Uridine Diphosphate Research Articles

Contribution of GalU to biofilm formation, motility, antibiotic and serum resistance, and pathogenicity of Salmonella Typhimurium.

Salmonella Typhimurium is the leading cause of foodborne illnesses in China, resulting in major epidemics and economic losses in recent years. Uridine diphosphate-glucose pyrophosphorylase galU plays an important role in thebiosynthesis of the bacterial envelope. Herein, we evaluated the role of galU in S. Typhimurium infection in chicken. A galU gene mutant was successfully constructed by red homologous recombination technology, and biological characteristics were studied. The galU mutant strain had a rough phenotype;was defective in biofilm formation, autoagglutination, and motility; exhibited greater sensitivity to most antibiotics, serum, and egg albumen; and had lowercapacity for adhesion to chicken embryo fibroblasts cell line (DF-1). The galU mutant showed dramatically attenuated pathogenicity in chicken embryos (100,000-fold), BALB/c mice (420-fold), and chicks (100-fold). The results imply that galU is an important virulence factor in the pathogenicity of S. Typhimurium, and it may serve a target for the development of veterinary drugs, providing a theoretical basis for the prevention and control of S. Typhimurium.

Protein O-GlcNAcylation impairment caused by N-acetylglucosamine phosphate mutase deficiency leads to growth variations in Arabidopsis thaliana.

As an essential enzyme in the uridine diphosphate (UDP)-GlcNAc biosynthesis pathway, the significant role of N-acetylglucosamine phosphate mutase (AGM) remains unknown in plants. In the present study, a functional plant AGM (AtAGM) was identified from Arabidopsis thaliana. AtAGM catalyzes the isomerization of GlcNAc-1-P and GlcNAc-6-P, and has broad catalytic activity on different phosphohexoses. UDP-GlcNAc contents were significantly decreased in AtAGM T-DNA insertional mutants, which caused temperature-dependent growth defects in seedlings and vigorous growth in adult plants. Further analysis revealed that protein O-GlcNAcylation but not N-glycosylation was dramatically impaired in Atagm mutants due to UDP-GlcNAc shortage. Combined with the results from O-GlcNAcylation or N-glycosylation deficient mutants, and O-GlcNAcase inhibitor all suggested that protein O-GlcNAcylation impairment mainly leads to the phenotypic variations of Atagm plants. In conclusion, based on the essential role in UDP-GlcNAc biosynthesis, AtAGM is important for plant growth mainly via protein O-GlcNAcylation-level regulation.

Coupled reaction of glycosyltransferase and sucrose synthase for high-yielding and cost-effective synthesis of rosin

Rosin has wide applications in food and pharmaceutical fields. Herein, 96% yield of rosin was synthesized using Arabidopsis thaliana glucosyltransferase (UGT73C5) from 2 mM cinnamyl alcohol (CA) and excess UDP-glucose (UDPG). Furthermore, replacing expensive UDPG by uridine diphosphate (UDP), coupled reaction by Arabidopsis thaliana sucrose synthase (AtSUS1) and UGT73C5 led to 100% yield of rosin. In addition, crude UGT73C5 and AtSUS1 resulted in rosin yield of 95% (10 mM CA) and 94% (20 mM CA) without UDP addition. With elevated CA concentration, whole-cell catalysis led to even higher rosin yield than crude enzymes preparations with a final yield of 91% from 50 mM CA. Furthermore, in cascaded reaction with a newly introduced Catharanthus roseus glucosyltransferase (CaUGT3), rosavin B was synthesized with a yield of 62% from 50 mM CA. This developed coupled pathway provides a feasible and cost-effective approach for the production of rosin and its glucosides from CA.

Comparative structural analysis of plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) in plant specialized metabolism: structures of plant UGTs for biosynthesis of steviol glycosides

Comparative structural analysis of plant uridine diphosphate (UDP)-dependent glycosyltransferases (UGTs) in plant specialized metabolism: structures of plant UGTs for biosynthesis of steviol glycosides

Nitrogen enhances the effect of pre-drought priming against post-anthesis drought stress by regulating starch and protein formation in wheat.

