes involved in polysaccharide biosynthesis were identified, and these genes encoded phosphoglucomutase (PGM), glucose phosphate isomerase (PGI), UDP-glucose pyrophosphorylase (UGP), glycoside hydrolase family proteins, glycosyltransferase family proteins and other proteins. Moreover, the putative pathway for the intracellular polysaccharide biosynthesis of H. erinaceus was analyzed. Additionally, the open reading frames (ORFs) and simple sequence repeats (SSRs) were predicted from the transcriptome data of the six strains.ConclusionsOverall, the present study may facilitate the discovery of polysaccharide biosynthesis processes in H. erinaceus and provide useful information for exploring the secondary metabolites in other members of the Basidiomycetes genus.

Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.

Physiological effects of nitrogen deficiency and recovery on the macroalga Gracilariopsis lemaneiformis (Rhodophyta).

Algal metabolites are the most promising feedstocks for bio-energy production. Gracilariopsis lemaneiformis seems to be a good candidate red alga for polysaccharide production, especially relating to the agar production industry. Nitrogen deficiency is an efficient environmental pressure used to increase the accumulation of metabolites in algae. However, there are no studies on the physiological effects of G.lemaneiformis in response to nitrogen deficiency and its subsequent recovery. Here we integrated physiological data with molecular studies to explore the response strategy of G.lemaneiformis under nitrogen deficiency and recovery. Physiological measurements indicated that amino acids and protein biosynthesis were decreased, while endogenous NH4+ and soluble polysaccharides levels were increased under nitrogen stress. The expression of key genes involved in these pathways further suggested that G.lemaneiformis responded to nitrogen stress through up-regulation or down-regulation of genes related to nitrogen metabolism, and increased levels of endogenous NH4+ to complement the deficiency of exogenous nitrogen. Consistent with the highest accumulation of soluble polysaccharides, the gene encoding UDP-glucose pyrophosphorylase, a molecular marker used to evaluate agar content, was dramatically up-regulated more than 4-fold compared to the relative expression of actin after 4d of nitrogen recovery. The present data provide important information on the mechanisms of nutrient balance in macroalgae.

Newborn Screening for Selected Disorders in Nepal: A Pilot Study.

The prevalence of metabolic disorders in Nepal is yet unknown, although many case reports occur in literature. Heel-prick blood samples from newborns were collected on Dried Blood Spot (DBS) collection cards and tested through Tandem Mass Spectroscopy and fluorescence assays for disorders included in the Swiss neonatal screening program; two cases of hypothyroidism and one case of cystic fibrosis were identified. Thyroid stimulating hormone (TSH), immuoreactive trypsinogen (IRT), hydroxyprogesterone (OHP), tyrosine (Tyr), and octanoylcarnitine (C8) showed significant differences with gestation age. Most of the parameters were positively correlated with each other except galactose, galactose 1 phosphate uridyl transferase (GALT), and biotinidase. First and ninety-ninth percentiles in the Nepalese newborns were found to be different when compared with the Swiss newborns. Congenital hypothyroidism and cystic fibrosis are candidates to be considered for a newborn screening program in Nepal. Differences between the Nepalese and Swiss newborns in parametric values that change with gestation age can be attributed to a higher survival rate of pre-term babies in Switzerland. Others could be explained in part by early and exclusive breastfeeding in Nepalese newborns.

Identification of Leishmania major UDP-Sugar Pyrophosphorylase Inhibitors Using Biosensor-Based Small Molecule Fragment Library Screening.

Leishmaniasis is a neglected disease that is caused by different species of the protozoan parasite Leishmania, and it currently affects 12 million people worldwide. The antileishmanial therapeutic arsenal remains very limited in number and efficacy, and there is no vaccine for this parasitic disease. One pathway that has been genetically validated as an antileishmanial drug target is the biosynthesis of uridine diphosphate-glucose (UDP-Glc), and its direct derivative UDP-galactose (UDP-Gal). De novo biosynthesis of these two nucleotide sugars is controlled by the specific UDP-glucose pyrophosphorylase (UGP). Leishmania parasites additionally express a UDP-sugar pyrophosphorylase (USP) responsible for monosaccharides salvage that is able to generate both UDP-Gal and UDP-Glc. The inactivation of the two parasite pyrophosphorylases UGP and USP, results in parasite death. The present study reports on the identification of structurally diverse scaffolds for the development of USP inhibitors by fragment library screening. Based on this screening, we selected a small set of commercially available compounds, and identified molecules that inhibit both Leishmania major USP and UGP, with a half-maximal inhibitory concentration in the 100 µM range. The inhibitors were predicted to bind at allosteric regulation sites, which were validated by mutagenesis studies. This study sets the stage for the development of potent USP inhibitors.

