Glucose-1,6-Bisphosphate, a Key Metabolic Regulator, Is Synthesized by a Distinct Family of α-Phosphohexomutases Widely Distributed in Prokaryotes.

The reactions of α-d-phosphohexomutases (αPHM) are ubiquitous, key to primary metabolism, and essential for several processes in all domains of life. The functionality of these enzymes relies on an initial phosphorylation step which requires the presence of α-d-glucose-1,6-bisphosphate (Glc-1,6-BP). While well investigated in vertebrates, the origin of this activator compound in bacteria is unknown. Here we show that the Slr1334 protein from the unicellular cyanobacterium Synechocysitis sp. PCC 6803 is a Glc-1,6-BP-synthase. Biochemical analysis revealed that Slr1334 efficiently converts fructose-1,6-bisphosphate (Frc-1,6-BP) and α-d-glucose-1-phosphate/α-d-glucose-6-phosphate into Glc-1,6-BP and also catalyzes the reverse reaction. As inferred from phylogenetic analysis, the slr1334 product belongs to a primordial subfamily of αPHMs that is present especially in deeply branching bacteria and also includes human commensals and pathogens. Remarkably, the homologue of Slr1334 in the human gut bacterium Bacteroides salyersiae catalyzes the same reaction, suggesting a conserved and essential role for the members of this αPHM subfamily. IMPORTANCE Glc-1,6-BP is known as an essential activator of phosphoglucomutase (PGM) and other members of the αPHM superfamily, making it a central regulator in glycogen metabolism, glycolysis, amino sugar formation as well as bacterial cell wall and capsule formation. Despite this essential role in carbon metabolism, its origin in prokaryotes has so far remained elusive. In this study we identify a member of a specific αPHM subfamily as the first bacterial Glc-1,6-BP synthase, forming free Glc-1,6-BP by using Frc-1,6-BP as phosphoryl-donor. PGMs of this subfamily are widely distributed among prokaryotes including human commensals and pathogens. By showing that a distinct subfamily member can also form Glc-1,6-BP, we provide evidence that Glc-1,6-BP synthase activity is a general feature of this group.

Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5'-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid.

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

Impaired glucose-1,6-biphosphate production due to bi-allelic PGM2L1 mutations is associated with a neurodevelopmental disorder.

We describe a genetic syndrome due to PGM2L1 deficiency. PGM2 and PGM2L1 make hexose-bisphosphates, like glucose-1,6-bisphosphate, which are indispensable cofactors for sugar phosphomutases. These enzymes form the hexose-1-phosphates crucial for NDP-sugars synthesis and ensuing glycosylation reactions. While PGM2 has a wide tissue distribution, PGM2L1 is highly expressed in the brain, accounting for the elevated concentrations of glucose-1,6-bisphosphate found there. Four individuals (three females and one male aged between 2 and 7.5 years) with bi-allelic inactivating mutations of PGM2L1 were identified by exome sequencing. All four had severe developmental and speech delay, dysmorphic facial features, ear anomalies, high arched palate, strabismus, hypotonia, and keratosis pilaris. Early obesity and seizures were present in three individuals. Analysis of the children's fibroblasts showed that glucose-1,6-bisphosphate and other sugar bisphosphates were markedly reduced but still present at concentrations able to stimulate phosphomutases maximally. Hence, the concentrations of NDP-sugars and glycosylation of the heavily glycosylated protein LAMP2 were normal. Consistent with this, serum transferrin was normally glycosylated in affected individuals. PGM2L1 deficiency does not appear to be a glycosylation defect, but the clinical features observed in this neurodevelopmental disorder point toward an important but still unknown role of glucose-1,6-bisphosphate or other sugar bisphosphates in brain metabolism.

Allomorphy as a mechanism of post-translational control of enzyme activity.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. ß-phosphoglucomutase (ßPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of ß-glucose 1-phosphate to glucose 6-phosphate via ß-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of ßPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In ßPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate ß-glucose 1,6-bisphosphate, whose concentration depends on the ß-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Impacts of rat hindlimb Fndc5/irisin overexpression on muscle and adipose tissue metabolism.