Drought stress is one of the most serious environmental stress factor constraining crop production across the globe. Among cereals, wheat grains are very sensitive to drought as a small degree of stress can affect the enzymatic system. This study aimed to investigate whether nitrogen and pre-anthesis drought priming could enhance the action of major regulatory enzymes involved in starch accumulation and protein synthesis in bread wheat (Triticum aestivum L.). For this purpose, cultivars YM-158 (medium gluten) and YM-22 (low gluten) were grown in rain-controlled conditions under two nitrogen levels, that is, N180 (N1) and N300 (N2). Drought priming was applied at the jointing stage and drought stress was applied 7 days after anthesis. Drought stress reduced starch content but enhanced protein content in grains. N2 and primed plants kept higher contents of nonstructural carbohydrates, fructans, and sucrose; with higher activity of sucrose-phosphate synthase in flag leaves. Furthermore, N2 and priming treatments showed higher sink ability to develop grains by showing higher sucrose-to-starch conversion activities of adenosine diphosphate-glucose pyrophosphorylase, uridine diphosphate glucose pyrophosphorylase, sucrose-synthase, soluble-starch synthase, starch branching enzyme, and granule-bound starch synthase as compared to N1 and non-primed treatments. The application of N2 and primed treatment showed a greater ability to maintain grain filling in both cultivars as compared to N1 and non-primed crops. Our study suggested that high nitrogen has the potential to enhance the effect of pre-drought priming to change source-sink relationships and grain yield of wheat under drought stress during the filling process.

Lipid nanoparticle-encapsulated mRNA therapy corrects serum total bilirubin level in Crigler-Najjar syndrome mouse model

Crigler-Najjar syndrome is a rare disorder of bilirubin metabolism caused by uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) mutations characterized by hyperbilirubinemia and jaundice. No cure currently exists; treatment options are limited to phototherapy, whose effectiveness diminishes over time, and liver transplantation. Here, we evaluated the therapeutic potential of systemically administered, lipid nanoparticle-encapsulated human UGT1A1 (hUGT1A1) mRNA therapy in a Crigler-Najjar mouse model. Ugt1 knockout mice were rescued from lethal post-natal hyperbilirubinemia by phototherapy. These adult Ugt1 knockout mice were then administered a single lipid nanoparticle-encapsulated hUGT1A1 mRNA dose. Within 24 h, serum total bilirubin levels decreased from 15 mg/dL (256μmol/L) to <0.5mg/dL (9μmol/L), i.e., slightly above wild-type levels. This reduction was sustained for 2weeks before bilirubin levels rose and returned to pre-treatment levels by day 42 post-administration. Sustained reductions in total bilirubin levels were achieved by repeated administration of the mRNA product in a frequency-dependent manner. We were also able to rescue the neonatal lethality phenotype seen in Ugt1 knockout mice with a single lipid nanoparticle dose, which suggests that this may be a treatment modality appropriate for metabolic crisis situations. Therefore, lipid nanoparticle-encapsulated hUGT1A1 mRNA may represent a potential treatment for Crigler-Najjar syndrome.

The Uridine diphosphate (UDP)-glycosyltransferases (UGTs) superfamily: the role in tumor cell metabolism.

UDP-glycosyltransferases (UGTs), important enzymes in biotransformation, control the levels and distribution of numerous endogenous signaling molecules and the metabolism of a wide range of endogenous and exogenous chemicals. The UGT superfamily in mammals consists of the UGT1, UGT2, UGT3, and UGT8 families. UGTs are rate-limiting enzymes in the glucuronate pathway, and in tumors, they are either overexpressed or underexpressed. Alterations in their metabolism can affect gluconeogenesis and lipid metabolism pathways, leading to alterations in tumor cell metabolism, which affect cancer development and prognosis. Glucuronidation is the most common mammalian conjugation pathway. Most of its reactions are mainly catalyzed by UGT1A, UGT2A and UGT2B. The body excretes UGT-bound small lipophilic molecules through the bile, urine, or feces. UGTs conjugate a variety of tiny lipophilic molecules to sugars, such as galactose, xylose, acetylglucosamine, glucuronic acid, and glucose, thereby inactivating and making water-soluble substrates, such as carcinogens, medicines, steroids, lipids, fatty acids, and bile acids. This review summarizes the roles of members of the four UGT enzyme families in tumor function, metabolism, and multiple regulatory mechanisms, and its Inhibitors and inducers. The function of UGTs in lipid metabolism, drug metabolism, and hormone metabolism in tumor cells are among the most important topics covered.

