Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Systematic characterization of Gossypium GLN family genes reveals a potential function of GhGLN1.1a regulates nitrogen use efficiency in cotton.

The enzyme glutamine synthetase (GLN) is mainly responsible for the assimilation and reassimilation of nitrogen (N) in higher plants. Although the GLN gene has been identified in various plants, there is little information about the GLN family in cotton (Gossypium spp.). To elucidate the roles of GLN genes in cotton, we systematically investigated and characterized the GLN gene family across four cotton species (G. raimondii, G. arboreum, G. hirsutum, and G. barbadense). Our analysis encompassed analysis of members, gene structure, cis-element, intragenomic duplication, and exploration of collinear relationships. Gene duplication analysis indicated that segmental duplication was the primary driving force for the expansion of the GhGLN gene family. Transcriptomic and quantitative real-time reverse-transcription PCR (qRT-PCR) analyses indicated that the GhGLN1.1a gene is responsive to N induction treatment and several abiotic stresses. The results of virus-induced gene silencing revealed that the accumulation and N use efficiency (NUE) of cotton were affected by the inactivation of GhGLN1.1a. This study comprehensively analyzed the GhGLN genes in Gossypium spp., and provides a new perspective on the functional roles of GhGLN1.1a in regulating NUE in cotton.

Post-Traumatic Expressions of Aromatase B, Glutamine Synthetase, and Cystathionine-Beta-Synthase in the Cerebellum of Juvenile Chum Salmon, Oncorhynchus keta.

In adult fish, neurogenesis occurs in many areas of the brain, including the cerebellum, with the ratio of newly formed cells relative to the total number of brain cells being several orders of magnitude greater than in mammals. Our study aimed to compare the expressions of aromatase B (AroB), glutamine synthetase (GS), and cystathionine-beta-synthase (CBS) in the cerebellum of intact juvenile chum salmon, Oncorhynchus keta. To identify the dynamics that determine the involvement of AroB, GS, and CBS in the cellular mechanisms of regeneration, we performed a comprehensive assessment of the expressions of these molecular markers during a long-term primary traumatic brain injury (TBI) and after a repeated acute TBI to the cerebellum of O. keta juveniles. As a result, in intact juveniles, weak or moderate expressions of AroB, GS, and CBS were detected in four cell types, including cells of the neuroepithelial type, migrating, and differentiated cells (graphic abstract, A). At 90 days post injury, local hypercellular areas were found in the molecular layer containing moderately labeled AroB+, GS+, and CBS+ cells of the neuroepithelial type and larger AroB+, GS+, and CBS+ cells (possibly analogous to the reactive glia of mammals); patterns of cells migration and neovascularization were also observed. A repeated TBI caused the number of AroB+, GS+, and CBS+ cells to further increase; an increased intensity of immunolabeling was recorded from all cell types (graphic abstract, C). Thus, the results of this study provide a better understanding of adult neurogenesis in teleost fishes, which is expected to clarify the issue of the reactivation of adult neurogenesis in mammalian species.

Kallistatin leads to cognition impairment via downregulating glutamine synthetase.

In many neurodegenerative disorders, such as Alzheimer's disease (AD), glutamate-mediated neuronal excitotoxicity is considered the basis for cognitive impairment. The mRNA and protein expression of SERPINA4(Kallistatin) are higher in patients with AD. However, whether Kallistatin plays a regulatory role in glutamate-glutamine cycle homeostasis remains unclear. In this study, we identified impaired cognitive function in Kallistatin transgenic (KAL-TG) mice. Baseline glutamate levels were elevated and miniature excitatory postsynaptic current (mEPSC) frequency was increased in the hippocampus, suggesting the impairment of glutamate homeostasis in KAL-TG mice. Mechanistically, we demonstrated that Kallistatin promoted lysine acetylation and ubiquitination of glutamine synthetase (GS) and facilitated its degradation via the proteasome pathway, thereby downregulating GS. Fenofibrate improved cognitive memory in KAL-TG mice by downregulating serum Kallistatin. Collectively, our study findings provide insights the mechanism by which Kallistatin regulates cognitive impairment, and suggest the potential of fenofibrate to prevente and treat of AD patients with high levels of Kallistatin.

Abundant urinary amino acids activate glutamine synthetase-encoding glnA by two different mechanisms in Escherichia coli.