Myokines, such as irisin, have been purported to exert physiological effects on skeletal muscle in an autocrine/paracrine fashion. In this study, we aimed to investigate the mechanistic role of in vivo fibronectin type III domain-containing 5 (Fndc5)/irisin upregulation in muscle. Overexpression (OE) of Fndc5 in rat hindlimb muscle was achieved by in vivo electrotransfer, i.e., bilateral injections of Fndc5 harboring vectors for OE rats (n = 8) and empty vector for control rats (n = 8). Seven days later, a bolus of D2O (7.2 mL/kg) was administered via oral gavage to quantify muscle protein synthesis. After an overnight fast, on day 9, 2-deoxy-d-glucose-6-phosphate (2-DG6P; 6 mg/kg) was provided during an intraperitoneal glucose tolerance test (2 g/kg) to assess glucose handling. Animals were euthanized, musculus tibialis cranialis muscles and subcutaneous fat (inguinal) were harvested, and metabolic and molecular effects were evaluated. Muscle Fndc5 mRNA increased with OE (~2-fold; P = 0.014), leading to increased circulating irisin (1.5 ± 0.9 to 3.5 ± 1.2 ng/mL; P = 0.049). OE had no effect on protein anabolism or mitochondrial biogenesis; however, muscle glycogen was increased, along with glycogen synthase 1 gene expression (P = 0.04 and 0.02, respectively). In addition to an increase in glycogen synthase activation in OE (P = 0.03), there was a tendency toward increased glucose transporter 4 protein (P = 0.09). However, glucose uptake (accumulation of 2-DG6P) was identical. Irisin elicited no endocrine effect on mitochondrial biogenesis or uncoupling proteins in white adipose tissue. Hindlimb overexpression led to physiological increases in Fndc5/irisin. However, our data indicate limited short-term impacts of irisin in relation to muscle anabolism, mitochondrial biogenesis, glucose uptake, or adipose remodeling.

Concurrent imaging of vascularization and metabolism in a mouse model of paraganglioma under anti-angiogenic treatment.

Rationale: Deregulation of metabolism and induction of vascularization are major hallmarks of cancer. Using a new multimodal preclinical imaging instrument, we explored a sequence of events leading to sunitinib-induced resistance in a murine model of paraganglioma (PGL) invalidated for the expression of succinate dehydrogenase subunit B (Sdhb-/-). Methods: Two groups of Sdhb-/- tumors bearing mice were treated with sunitinib (6 weeks) or vehicle (3 weeks). Concurrent Positron Emission Tomography (PET) with 2' -deoxy-2'-[18F]fluoro-D-glucose (FDG), Computed Tomography (CT) and Ultrafast Ultrasound Imaging (UUI) imaging sessions were performed once a week and ex vivo samples were analyzed by western blots and histology. Results: PET-CT-UUI enabled to detect a rapid growth of Sdhb-/- tumors with increased glycolysis and vascular development. Sunitinib treatment prevented tumor growth, vessel development and reduced FDG uptake at week 1 and 2 (W1-2). Thereafter, imaging revealed tumor escape from sunitinib treatment: FDG uptake in tumors increased at W3, followed by tumor growth and vessel development at W4-5. Perfused vessels were preferentially distributed in the hypermetabolic regions of the tumors and the perfused volume increased during escape from sunitinib treatment. Finally, initial changes in total lesion glycolysis and maximum vessel length at W1 were predictive of resistance to sunitinib. Conclusion: These results demonstrate an adaptive resistance of Sdhb-/- tumors to six weeks of sunitinib treatment. Early metabolic changes and delayed vessel architecture changes were detectable and predictable in vivo early during anti-angiogenic treatment. Simultaneous metabolic, anatomical and functional imaging can monitor precisely the effects of anti-angiogenic treatment of tumors.

A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.

Few genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for gene knockout, gene knock-in, gene complementation, and endogenous gene tagging. Riboswitches are naturally occurring self-cleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter gene expression in response to exogenous glucosamine, for gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential genes in T. cruzi and to validate potential drug targets in this parasite.

Small RNA-binding protein RapZ mediates cell envelope precursor sensing and signaling in Escherichia coli.