Comparative proteomic analysis revealed the metabolic mechanism of excessive exopolysaccharide synthesis by Bacillus mucilaginosus under CaCO3 addition

The metabolic mechanism of excessive exopolysaccharide (BMPS) synthesis by Bacillus mucilaginosus CGMCC5766 under CaCO3 addition was investigated. Under CaCO3 (5 g/L), the maximum BMPS concentration reached 28.4 g/L, which was 11.2 folds higher than that of the control. Proteomics was then used to analyze the proteins with substantial differences expressed by B. mucilaginosus with and without CaCO3 addition. The proteomic results revealed that the enzymes related to the central metabolic pathway, amino acid biosynthesis, and nucleotide metabolism were depressed. By contrast, the UDP–glucose pyrophosphorylase involved in BMPS biosynthesis was overexpressed and converted metabolic flux from the biomass accumulation to the biosynthesis of BMPS. This research provides a new and widened perspective into understanding the mechanism of BMPS biosynthesis and applying theoretical and practical significance for the improvement of BMPS production from B. mucilaginosus.

Identification of UGP2 as a progression marker that promotes cell growth and motility in human glioma.

Glioblastoma (GBM) is one of the most common highly malignant primary brain tumor with poor prognosis. This study aimed to explore the possible mechanism by bioinformatics method and detect potential function of UGP2 of GBM. Gene expression microarray data of GSE4412 and messenger RNA-sequencing data of GBM with samples clinical information were downloaded from the Gene Expression Omnibus database and The Cancer Genome Atlas database, respectively. Differentially expressed genes (DEGs) analysis using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology based on R language. A total of 1000 common DEGs were identified in GBM samples, including 353 upregulated and 647 downregulated genes. Based on the random survival forest model, we identified UDP-glucose pyrophosphorylase 2 (UGP2) (upregulated gene) had a significant effect on GBM prognosis. Functional enrichment showed that UGP2 was enriched in the biological progresses of cell proliferation, migration, and invasion. Furthermore, UGP2 expression is aberrantly overexpressed in human glioma and positively correlated with pathologic grade. A loss-of-function study showed that knockdown of UGP2 decreases U251 cell growth, migration, and invasion in vivo and vitro. We proposed the development and progression of human glioma were associated with survival based on bioinformatics analysis. We also found that UGP2 might function as prognostic markers in the pathogenesis of GBM.

Mast Cells Induce Blood Brain Barrier Damage in SCD by Causing Endoplasmic Reticulum Stress in the Endothelium.

Endothelial dysfunction underlies the pathobiology of cerebrovascular disease. Mast cells are located in close proximity to the vasculature, and vasoactive mediators released upon their activation can promote endothelial activation leading to blood brain barrier (BBB) dysfunction. We examined the mechanism of mast cell-induced endothelial activation via endoplasmic reticulum (ER) stress mediated P-selectin expression in a transgenic mouse model of sickle cell disease (SCD), which shows BBB dysfunction. We used mouse brain endothelial cells (mBECs) and mast cells-derived from skin of control and sickle mice to examine the mechanisms involved. Compared to control mouse mast cell conditioned medium (MCCM), mBECs incubated with sickle mouse MCCM showed increased, structural disorganization and swelling of the ER and Golgi, aggregation of ribosomes, ER stress marker proteins, accumulation of galactose-1-phosphate uridyl transferase, mitochondrial dysfunction, reactive oxygen species (ROS) production, P-selectin expression and mBEC permeability. These effects of sickle-MCCM on mBEC were inhibited by Salubrinal, a reducer of ER stress. Histamine levels in the plasma, skin releasate and in mast cells of sickle mice were higher compared to control mice. Compared to control BBB permeability was increased in sickle mice. Treatment of mice with imatinib, Salubrinal, or P-selectin blocking antibody reduced BBB permeability in sickle mice. Mast cells induce endothelial dysfunction via ER stress-mediated P-selectin expression. Mast cell activation contributes to ER stress mediated endothelial P-selectin expression leading to increased endothelial permeability and impairment of BBB. Targeting mast cells and/or ER stress has the potential to ameliorate endothelial dysfunction in SCD and other pathobiologies.