Growth of uropathogenic Escherichia coli in the bladder induces transcription of glnA which codes for the ammonia-assimilating glutamine synthetase (GS) despite the normally suppressive high ammonia concentration. We previously showed that the major urinary component, urea, induces transcription from the Crp-dependent glnAp1 promoter, but the urea-induced transcript is not translated. Our purpose here was to determine whether the most abundant urinary amino acids, which are known to inhibit GS activity in vitro, also affect glnA transcription in vivo. We found that the abundant amino acids impaired growth, which glutamine and glutamate reversed; this implies inhibition of GS activity. In strains with deletions of crp and glnG that force transcription from the glnAp2 and glnAp1 promoters, respectively, we examined growth and glnA transcription with a glnA-gfp transcriptional fusion and quantitative reverse transcription PCR with primers that can distinguish transcription from the two promoters. The abundant urinary amino acids stimulated transcription from the glnAp2 promoter in the absence of urea but from the glnAp1 promoter in the presence of urea. However, transcription from glnAp1 did not produce a translatable mRNA or GS as assessed by a glnA-gfp translational fusion, enzymatic assay of GS, and Western blot to detect GS antigen in urea-containing media. We discuss these results within the context of the extremely rapid growth of uropathogenic E. coli in urine, the different factors that control the two glnA promoters and possible mechanisms that either overcome or bypass the urea-imposed block of glutamine synthesis during bacterial growth in urine.IMPORTANCEKnowledge of the regulatory mechanisms for genes expressed at the site of infection provides insight into the virulence of pathogenic bacteria. During urinary tract infections-most often caused by Escherichia coli-growth in urine induces the glnA gene which codes for glutamine synthetase. The most abundant urinary amino acids amplified the effect of urea which resulted in hypertranscription from the glnAp1 promoter and, unexpectedly, an untranslated transcript. E. coli must overcome this block in glutamine synthesis during growth in urine, and the mechanism of glutamine acquisition or synthesis may suggest a possible therapy.

Metabolic changes during wheat microspore embryogenesis induction using the highly responsive cultivar Svilena.

Androgenetically-derived haploids can be obtained by inducing embryogenesis in microspores. Thus, full homozygosity is achieved in a single generation, oppositely to conventional plant breeding programs. Here, the metabolite profile of embryogenic microspores of Triticum aestivum was acquired and integrated with transcriptomic existing data from the same samples in an effort to identify the key metabolic processes occurring during the early stages of microspore embryogenesis. Primary metabolites and transcription profiles were identified at three time points: prior to and immediately following a low temperature pre-treatment given to uninuclear microspores, and after the first nuclear division. This is the first time an integrative -omics analysis is reported in microspore embryogenesis in T. aestivum. The key findings were that the energy produced during the pre-treatment was obtained from the tricarboxylic acid (TCA) cycle and from starch degradation, while starch storage resumed after the first nuclear division. Intermediates of the TCA cycle were highly demanded from a very active amino acid metabolism. The transcription profiles of genes encoding enzymes involved in amino acid synthesis differed from the metabolite profiles. The abundance of glutamine synthetase was correlated with that of glutamine. Cytosolic glutamine synthetase isoform 1 was found predominantly after the nuclear division. Overall, energy production was shown to represent a major component of the de-differentiation process induced by the pre-treatment, supporting a highly active amino acid metabolism.

Nitrogen application in pod zone improves yield and quality of two peanut cultivars by modulating nitrogen accumulation and metabolism.

Cultivated peanut (Arachis hypogaea L.) represents one of the most important oil and cash crops world-widely. Unlike many other legumes, peanuts absorb nitrogen through their underground pods. Despite this unique feature, the relationship between yield and nitrogen uptake within the pod zone remains poorly understood. In our pot experiment, we divided the underground peanut part into two zones-pod and root-and investigated the physiological and agronomic traits of two peanut cultivars, SH11 (large seeds, LS) and HY23 (small seeds, SS), at 10 (S1), 20 (S2), and 30 (S3) days after gynophores penetrated the soil, with nitrogen application in the pod zone. Results indicated that nitrogen application increased pod yield, kernel protein content, and nitrogen accumulation in plants. For both LS and SS peanut cultivars, optimal nitrogen content was 60 kg·hm- 2, leading to maximum yield. LS cultivar exhibited higher yield and nitrogen accumulation increases than SS cultivar. Nitrogen application up-regulated the expression of nitrogen metabolism-related genes in the pod, including nitrate reductase (NR), nitrite reductase (NIR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), ATP binding cassette (ABC), and nitrate transporter (NRT2). Additionally, nitrogen application increased enzyme activity in the pod, including NR, GS, and GOGAT, consistent with gene expression levels. These nitrogen metabolism traits exhibited higher up-regulations in the large-seeded cultivar than in the small-seeded one and showed a significant correlation with yield in the large-seeded cultivar at S2 and S3. Our findings offer a scientific basis for the judicious application and efficient utilization of nitrogen fertilization in peanuts, laying the groundwork for further elucidating the molecular mechanisms of peanut nitrogen utilization.

Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations.

Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50'-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

Integrated proteome and physiological traits reveal interactive mechanisms of new leaf growth and storage protein degradation with mature leaves of evergreen citrus trees.

The growth of fruit trees depends on the nitrogen (N) remobilization in mature tissues and N acquisition from the soil. However, in evergreen mature citrus (Citrus reticulata Blanco) leaves, proteins with N storage functions and hub molecules involved in driving N remobilization remain largely unknown. Here, we combined proteome and physiological analyses to characterize the spatiotemporal mechanisms of growth of new leaves and storage protein degradation in mature leaves of citrus trees exposed to low-N and high-N fertilization in the field. Results show that the growth of new leaves is driven by remobilization of stored reserves, rather than N uptake by the roots. In this context, proline and arginine in mature leaves acted as N sources supporting the growth of new leaves in spring. Time-series analyses with gel electrophoresis and proteome analysis indicated that the mature autumn shoot leaves are probably the sites of storage protein synthesis, while the aspartic endopeptidase protein is related to the degradation of storage proteins in mature citrus leaves. Furthermore, bioinformatic analysis based on protein-protein interactions indicated that glutamate synthetase and ATP-citrate synthetase are hub proteins in N remobilization from mature citrus leaves. These results provide strong physiological data for seasonal optimization of N fertilizer application in citrus orchards.

Portosinusoidal Vascular Disorder: A Heretofore Unrecognized Manifestation of Sickle Cell Disease?

Portosinusoidal vascular disorder (PSVD) is a recently proposed histopathologic entity that encompasses a spectrum of often subtle hepatic microvascular lesions and related microarchitectural abnormalities. Clinical manifestations may arise years after histologic diagnosis and include extrahepatic portal vein thrombosis and portal hypertension. While the histopathologic features of PSVD have been associated with numerous clinical conditions, most notably prothrombotic/vasculopathic disorders, PSVD has not yet been described in sickle cell disease. This gap is striking given the central role of microvascular dysfunction in sickle cell disease and well-described patterns of hepatic injury and dysfunction in this population. This case series is the first to explore the prevalence and pathogenesis of PSVD in sickle cell disease. Forty-one diagnostically adequate liver biopsies from patients with sickle cell disease were identified across the archives of 5 tertiary medical centers. All biopsies exhibited at least 1 histopathologic feature associated with PSVD (mean 3.8 features/case). Overall, 90.2% of patients met the criteria for a diagnosis of PSVD based on the presence of specific histopathologic and/or clinical findings. Immunohistochemical stains for von Willebrand factor, CD34, and glutamine synthetase were performed on 36 cases (87.8%). Aberrant (centrilobular sinusoidal) CD34 and von Willebrand factor staining was present in 97.2% and 86.1% of cases, respectively. Glutamine synthetase reactivity was at least mildly decreased in zone 3 hepatocytes in 52.8% of cases. We posit that chronic erythrocyte sickling results in dysfunction and remodeling of the portal microvasculature, culminating in regression of zone 3 hepatocytes. The presence of PSVD may explain, at least in part, the hepatic dysfunction observed in this patient population. These patients may also benefit from extended clinical surveillance for portal hypertension and other complications. While subtle and prone to overdiagnosis, the features of PSVD should be carefully considered when interpreting liver biopsies from patients with sickle cell disease.

Leat-associated seizures the possible role of EAAT2, pyruvate carboxylase and glutamine synthetase.

