Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible ...glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.
Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells
. Macrophages and satellite cells communicate in different ways
, but their metabolic ...interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.
c-Myc is known to promote glutamine usage by upregulating glutaminase (GLS), which converts glutamine to glutamate that is catabolized in the TCA cycle. Here we report that in a number of human and ...murine cells and cancers, Myc induces elevated expression of glutamate-ammonia ligase (GLUL), also termed glutamine synthetase (GS), which catalyzes the de novo synthesis of glutamine from glutamate and ammonia. This is through upregulation of a Myc transcriptional target thymine DNA glycosylase (TDG), which promotes active demethylation of the GS promoter and its increased expression. Elevated expression of GS promotes cell survival under glutamine limitation, while silencing of GS decreases cell proliferation and xenograft tumor growth. Upon GS overexpression, increased glutamine enhances nucleotide synthesis and amino acid transport. These results demonstrate an unexpected role of Myc in inducing glutamine synthesis and suggest a molecular connection between DNA demethylation and glutamine metabolism in Myc-driven cancers.
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•Myc induces expression of glutamine synthetase (GS)•Increased GS expression is mediated by TDG-mediated promoter demethylation•GS promotes biosynthesis of asparagine and ribonucleotides, and amino acid transport•Suppression of GS decreases cell survival, proliferation, and oncogenesis
Bott et al. reveal that oncogenic Myc induces active DNA demethylation to increase glutamine synthetase (GS) expression and boost glutamine-dependent anabolic processes, including nucleotide synthesis and amino acid transport. GS modulates cell sensitivity to glutamine shortage, as well as proliferation and tumorigenesis.
Summary
Glutamine synthetase (GS; E.C.6.3.1.2) is a key enzyme in higher plants with two isozymes, cytosolic GS1 and plastidic GS2, and involves in the assimilation and recycling of NH4+ ions and ...maintenance of complex traits such as crop nitrogen‐use efficiency and yield. Our present understanding of crop nitrogen‐use efficiency and its correlation with the functional role of the GS family genes is inadequate, which delays harnessing the benefit of this key enzyme in crop improvement. In this report, we performed a comprehensive investigation on the phylogenetic relationship, structural properties, complex multilevel gene regulation, and expression patterns of the GS genes to enrich present understanding about the enzyme. Our Gene Ontology and protein–protein interactions analysis revealed the functional aspects of GS isozymes in stress mitigation, aging, nucleotide biosynthesis/transport, DNA repair and response to metals. The insight gained here contributes to the future research strategies in developing climate‐smart crops for global sustainability.
Significance Statement
Glutamine synthetase (GS) is a key enzyme involved in crop nitrogen‐use efficiency and is a determining factor for yield. Our present understanding of crop nitrogen‐use efficiency and its correlation with the functional role of GS family genes is inadequate, which delays harnessing the benefit of this key enzyme. Present report explores the extent of structural and functional properties of GS isozymes through bioinformatics approaches. In conclusion, this study summarize the present understanding as well as future perspectives of research and manipulation of GS genes/proteins.
Glutamine synthetase (GS) isozymes play critical roles in nitrogen (N) metabolism. However, the exact relationship between GS and nitrogen use efficiency (NUE) remain unclear. We have selected and ...compared two wheat cultivars, YM49 and XN509, which were identified as the N-efficient and N-inefficient genotypes, respectively. In this study, agronomical, morphological, physiological and biochemical approaches were performed. The results showed that TaGS1 was high expressed post-anthesis, and TaGS2 was highly expressed pre-anthesis in N-efficient genotype compared to N-inefficient genotype. GS1 and GS2 isozymes were also separated by native-PAGE and found that the spatial and temporal distribution of GS isozymes, their expression of gene and protein subunits in source-sink-flow organs during development periods triggered the pool strength and influenced the N flow. According to the physiological role of GS isozymes, we illustrated four metabolic regulation points, by which acting collaboratively in different organs, accelerating the transport of nutrients to the grain. It suggested that the regulation of GS isozymes may promote flow strength and enhance NUE by a complex C-N metabolic mechanism. The relative activity or amount of GS1 and GS2 isozymes could be a potential marker to predict and select wheat genotypes with enhanced NUE.
In cnidarian-Symbiodiniaceae symbioses, algal endosymbiont population control within the host is needed to sustain a symbiotic relationship. However, the molecular mechanisms that underlie such ...population control are unclear. Here we show that a cnidarian host uses nitrogen limitation as a primary mechanism to control endosymbiont populations. Nitrogen acquisition and assimilation transcripts become elevated in symbiotic Breviolum minutum algae as they reach high-densities within the sea anemone host Exaiptasia pallida. These same transcripts increase in free-living algae deprived of nitrogen. Symbiotic algae also have an elevated carbon-to-nitrogen ratio and shift metabolism towards scavenging nitrogen from purines relative to free-living algae. Exaiptasia glutamine synthetase and glutamate synthase transcripts concomitantly increase with the algal endosymbiont population, suggesting an increased ability of the host to assimilate ammonium. These results suggest algal growth and replication in hospite is controlled by access to nitrogen, which becomes limiting for the algae as their population within the host increases.