Effect of 2-deoxy-D-glucose on gellan gum biosynthesis by Sphingomonas paucimobilis.

2-Deoxy-D-glucose (2-DG) is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis. Effect of 2-DG on gellan gum biosynthesis by Sphingomonas paucimobilis ATCC31461 were studied in this research. The concentrationand the addition time of 2-DG significantly affected the biomass and gellan gum accumulation. The maximum gellan gum yield of 20.78g/L was obtained with the addition of 50µg/L of 2-DG at 24h. The mechanism of 2-DG addition favoring to gellan production was revealed by determining the activities of key enzymes. Results indicated that 2-DG addition increased the activities of glucosyltransferase and inhibited UDP-glucose pyrophosphorylase activity. The result indicated that 2-DG inhibited glycolysis and changed metabolic driving force to activate gellan gum biosynthesis metabolism pathways.

UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions.

Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.

Effects of (S)-Carvone and Gibberellin on Sugar Accumulation in Potatoes during Low Temperature Storage.

Potato tubers (Solanum tuberosum L.) are usually stored at low temperature, which can suppress sprouting and control the occurrence of diseases. However, low temperatures lead potatoes to easily suffer from cold-induced sweetening (CIS), which has a negative effect on food processing. The aim of this research was to investigate potential treatments on controlling CIS in potatoes during postharvest storage. “Atlantic” potatoes were treated with gibberellin and (S)-carvone, respectively, and stored at 4 °C for 90 days. The results showed that gibberellin can significantly accelerate sprouting and sugar accumulation by regulating expressions of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), β-amylase (BAM1/2), UDP-glucose pyrophosphorylase (UGPase) and invertase inhibitor (INH1/2) genes. The opposite effects were found in the (S)-carvone treatment group, where CIS was inhibited by modulation of the expressions of GBSS and INH1/2 genes. In summary, gibberellin treatment can promote sugar accumulation while (S)-carvone treatment has some effects on alleviating sugar accumulation. Thus, (S)-carvone can be considered as a potential inhibitor of some of the sugars which are vital in controlling CIS in potatoes. However, the chemical concentration, treatment time, and also the treatment method needs to be optimized before industrial application.

Sucrose synthesis in Unpollinated ovaries of pomegranate (Punica granatum L.), as well as, in reproductive and vegetative shoot apices

Sugar levels, the enzyme activity of UDP-Glucose Pyrophosphorylase (UGPase) and Sucrose Phosphate Synthase (SPS), key enzymes for sucrose synthesis, and the expression of their respective genes were examined in developing vegetative and reproductive shoot apices and in developing unpollinated ovaries of Pomegranate trees in order to detect differences possibly important for fruit production and plant vigor. The level of total sugars was higher in ovaries, followed by reproductive apices and vegetative apices, suggesting higher energy requirements in the reproductive organs. Moreover, the activities of SPS and UGPase were found to be elevated in the vegetative apices, indicating that sucrose synthesis is possibly differently regulated in comparison to the reproductive organs. Our results suggest that the sink power of each organ primarily regulates the levels of both transported (sucrose, mannitol) and non-transported (fructose, glucose) sugars. However, the endogenous production of sucrose may be a regulatory factor contributing to organ development.