The RNA-binding protein RapZ cooperates with small RNAs (sRNAs) GlmY and GlmZ to regulate the glmS mRNA in Escherichia coli. Enzyme GlmS synthesizes glucosamine-6-phosphate (GlcN6P), initiating cell envelope biosynthesis. GlmZ activates glmS expression by base-pairing. When GlcN6P is ample, GlmZ is bound by RapZ and degraded through ribonuclease recruitment. Upon GlcN6P depletion, the decoy sRNA GlmY accumulates through a previously unknown mechanism and sequesters RapZ, suppressing GlmZ decay. This circuit ensures GlcN6P homeostasis and thereby envelope integrity. In this work, we identify RapZ as GlcN6P receptor. GlcN6P-free RapZ stimulates phosphorylation of the two-component system QseE/QseF by interaction, which in turn activates glmY expression. Elevated GlmY levels sequester RapZ into stable complexes, which prevents GlmZ decay, promoting glmS expression. Binding of GlmY also prevents RapZ from activating QseE/QseF, generating a negative feedback loop limiting the response. When GlcN6P is replenished, GlmY is released from RapZ and rapidly degraded. We reveal a multifunctional sRNA-binding protein that dynamically engages into higher-order complexes for metabolite signaling.

Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis.

Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.

Overexpression of the HECT ubiquitin ligase PfUT prolongs the intraerythrocytic cycle and reduces invasion efficiency of Plasmodium falciparum.

The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3' untranslated region of a gene encoding a HECT E3 ubiquitin ligase, termed Plasmodium falciparum ubiquitin transferase (PfUT), increased steady state RNA and protein levels 2.5-fold in the human malaria parasite P. falciparum. Overexpression of pfut resulted in an S/M phase-associated lengthening of the parasite's intraerythrocytic developmental cycle and a reduced merozoite invasion efficiency. The addition of glucosamine partially restored the wild type phenotype. Our study suggests a role of PfUT in controlling cell cycle progression and merozoite invasion. Our study further raises awareness regarding unexpected effects on gene expression when inserting the glms ribozyme sequence into a gene locus.

ß-Glucose-1,6-Bisphosphate Stabilizes Pathological Phophomannomutase2 Mutants In Vitro and Represents a Lead Compound to Develop Pharmacological Chaperones for the Most Common Disorder of Glycosylation, PMM2-CDG.

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, ß-glucose-1,6-bisphosphate, was synthesized and characterized via 31P-NMR. ß-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. ß-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.

Rapid radiochemical filter paper assay for determination of hexokinase activity and affinity for glucose-6-phosphate.

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.NEW & NOTEWORTHY Traditionally, hexokinase activity has been analyzed spectrophotometrically in which the product formation of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH formation during conversion of G6P to 6-P gluconolactone. By nature, this assay prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have developed a rapid radiochemical filter paper assay to study HK affinity for G6P by use of radiolabeled 2-deoxyglucose as substrate to study physiological regulation of HK affinity for G6P-induced inhibition.

Feedback regulation of small RNA processing by the cleavage product.

Many bacterial small RNAs (sRNAs) are processed resulting in variants with roles potentially distinct from the primary sRNAs. In Enterobacteriaceae sRNA GlmZ activates expression of glmS by base-pairing when the levels of glucosamine-6-phosphate (GlcN6P) are low. GlmS synthesizes GlcN6P, which is required for cell envelope biosynthesis. When dispensable, GlmZ is cleaved by RNase E in the base-pairing sequence. Processing requires protein RapZ, which binds GlmZ and recruits RNase E by interaction. Cleavage is counteracted by the homologous sRNA GlmY, which accumulates upon GlcN6P scarcity and sequesters RapZ. Here, we report a novel role for a processed sRNA. We observed that processing of GlmZ is never complete in vivo. Even upon RapZ overproduction, a fraction of GlmZ remains full-length, while the 5' cleavage product (GlmZ*) accumulates. GlmZ* retains all elements required for RapZ binding. Accordingly, GlmZ* can displace full-length GlmZ from RapZ and counteract processing in vitro. To mimic GlmZ* in vivo, sRNA chimeras were employed consisting of foreign 3' ends including a terminator fused to the 3' end of GlmZ*. In vitro, these chimeras perform indistinguishable from GlmZ*. Expression of the chimeras in vivo inhibited processing of endogenous GlmZ, causing moderate upregulation of GlmS synthesis. Hence, accumulation of GlmZ* prevents complete GlmZ turnover. This mechanism may serve to adjust a robust glmS basal expression level that is buffered against fluctuations in RapZ availability.