Identifying a Detoxifying Uridine Diphosphate Glucosyltransferase (UGT), MdUGT83K2, Which Can Glycosylate the Aryloxyphenoxypropionate Herbicide

Glycosylation is a common modification reaction in plants. The products obtained upon glycosylation have different biological functions, making glycosylation an important mechanism affecting and regulating the balance of plant growth and metabolism. In this study, we first speculated that Group I in the apple glycosyltransferase family may have a predicted function like UGT83A1, according to gene chip data published online. Subsequently, by real-time PCR (polymerase chain reaction), we analyzed whether the expression of nine glycosyltransferase genes in Group I was induced by our previously reported ACCase (Acetyl-CoA carboxylase) inhibition-based herbicide QPP ((R)-ethyl·2-(4-((6-fluoro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)oxy) phenoxy) propanoate). It was found that expression of the MdUGT83K2 gene in Group I was significantly increased by QPP. In order to determine whether MdUGT83K2 can glycosylate QPP, we confirmed the enzymatic reaction of MdUGT83K2 in vitro and the presence of QPP glycosides in MdUGT83K2 transgenic apple seedlings by HPLC (High Performance Liquid Chromatography), and found that MdUGT83K2 can transfer glucose to QPP in vivo, which is glycosylated. In this work, we identified a novel apple glycosyltransferase, MdUGT83K2, which functions to glycosylate the ACCase-inhibiting herbicide QPP and may be involved in plant detoxification. Key Contribution: A novel apple glycosyltransferase, MdUGT83K2, was identified, which may be involved in plant detoxification by glycosylation modification of the ACCase-inhibiting herbicide.

Inhibition of protein phosphatase 2A by okadaic acid induces translocation of nucleocytoplasmic O-GlcNAc transferase

Post-translational modification (PTM) is crucial for many biological events, such as the modulation of bone metabolism. Phosphorylation and O-GlcNAcylation are two examples of PTMs that can occur at the same site in the protein: serine and threonine residues. This phenomenon may cause crosstalk and possible interactions between the molecules involved. Protein phosphatase 2 A (PP2A) is widely expressed throughout the body and plays a major role in dephosphorylation. At the same location where PP2A acts, O-GlcNAc transferase (OGT) can introduce uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) molecules and mediates O-GlcNAc modifications. To examine the effects of PP2A inhibition on OGT localization and expression, osteoblastic MC3T3-E1 cells were treated with Okadaic Acid (OA), a potent PP2A inhibitor. In the control cells, OGT was strictly localized in the nucleus. However, OGT was observed diffusely in the cytoplasm of the OA-treated cells. This change in localization from the nucleus to the cytoplasm resulted from an increase in mitochondrial OGT expression and translocation of the nucleocytoplasmic isoform. Furthermore, knockdown of PP2A catalytic subunit α isoform (PP2A Cα) significantly affected OGT expression (p < 0.05), and there was a correlation between PP2A Cα and OGT expression (r = 0.93). These results suggested a possible interaction between PP2A and OGT, which strengthens the notion of an interaction between phosphorylation and O-GlcNAcylation.

A new function of a putative UDP-glucose 4-epimerase on the expression of glycoside hydrolase genes in Aspergillus aculeatus.

In order to figure out the induction mechanisms of glycoside hydrolase genes in Aspergillus aculeatus, we screened approximately 9,000 transfer DNA (T-DNA)-inserted mutants for positive regulators involved in the induction. Since the mutants possess the orotidine 5'-monophosphate decarboxylase gene as a reporter gene to monitor the cellulose-responsive expression of the cellobiohydrolase I gene (cbhI), candidate strains were isolated by counterselection against 5-fluoroorotic acid (5-FOA). One 5-FOA-resistant mutant harboring the T-DNA at the uge5 locus showed reduced cellulose utilization and cbhI expression. A. aculeatus Uge5 is homologous to Aspergillus fumigatus uge5 (Afu5g10780; E-value, 0.0; identities, 93%), which catalyzes the conversion of uridine diphosphate (UDP)-glucose to UDP-galactopyranose. The uge5 deletion mutant in A. aculeatus (Δuge5) showed reduced conidium formation on minimal media supplemented with galactose, locust bean gum (LBG), and guar gum as a carbon source. β-1,4-Endoglucanase and β-1,4-mannanase production in submerged culture containing LBG was reduced to 10% and 6% of the control strain at day 5, respectively, but no difference was observed in cultures containing wheat bran. The expression of major cellulolytic and mannolytic genes in the presence of mannobiose in Δuge5 was reduced to less than 15% of the control strain, while cellobiose-responsive expression was only modestly reduced at early inducing time points. Since all test genes were controlled by a transcription factor ManR, these data demonstrate that Uge5 is involved in inducer-dependent selective expression of genes controlled via ManR. KEY POINTS: • UDP-glucose 4-epimerase (Uge5) regulates expression of glycosyl hydrolase genes. • ManR regulates both cellobiose- and mannobiose-responsive expression. • Uge5 plays a key role in mannobiose-responsive expression.