Glutamine is thought to play an important role in cancer cells by being deaminated via glutaminolysis to α-ketoglutarate (aKG) to fuel the tricarboxylic acid (TCA) cycle. Supporting this notion, aKG ...supplementation can restore growth/survival of glutamine-deprived cells. However, pancreatic cancers are often poorly vascularized and limited in glutamine supply, in alignment with recent concerns on the significance of glutaminolysis in pancreatic cancer. Here, we show that aKG-mediated rescue of glutamine-deprived pancreatic ductal carcinoma (PDAC) cells requires glutamate ammonia ligase (GLUL), the enzyme responsible for de novo glutamine synthesis. GLUL-deficient PDAC cells are capable of the TCA cycle but defective in aKG-coupled glutamine biosynthesis and subsequent nitrogen anabolic processes. Importantly, GLUL expression is elevated in pancreatic cancer patient samples and in mouse PDAC models. GLUL ablation suppresses the development of KrasG12D-driven murine PDAC. Therefore, GLUL-mediated glutamine biosynthesis couples the TCA cycle with nitrogen anabolism and plays a critical role in PDAC.
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•GLUL expression is high in human pancreatic cancers and in mouse PDAC models•aKG rescue of glutamine-deprived cells requires GLUL•De novo glutamine synthesis is essential for nitrogen anabolic processes•Genetic ablation of GLUL suppresses mouse PDAC development
Bott et al. demonstrate that GLUL-mediated glutamine synthesis plays a critical role in converging the TCA cycle and nitrogen metabolism to promote nitrogen-dependent anabolic processes in pancreatic cancer. Ablation of GLUL suppresses PDAC development and may have important clinical implications.
Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in ...pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia.
GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced.
Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target.
Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351-363.
Abstract
As the key enzyme of bacterial nitrogen assimilation, glutamine synthetase (GS) is tightly regulated. In cyanobacteria, GS activity is controlled by the interaction with inactivating protein ...factors IF7 and IF17 encoded by the genes gifA and gifB, respectively. We show that a glutamine-binding aptamer within the gifB 5′ UTR of Synechocystis sp. PCC 6803 is critical for the expression of IF17. Binding of glutamine induced structural re-arrangements in this RNA element leading to enhanced protein synthesis in vivo and characterizing it as a riboswitch. Mutagenesis showed the riboswitch mechanism to contribute at least as much to the control of gene expression as the promoter-mediated transcriptional regulation. We suggest this and a structurally related but distinct element, to be designated type 1 and type 2 glutamine riboswitches. Extended biocomputational searches revealed that glutamine riboswitches are exclusively but frequently found in cyanobacterial genomes, where they are primarily associated with gifB homologs. Hence, this RNA-based sensing mechanism is common in cyanobacteria and establishes a regulatory feedback loop that couples the IF17-mediated GS inactivation to the intracellular glutamine levels. Together with the previously described sRNA NsiR4, these results show that non-coding RNA is an indispensable component in the control of nitrogen assimilation in cyanobacteria.
The roles of two cytosolic maize glutamine synthetase isoenzymes (GS1), products of the Gln1-3 and Gln1-4 genes, were investigated by examining the impact of knockout mutations on kernel yield. In ...the gln1-3 and gln1-4 single mutants and the gln1-3 gln1-4 double mutant, GS mRNA expression was impaired, resulting in reduced GS1 protein and activity. The gln1-4 phenotype displayed reduced kernel size and gln1-3 reduced kernel number, with both phenotypes displayed in gln1-3 gln1-4. However, at maturity, shoot biomass production was not modified in either the single mutants or double mutants, suggesting a specific impact on grain production in both mutants. Asn increased in the leaves of the mutants during grain filling, indicating that it probably accumulates to circumvent ammonium buildup resulting from lower GS1 activity. Phloem sap analysis revealed that unlike Gln, Asn is not efficiently transported to developing kernels, apparently causing reduced kernel production. When Gln1-3 was overexpressed constitutively in leaves, kernel number increased by 30%, providing further evidence that GS1-3 plays a major role in kernel yield. Cytoimmunochemistry and in situ hybridization revealed that GS1-3 is present in mesophyll cells, whereas GS1-4 is specifically localized in the bundle sheath cells. The two GS1 isoenzymes play nonredundant roles with respect to their tissue-specific localization.
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