Molecular Fingerprints for a Novel Enzyme Family in Actinobacteria with Glucosamine Kinase Activity.

Actinobacteria have long been the main source of antibiotics, secondary metabolites with tightly controlled biosynthesis by environmental and physiological factors. Phosphorylation of exogenous glucosamine has been suggested as a mechanism for incorporation of this extracellular material into secondary metabolite biosynthesis, but experimental evidence of specific glucosamine kinases in Actinobacteria is lacking. Here, we present the molecular fingerprints for the identification of a unique family of actinobacterial glucosamine kinases. Structural and biochemical studies on a distinctive kinase from the soil bacterium Streptacidiphilus jiangxiensis unveiled its preference for glucosamine and provided structural evidence of a phosphoryl transfer to this substrate. Conservation of glucosamine-contacting residues across a large number of uncharacterized actinobacterial proteins unveiled a specific glucosamine binding sequence motif. This family of kinases and their genetic context may represent the missing link for the incorporation of environmental glucosamine into the antibiotic biosynthesis pathways in Actinobacteria and can be explored to enhance antibiotic production.IMPORTANCE The discovery of novel enzymes involved in antibiotic biosynthesis pathways is currently a topic of utmost importance. The high levels of antibiotic resistance detected worldwide threaten our ability to combat infections and other 20th-century medical achievements, namely, organ transplantation or cancer chemotherapy. We have identified and characterized a unique family of enzymes capable of phosphorylating glucosamine to glucosamine-6-phosphate, a crucial molecule directly involved in the activation of antibiotic production pathways in Actinobacteria, nature's main source of antimicrobials. The consensus sequence identified for these glucosamine kinases will help establish a molecular fingerprint to reveal yet-uncharacterized sequences in antibiotic producers, which should have an important impact in biotechnological and biomedical applications, including the enhancement and optimization of antibiotic production.

Dual-tracer PET/CT scan after injection of combined [18 F]NaF and [18 F]FDG outperforms MRI in the detection of myeloma lesions.

The detection rates of whole-body combined [18 F]NaF/[18 F]FDG positron emission tomography combined with computed tomography (PET/CT), CT alone, whole-body magnetic resonance imaging (WB-MRI), and X-ray were prospectively studied in patients with treatment-requiring plasma cell disorders The detection rates of imaging techniques were compared, and focal lesions were classified according to their anatomic location. Twenty-six out of 30 initially included patients were assessable. The number of focal lesions detected in newly diagnosed patients (n = 13) and in relapsed patients (n = 13) were 296 and 234, respectively. The detection rate of PET/CT was significantly higher than those of WB-MRI (P < 0.05) and CT (P < 0.0001) both in patients with newly diagnosed and in those with relapsed multiple myeloma (MM). The X-ray detection rate was significantly lower than those of all other techniques, while CT detected more lesions compared with WB-MRI at diagnosis (P = 0.025). With regard to the infiltration patters, relapsed patients presented more diffuse patterns, and more focal lesions located in the limbs compared with newly diagnosed patients. In conclusion, the detection rate of [18 F]NaF/[18 F]FDG PET/CT was significantly higher than those of CT, MRI, and X-ray, while the detection rate of X-rays was significantly lower than those of all other imaging techniques except for focal lesions located in the skull.

FDG PET/CT as a diagnostic and prognostic tool for the evaluation of marginal zone lymphoma.