Anticancer Properties of Hexosamine Analogs Designed to Attenuate Metabolic Flux through the Hexosamine Biosynthetic Pathway.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac4Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac4Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac4Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac4Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac4Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

Overexpression of the Persimmon Abscisic Acid DkUGT3 Gene Alters Plant/Fruit Development in Transgenic Tomato

Persimmon (Diospyros kaki Thunb.) contains numerous uridine diphosphate glucosyltransferases (UGT), and their roles in fruit development and quality formation have not been well studied because of limited genetic information. This study investigated a persimmon DkUGT3 which is highly expressed in young fruits and leaves during development. DkUGT3 can catalyze ABA glycosylation to form ABA-GE, thereby reducing free ABA. Tomato with overexpressed (OE) DkUGT3 significantly induces pale green color phenotypes in both transgenic young plants and fruits. DkUGT3-OE significantly weakens the tomato ABA signaling which affects the expressions of ABA-inducible transcriptional factors (TFs), such as GLK1 and GLK2 and their downstream target genes involved in chlorophyll synthesis, chloroplast development, sugar metabolism and transport, and photosynthesis, thereby impeding leaf and fruit development and quality. Conversely, DkUGT3-RNAi-treatment recovered the OE tomato fruits from yellowing phenotype to green color. This study found that chlorophyll accumulations and ABA level were increased by DkUGT3-RNAi-treatment in young persimmon leaves. These results demonstrate that DkUGT3 plays crucial roles in ABA-mediated leaf and fruit development. This study provides new evidence for the regulation of ABA in early development.

Multienzyme Synthesis of Glycyrrhetic Acid 3-O-mono-β-d-glucuronide by Coupling UGT73F15 to UDP-Glucuronic Acid Regeneration Module

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG), a rare and innovative compound in licorice, exhibits high-potency sweetness and improved physiological activities. However, low amounts of GAMG from plants cannot meet the demands of growing markets. In this study, an efficient one-pot multienzyme cascade reaction for GAMG biosynthesis was constructed using a coupled catalysis of glycosyltransferase and uridine 5′-diphosphate (UDP) glucuronic acid (GlcA) regeneration system. The Glycyrrhiza uralensis glycosyltransferase UGT73F15 was expressed in Escherichia coli BL21 (DE3). The optimal reaction conditions of UGT73F15 were found to be pH 7.5 and 35 °C. The catalytic efficiency (kcat/Km) for glycyrrhetic acid (GA) was 2.14 min−1 mM−1 when using UDP-GlcA as sugar donor. To regenerate costly UDP-GlcA, the one-pot multienzyme cascade reaction including UGT73F15, sucrose synthase, UDP-glucose dehydrogenase, and lactate dehydrogenase was adopted to synthesize GAMG from GA on the basis of the UDP-GlcA regeneration system. By optimizing the cascade reaction conditions, the GAMG production successfully achieved 226.38 mg/L. Our study developed an economical and efficient one-pot multienzyme cascade method for facile synthesis of GAMG and other bioactive glucuronosides.

UGT2B7 c.-161C>T polymorphism frequency in Croatian population.

Uridine diphosphate glucuronosyltransferase-2B7 (UGT2B7), enzyme responsible for the elimination of a number of xenobiotics through glucuronidation, is expressed in the gut, kidneys, intestines, and brain. However, data on the frequency of UGT2B7 polymorphisms in the Croatian population are limited. The aim of this study was to assess the frequency of the UGT2B7 c.-161C>T (rs7668258) polymorphism in the Croatian population and to compare it with reported frequencies in other populations. This polymorphism is in complete linkage disequilibrium with the UGT2B7 c.802C>T (UGT2B7*2, rs7439366) variant, which is important in clinical medicine. The study reports data of 501 participants from University Hospital Centre Zagreb. All data were collected and analysed retrospectively. Genotyping was performed by real-time polymerase chain reaction (PCR) using the TaqMan® Drug Metabolism Genotyping Assay for UGT2B7 c.-161C>T (rs7668258). We found that 120 (23.95 %) participants were carriers of the UGT2B7 c.-161CC genotype and 255 (50.9 %) were heterozygous carriers (UGT2B7 c.-161CT), while 126 (25.15 %) were homozygous carriers of the variant allele (UGT2B7 c.-161TT). The frequency of the variant UGT2B7 c.-161C>T allele in this study was T=0.506. The frequency of the UGT2B7 c.-161C>T allelic variants and genotypes in the Croatian population is similar to other European populations.