BACKGROUND: Drug-resistant epilepsy is a common condition in patients with brain neoplasms. The pathogenesis of tumor-associated seizures is poorly understood. Among the possible pathogenetic mechanisms, the increase in glutamate concentration has been proposed. Glutamate transporters, glutamine synthetase and pyruvate carboxylase are involved in maintaining the physiological concentration of glutamate in the intersynaptic spaces. In our previous research on angiocentric gliomas, we demonstrated that all tumors lacked the expression of the main glutamate transporter EAAT2, while the expression of glutamine synthetase and pyruvate carboxylase was mostly preserved. METHODS: In the present study, we evaluated the immunohistochemical expression of EAAT2, glutamine synthetase and pyruvate carboxylase in a heterogeneous series of 25 long-term epilepsy-associated tumors (10 dysembryoplastic neuroepithelial tumors, 7 gangliogliomas, 3 subependymal giant cell astrocytomas, 3 rosette forming glioneuronal tumors, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). In order to evaluate the incidence of variants in the SLC1A2 gene, encoding EAAT2, in a large number of central nervous system tumors we also queried the PedcBioPortal. RESULTS: EAAT2 protein expression was lost in 9 tumors (36 %: 3 dysembryoplastic neuroepithelial tumors, 1 ganglioglioma, 3 subependymal giant cell astrocytomas, 1 diffuse astrocytoma MYB- or MYBL1-altered and 1 angiocentric glioma). Glutamine synthetase protein expression was completely lost in 2 tumors (8 %; 1 ganglioglioma and 1 diffuse astrocytoma MYB- or MYBL1-altered). All tumors of our series but rosette forming glioneuronal tumors (in which neurocytic cells were negative) were diffusely positive for pyruvate carboxylase. Consultation of the PedcBioPortal revealed that of 2307 pediatric brain tumors of different histotype and grade, 20 (< 1%) had variants in the SLC1A2 gene. Among the SLC1A2-mutated tumors, there were no angiocentric gliomas or other LEATs CONCLUSIONS: In conclusion, unlike angiocentric gliomas where the EAAT2 loss is typical and constant, the current study shows the loss of EAAT2 expression only in a fraction of the LEATs. In these cases, we may hypothesize some possible epileptogenic role of the EAAT2 loss. The retained expression of pyruvate carboxylase may contribute to determining a pathological glutamate excess unopposed by glutamine synthetase that resulted expressed to a variable extent in the majority of the tumors. Furthermore, we can assume that the EAAT2 loss in brain tumors in general and in LEATs in particular is more conceivably epigenetic.

Astrocyte S100ß expression and selective differentiation to GFAP and GS in the entorhinal cortex during ageing in the 3xTg-Alzheimer's disease mouse model.

The study of astrocytes and its role in the development and evolution of neurodegenerative diseases, including Alzheimer's disease (AD) is essential to fully understand their aetiology. The aim if this study is to deepen into the concept of the heterogeneity of astrocyte subpopulations in the EC and in particular the identification of differentially functioning astrocyte subpopulations that respond differently to AD progression. S100ß protein belongs to group of small calcium regulators of cell membrane channels and pumps that are expressed by astrocytes and is hypothesised to play and have a relevant role in AD development. We analysed the selective differentiation of S100ß-positive astrocytes into Glutamine synthetase (GS) and Glial fibrillary acidic protein (GFAP)-positive sub-groups in the entorhinal cortex (EC) of AD triple transgenic animal model (3xTg-AD). EC is the brain region earliest affected in humans AD but also in this closest animal model regarding their pathology and time course. We observed no changes in the number of S100ß-positive astrocytes between 1 and 18 months of age in the EC of 3xTg-AD mice. However, we identified relevant morphological changes in S100ß/GFAP positive astrocytes showing a significant reduction in their surface and volume whilst an increase in number and percentage. Furthermore, the percentage of S100ß/GS positive astrocyte population was also increased in 18 months old 3xTg-AD mice compared to the non-Tg mice. Our findings reveal the presence of differentially controlled astrocyte populations that respond differently to AD progression in the EC of 3xTg-AD mice. These results highpoints the major astrocytic role together with its early and marked affection in AD and arguing in favour of its importance in neurogenerative diseases and potential target for new therapeutic approaches.

Nicotinamide prevention in diabetes-induced alterations in the rat liver.

Objective. The study was performed to elucidate whether nicotinamide (NAm) can attenuate the diabetes-induced liver damage by correction of ammonia detoxifying function and disbalance of NAD-dependent processes in diabetic rats. Methods. After four weeks of streptozotocin-induced diabetes, Wistar male rats were treated for two weeks with or without NAm. Urea concentration, arginase, and glutamine synthetase activities, NAD+ levels, and NAD+/NADH ratio were measured in cytosolic liver extracts. Expression of parp-1 gene in the liver was estimated by quantitative polymerase chain reaction and PARP-1 cleavage evaluated by Western blotting. Results. Despite the blood plasma lipid peroxidation products in diabetic rats were increased by 60%, the activity of superoxide dismutase (SOD) was reduced. NAm attenuated the oxidative stress, but did not affect the enzyme activity in diabetic rats. In liver of the diabetic rats, urea concentration and arginase activity were significantly higher than in the controls. The glutamine synthetase activity was decreased. Decline in NAD+ level and cytosolic NAD+/NADH ratio in the liver of diabetic rats was observed. Western blot analysis demonstrated a significant up-regulation of PARP-1 expression accompanied by the enzyme cleavage in the diabetic rat liver. However, no correlation was seen between mRNA expression of parp-1 gene and PARP-1 protein in the liver of diabetic rats. NAm markedly attenuated PARP-1 cleavage induced by diabetes, but did not affect the parp-1 gene expression. Conclusions. NAm counteracts diabetes-induced impairments in the rat liver through improvement of its detoxifying function, partial restoration of oxidative stress, NAD+ level, normalization of redox state of free cytosolic NAD+/NADH-couples, and prevention of PARP-1 cleavage.