We evaluated the role of 18-fluoro-2-deoxy-d-glucose positron emission tomography ([18F] FDG-PET) with computed tomography (CT) (PET/CT) as a diagnostic and prognostic tool in newly diagnosed marginal zone lymphoma (MZL) patients. This is a retrospective cohort study of patients with newly diagnosed MZL, treated with immunotherapy, chemotherapy regimens, surgery, or Helicobacter pylori eradication between 2008 and 2016 in a single tertiary center. Only patients who had a pretreatment PET/CT (P-PET/CT) were included. P-PET/CT, interim (I-PET/CT), and end-of-treatment PET/CT (E-PET/CT) studies were reviewed. P-PET/CT results were reported using two methods of evaluation, qualitative and semi quantitative: visual assessment (VAS) and maximal standardized uptake value (SUVmax), and I-PET and E-PET results were reported by Deauville 5-point score (DS) evaluation as well. Avidity of PET/CT was defined as abnormal uptake in any of these methods. The primary outcome was the prognostic role of P-PET/CT, I-PET/CT, and E-PET/CT on progression-free survival (PFS) and overall survival (OS). Data of 196 patients with MZL were identified, 110 of which had P-PET/CT and were included in this analysis. Median age was 67 years (range 18-93). The median follow-up period was 63 months (range 3-278). The median OS and PFS for the whole cohort were 63 (interquartile range 39-85) and 60 (interquartile range 37-76) months, respectively. The avidity of PET at baseline for the whole cohort was 70% (77/110 patients), for MALT lymphoma, 62.5% (40/64 patients), for NMZL, 76.4% (13/17 patients), and for SMZL, 82.7% (24/29 patients). When adjusted for IPI, sex, and comorbidities, positive E-PET/CT was associated with reduced PFS with a hazard ratio (HR) of 3.4 (95% CI, 1.27-9.14, P = 0.02). Positive E-PET/CT did not correlate with OS. However, there were only three events. P-PET/CT was not predictive of PFS or OS. Our study demonstrates that above 70% of MZL are FDG avid. Positive E-PET/CT is a strong prognostic factor for PFS.

Mammalian Target of Rapamycin 2 (MTOR2) and C-MYC Modulate Glucosamine-6-Phosphate Synthesis in Glioblastoma (GBM) Cells Through Glutamine: Fructose-6-Phosphate Aminotransferase 1 (GFAT1).

Glucose and glutamine are two essential ingredients for cell growth. Glycolysis and glutaminolysis can be linked by glutamine: fructose-6-phosphate aminotransferase (GFAT, composed of GFAT1 and GFAT2) that catalyzes the synthesis of glucosamine-6-phosphate and glutamate by using fructose-6-phosphate and glutamine as substrates. The role of mammalian target of rapamycin (MTOR, composed of MTOR1 and MTOR2) in regulating glycolysis has been explored in human cancer cells. However, whether MTOR can interact with GFAT to regulate glucosamine-6-phosphate is poorly understood. In this study, we report that GFAT1 is essential to maintain the malignant features of GBM cells. And MTOR2 rather than MTOR1 plays a robust role in promoting GFAT1 protein activity, and accelerating the progression of glucosamine-6-phosphate synthesis, which is not controlled by the PI3K/AKT signaling. Intriguingly, high level of glucose or glutamine supply promotes MTOR2 protein activity. In turn, up-regulating glycolytic and glutaminolytic metabolisms block MTOR dimerization, enhancing the release of MTOR2 from the MTOR complex. As a transcriptional factor, C-MYC, directly targeted by MTOR2, promotes the relative mRNA expression level of GFAT1. Notably, our data reveal that GFAT1 immunoreactivity is positively correlated with the malignant grades of glioma patients. Kaplan-Meier assay reveals the correlations between patients' 5-year survival and high GFAT1 protein expression. Taken together, we propose that the MTOR2/C-MYC/GFAT1 axis is responsible for the modulation on the crosstalk between glycolysis and glutaminolysis in GBM cells. Under the condition of accelerated glycolytic and/or glutaminolytic metabolisms, the MTOR2/C-MYC/GFAT1 axis will be up-regulated in GBM cells.

Combinatorial Fine-Tuning of GNA1 and GlmS Expression by 5'-Terminus Fusion Engineering Leads to Overproduction of N-Acetylglucosamine in Bacillus subtilis.