NOVEL ACTIVE SITE INHIBITION OF O-LINKED N-ACETYL GLUCOSAMINE TRANSFERASE (OGT) FROM ALKALOID LIBRARY

O-linked-N- acetyl glucosaminetransferase (OGT) is an important enzyme which regulates many important cellular processes through posttranscriptional modification of the nuclear and cytosolic proteins. OGT catalyzes the attachment of N- acetylglucosamine portion of uridine diphosphate N- acetyl glucosamine (UDP- GlcNac) to the serine and threonine residues of nuclear and cytoplasmic proteins of multicellular eukaryote in protein O-GlcNacylation process. Hyperglycemia increases GlcNacylation of proteins. GlcNacylation has been linked to some human diseases, such as cardiovascular diseases, diabetes mellitus, neurodegenerative diseases, and cancer. Several inhibitors of the enzyme have been explored in the treatment of the diseases associated with it. In this study, alkaloids were mined from online database and screened computationally using molecular docking approach. 290 alkaloids were retrieved and docked against OGT, using OGT complexed with a thio – linked substrate as the control, and the docking approach predicted 14-norpseurotinA (14-NSA), Protoveratrine B (PB) and 3-(4-methoxyphenyl)-3-[[2-(4-methoxyphenyl)-1-oxoethyl] amino] propanoic acid (MPOAPA) to be suitable inhibitors of OGT, through their interaction and binding pose with the amino acid residues at the substrate-binding site of the protein, with a docking score of -11.106, -9.176 and -8.269 respectively. Additionally, the hit compounds were then subjected to ADMETox screening to predict their drug – likeness. The ADMETox analysis predicted that 14-NSA, and MPOAPA shows more drug-like characteristics compared to PB and the co crystallised ligand by obeying the rule of five for drug development. However, natural bioactive compounds 14-NSA, and MPOAPA could be a potential active site inhibitor of OGT in the treatment of cancer, diabetes, neurodegenerative diseases and cardiovascular diseases caused by aberrant O- GlcNacylation.

Whole-crop biorefinery of corn biomass for pullulan production by Aureobasidium pullulans

In the present study, corn starch, cob, and straw were biorefined and used as feedstocks for the production of pullulan. The titer and molecular weight (Mw) of pullulan significantly decreased when corn cob and straw hydrolysates were utilized by the parental strain Aureobasidium pullulans CCTCC M 2012259 (PS). Based on adaptive laboratory evolution of PS, an evolved strain A. pullulans EV6 with strong adaptability to the whole corn biomass hydrolysate and high capability of pullulan biosynthesis was screened. Batch pullulan fermentation results indicated that EV6 produced an increased titer of pullulan with a higher Mw than PS. The underlying reasons for these increases were revealed by assaying key enzymes activities and measuring intracellular uridine diphosphate glucose levels. Subsequently, whole-crop biorefinery of corn biomass was conducted, and the results confirmed that whole corn crop has immense potential for efficient pullulan production.

Contribution of UDP-glycosyltransferases to chlorpyrifos resistance in Nilaparvata lugens

As a multigene superfamily of Phase II detoxification enzymes, uridine diphosphate (UDP)-glycosyltransferases (UGTs) play important roles in the metabolism of xenobiotics including insecticides. In this study, 5-nitrouracil, an inhibitor of UGT enzyme activity, effectively increased the toxicity of chlorpyrifos to the chlorpyrifos-resistant strain of Nilaparvata lugens, one of the most resistant rice pests. The enzyme content of UGT in the resistant strain was significantly higher than that in the susceptible strain. Among 20 identified UGT genes, UGT386H2, UGT386J2, UGT386N2 and UGT386P1 were found significantly overexpressed in the resistant strain and can be effectively induced by chlorpyrifos. These four UGT genes were most highly expressed in the midgut and/or fat body, two main insect detoxification tissues. Amino acid sequence alignments revealed that these four UGTs contained a variable N-terminal substrate-binding domain and a conserved C-terminal sugar donor-binding domain. Furthermore, homology modeling and molecular docking analyses showed that these UGTs could stably bind to chlorpyrifos and chlorpyrifos oxon, with the binding free energies from −19.4 to −110.62 kcal mol−1. Knockdown of UGT386H2 or UGT386P1 by RNA interference dramatically increased the susceptibility of the resistant strain to chlorpyrifos. These findings suggest that overexpression of these two UGT genes contributes to chlorpyrifos resistance in N. lugens.