Elucidating paramylon and other carbohydrate metabolism in Euglena gracilis: Kinetic characterization, structure and cellular localization of UDP-glucose pyrophosphorylase

Many oligo and polysaccharides (including paramylon) are critical in the Euglena gracilis life-cycle and they are synthesized by glycosyl transferases using UDP-glucose as a substrate. Herein, we report the molecular cloning of a gene putatively coding for a UDP-glucose pyrophosphorylase (EgrUDP-GlcPPase) in E. gracilis. After heterologous expression of the gene in Escherichia coli, the recombinant enzyme was characterized structural and functionally. Highly purified EgrUDP-GlcPPase exhibited a monomeric structure, able to catalyze synthesis of UDP-glucose with a Vmax of 3350 U.mg−1. Glucose-1P and UTP were the preferred substrates, although the enzyme also used (with lower catalytic efficiency) TTP, galactose-1P and mannose-1P. Oxidation by hydrogen peroxide inactivated the enzyme, an effect reversed by reduction with dithiothreitol or thioredoxin. The redox process would involve sulfenic acid formation, since no pair of the 7 cysteine residues is close enough in the 3D structure of the protein to form a disulfide bridge. Electrophoresis studies suggest that, after oxidation, the enzyme arranges in many enzymatically inactive structural conformations; which were also detected in vivo. Finally, confocal fluorescence microscopy provided evidence for a cytosolic (mainly in the flagellum) localization of the enzyme.

Streptococcus pneumoniae galU gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo.

Streptococcus pneumoniae, the most common cause of bacterial pneumonia, has developed a wide range of virulence factors to evade the immune system of which the polysaccharide capsule is the most important one. Formation of this capsule is dependent on the cps gene locus, but also involves other genes-like galU. The pyrophosphorylase encoded by galU plays a role in the UDP-glucose metabolism of prokaryotes and is required for the biosynthesis of capsular polysaccharides. In this paper, the effect of a galU mutation leading to a dysfunctional UDP-glucose pyrophosphorylase (UDPG:PP) on in vitro biofilm biomass, adherence to lung epithelial cells and macrophage phagocytosis is studied. Last, in vivo virulence using a Galleria mellonella model has been studied. We show that the mutation improves streptococcal adherence to epithelial cells and macrophage phagocytosis in vitro, while there is no definitive correlation on biofilm formation between parent and mutant strains. Moreover, in vivo virulence is attenuated for all mutated strains. Together, these results demonstrate that a galU mutation in S. pneumoniae influences host cell interactions in vitro and in vivo and can strongly influence the outcome of a streptococcal infection. As such, UDPG:PP is worth investigating further as a potential drug target.

Quantitative Proteomics and Phosphoproteomics Analysis Revealed Different Regulatory Mechanisms of Halothane and Rendement Napole Genes in Porcine Muscle Metabolism.

Pigs with the Halothane (HAL) or Rendement Napole (RN) gene mutations demonstrate abnormal muscle energy metabolism patterns and produce meat with poor quality, classified as pale, soft, and exudative (PSE) meat, but it is not well understood how HAL and RN mutations regulate glucose and energy metabolism in porcine muscle. To investigate the potential signaling pathways and phosphorylation events related to these mutations, muscle samples were collected from four genotypes of pigs, wild type, RN, HAL, and RN-HAL double mutations, and subjected to quantitative proteomic and phosphoproteomic analysis using the TiO2 enrichment strategy. The study led to the identification of 932 proteins from the nonmodified peptide fractions and 1885 phosphoproteins with 9619 phosphorylation sites from the enriched fractions. Among them, 128 proteins at total protein level and 323 phosphosites from 91 phosphoproteins were significantly regulated in mutant genotypes. The quantitative analysis revealed that the RN mutation mainly affected the protein expression abundance in muscle. Specifically, high expression was observed for proteins related to mitochondrial respiratory chain and energy metabolism, thereby enhancing the muscle oxidative capacity. The high content of UDP-glucose pyrophosphorylase 2 (UGP2) in RN mutant animals may contribute to high glycogen storage. However, the HAL mutation mainly contributes to the up-regulation of phosphorylation in proteins related to calcium signaling, muscle contraction, glycogen, glucose, and energy metabolism, and cellular stress. The increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CAMK2) in HAL mutation may act as a key regulator in these processes of muscle. Our findings indicate the different regulatory mechanisms of RN and HAL mutations in relation to porcine muscle energy metabolism and meat quality.