Glucosamine-6-phosphate N-acetyltransferase (GNA1) that catalyzes acetyl transfer from acetyl-coenzyme A to glucosamine-6-phosphate (GlcN-6P), and glutamine-fructose-6-phosphate aminotransferase (GlmS) that catalyzes the formation of GlcN-6P from fructose-6-phosphate (Fru-6P), are two key enzymes in Bacillus subtilis for the bioproduction of N-acetylglucosamine (GlcNAc), a nutraceutical that has various applications in healthcare. In this study, the expression of GNA1 and GlmS is fine-tuned by 5'-terminus fusion engineering to improve GlcNAc production. Specifically, the expression level of GNA1 is enhanced at the translational level via fusion of an epitope tag to the 5'-terminus of GNA1 gene and ribosome binding site (RBS) sequence engineering. Next, enhanced expression of GlmS is achieved at the transcriptional and translational levels by fusing an mRNA stabilizer to the 5'-terminus of GlmS gene. Under the control of GNA1 (fusion with cMyc tag and with the optimum RBS M-Rm) and GlmS (fusion with mRNA stabilizer ΔermC+14/7A), the GlcNAc titer and yield in the shake flask increase to 18.5 g L-1 and 0.37 g GlcNAc/g glucose, which are 2.9-fold and 2.3-fold that of the control, respectively. This synthetic pathway fine-tuning method at the transcriptional and translational levels by combinatorial modulation of regulatory elements, including epitope tag, RBS sequence, and mRNA stabilizer, might represent a general and effective approach for the construction of microbial cell factories.

The Action of the Hexokinase Inhibitor 2-deoxy-d-glucose on Cryptosporidium parvum and the Discovery of Activities against the Parasite Hexokinase from Marketed Drugs.

Cryptosporidium parvum is one of the major species causing mild to severe cryptosporidiosis in humans and animals. We have previously observed that 2-deoxy-d-glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG-6-phosphate (2DG6P), the anti-cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme glucose-6-phosphate isomerase (CpGPI) nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. We also screened 1,200 small molecules consisting of marketed drugs against CpHK, from which four drugs were identified as CpHK inhibitors with micromolar level of anti-cryptospordial activities at concentrations nontoxic to the host cells (i.e. hexachlorphene, thimerosal, alexidine dihydrochloride, and ebselen with EC50  = 0.53, 1.77, 8.1 and 165 µM, respectively). The anti-CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase.

Clinical utility of FDG uptake within reticuloendothelial system on F-18 FDG PET/CT for prediction of tumor recurrence in breast cancer.

BACKGROUND: The aim of this study was to investigate the metabolism of the spleen, bone marrow (BM), and liver from preoperative F-18 FDG PET/CT scans for the prediction of recurrence in breast cancer. METHODS: We retrospectively included 153 patients diagnosed with invasive ductal carcinoma (IDC) of the breast who underwent preoperative F-18 FDG PET/CT scan and a curative operation. The mean standardized uptake value (SUVmean) of the spleen, liver, and BM and maximum SUV (SUVmax) of primary tumors were measured. The relationships between spleen, BM, and liver metabolism and clinicopathologic parameters were evaluated, and possible prognostic parameters predicting recurrence were assessed using disease-free survival (DFS). RESULTS: Spleen SUVmean was significantly correlated with primary tumor SUVmax, pathologic T (pT) stage, and histologic grade of primary tumor. BM SUVmean also showed a positive correlation with primary tumor SUVmax. Spleen SUVmean were significantly associated with recurrence from binary logistic regression analysis (P = 0.004). Spleen, BM, liver, and primary tumor SUVs were all significant prognostic factors for DFS in univariate Cox regression analysis (all P<0.024). Among all PET parameters analyzed, spleen SUVmean ≥ 2.21 (P = 0.032) was in the multivariable analysis the powerful poor prognostic factor predicting DFS that was independent of other clinicopathological features like T stage (pT >2; P = 0.009) and estrogen receptor (ER) status (ER negativity; P = 0.001). CONCLUSION: Splenic metabolism together with pT stage and ER status was an independent prognostic factor for predicting recurrence in breast cancer. Metabolic activity of reticuloendothelial system such as spleen, liver or BM on preoperative F-18 FDG PET/CT can be a meritorious imaging factor for discriminating patients with IDC that require adjunctive therapy to prevent recurrence.