Selective involvement of UGGT variant: UGGT2 in protecting mouse embryonic fibroblasts from saturated lipid-induced ER stress

Secretory proteins and lipids are biosynthesized in the endoplasmic reticulum (ER). The "protein quality control" system (PQC) monitors glycoprotein folding and supports the elimination of terminally misfolded polypeptides. A key component of the PQC system is Uridine diphosphate glucose:glycoprotein glucosyltransferase 1 (UGGT1). UGGT1 re-glucosylates unfolded glycoproteins, to enable the re-entry in the protein-folding cycle and impede the aggregation of misfolded glycoproteins. In contrast, a complementary "lipid quality control" (LQC) system that maintains lipid homeostasis remains elusive. Here, we demonstrate that cytotoxic phosphatidic acid derivatives with saturated fatty acyl chains are one of the physiological substrates of UGGT2, an isoform of UGGT1. UGGT2 produces lipid raft-resident phosphatidylglucoside regulating autophagy. Under the disruption of lipid metabolism and hypoxic conditions, UGGT2 inhibits PERK-ATF4-CHOP-mediated apoptosis in mouse embryonic fibroblasts. Moreover, the susceptibility of UGGT2 KO mice to high-fat diet-induced obesity is elevated. We propose that UGGT2 is an ER-localized LQC component that mitigates saturated lipid-associated ER stress via lipid glucosylation.

The galU gene is required for in vivo survival of pseudomonas plecoglossicida in large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is an important pathogenic bacterium in aquaculture that causes visceral granulomas in large yellow croaker (Larimichthys crocea). Uridine diphosphate glucose phosphorylase encoded by galU plays a key role in biosynthesis of the bacterial envelope, particularly lipopolysaccharide and the capsule. In this study, we inactivated the galU gene in the P. plecoglossicida isolate XSDHY-P. The galU mutant strain showed impaired growth in the early exponential stage and lacked the O polysaccharide side chain in lipopolysaccharide, but almost no defect in biofilm formation was detected. The galU mutant strain also exhibited significantly more sensitivity to the bactericidal action of normal fish serum mediated by the complement system compared to the wild-type strain. In a cell model originating from the head kidney of large yellow croaker, the galU mutant strain showed lower capacities of adhesion, invasion, and intracellular survival compared to the wild-type strain. In addition, the deficiency of the galU mutant drastically decreased bacterial loads in tissues and attenuated P. plecoglossicida virulence in fish. These results suggest that the galU gene of P. plecoglossicida is required for in vivo survival in large yellow croaker.

Characterisation of UDP-glucuronosyltransferase activity in sea turtle Chelonia mydas

Uridine diphosphate glucuronosyltransferase (UGT) enzymes conjugate many lipophilic chemicals, such as drugs, environmental contaminants, and endogenous compounds, promoting their excretion. The complexity of UGT kinetics, and the location of enzyme active site in endoplasmic reticulum lumen, requires an accurate optimisation of enzyme assays. In the present study, we characterised UGT activity in liver microsomes of green turtles (Chelonia mydas), an endangered species. The conditions for measuring UGT activity were standardised through spectrofluorimetric methods, using the substrates 4-methylumbelliferone (4-MU) and uridine diphosphate glucuronic acid (UDPGA) at 30 °C and pH 7.4. The green turtles showed UGT activity at the saturating concentrations of substrates of 250 µM to 4-MU and 7 mM to UDPGA. The alamethicin, Brij®58, bovine serum albumin (BSA), and magnesium increased UGT activity. The assay using alamethicin (22 µg per mg of protein), magnesium (1 mM), and BSA (0.25%) reached the highest V max (1203 pmol·min−1mg·protein−1). Lithocholic acid and diclofenac inhibited UGT activity in green turtles. This study is the first report of UGT activity in the liver of green turtles and provides a base for future studies to understand the mechanisms of toxicity by exposure to contaminants in this charismatic species.