Responses of root characteristics and nitrogen absorption and assimilation to different pH gradients of winter wheat at seedling stage.

Nitrogen (N) and rhizosphere pH are the two main factors restricting the growth of winter wheat (Triticum aestivum L.) in North China Plain. Soil nutrient availability is affected by soil acidity and alkalinity. In order to understand the effect of rhizosphere pH value on wheat nitrogen metabolism and the response of wheat growth to pH value at seedling stage, winter wheat varieties 'Aikang 58' (AK58) and 'Bainong 4199' (BN4199) were tested in hydroponics under three pH treatments (pH = 4.0, 6.5, and 9.0). The results showed that the accumulation of dry matter in root and above ground under pH 4.0 and pH 9.0 treatments was lower than that under pH 6.5 treatments, and the root/shoot ratio increased with the increase of pH value. Regardless of pH value, 'BN4199' had higher root dry weight, root length, root surface area, root activity and root tip than 'AK58'. Therefore, wheat that is tolerant to extreme pH is able to adapt to the acid-base environment by changing root characteristics. At pH 4.0, the net H+ outflow rate of wheat roots was significantly lower than that of the control group, and the net NO3- flux of wheat roots was also low. The net H+ outflow occurred at pH 6.5 and 9.0, and at the same time, the net NO3- flux of roots also increased, and both increased with the increase of pH. The activity of nitrate reductase (NR) in stem of pH 9.0 treatment was significantly higher than that of other treatments, while the activity of glutamine synthetase (GS) in root and stem of pH 6.5 treatment was significantly higher than that of other treatments. Under pH 4.0 and pH 9.0 treatments, the activities of NR and GS in 'BN4199' were higher than those in 'AK58', The root respiration of 'BN4199' was significantly higher than that of 'AK58' under pH 4.0 and pH 9.0 treatment, and 'BN4199' had higher NO3- net flux, key enzyme activity of root nitrogen metabolism and root respiration. Therefore, we believe that 'BN4199' has strong resistance ability to extreme pH stress, and high root/shoot ratio and strong root respiration can be used as important indicators for wheat variety screening adapted to the alkaline environment at the seedling stage.

Hormonal Status of Transgenic Birch with a Pine Glutamine Synthetase Gene during Rooting In Vitro and Budburst Outdoors.

Improving nitrogen use efficiency (NUE) is one of the main ways of increasing plant productivity through genetic engineering. The modification of nitrogen (N) metabolism can affect the hormonal content, but in transgenic plants, this aspect has not been sufficiently studied. Transgenic birch (Betula pubescens) plants with the pine glutamine synthetase gene GS1 were evaluated for hormone levels during rooting in vitro and budburst under outdoor conditions. In the shoots of the transgenic lines, the content of indoleacetic acid (IAA) was 1.5-3 times higher than in the wild type. The addition of phosphinothricin (PPT), a glutamine synthetase (GS) inhibitor, to the medium reduced the IAA content in transgenic plants, but it did not change in the control. In the roots of birch plants, PPT had the opposite effect. PPT decreased the content of free amino acids in the leaves of nontransgenic birch, but their content increased in GS-overexpressing plants. A three-year pot experiment with different N availability showed that the productivity of the transgenic birch line was significantly higher than in the control under N deficiency, but not excess, conditions. Nitrogen availability did not affect budburst in the spring of the fourth year; however, bud breaking in transgenic plants was delayed compared to the control. The IAA and abscisic acid (ABA) contents in the buds of birch plants at dormancy and budburst depended both on N availability and the transgenic status. These results enable a better understanding of the interaction between phytohormones and nutrients in woody plants.

The Role of Glutamine Synthetase (GS) and Glutamate Synthase (GOGAT) in the Improvement of Nitrogen Use Efficiency in Cereals.

Cereals are the most broadly produced crops and represent the primary source of food worldwide. Nitrogen (N) is a critical mineral nutrient for plant growth and high yield, and the quality of cereal crops greatly depends on a suitable N supply. In the last decades, a massive use of N fertilizers has been achieved in the desire to have high yields of cereal crops, leading to damaging effects for the environment, ecosystems, and human health. To ensure agricultural sustainability and the required food source, many attempts have been made towards developing cereal crops with a more effective nitrogen use efficiency (NUE). NUE depends on N uptake, utilization, and lastly, combining the capability to assimilate N into carbon skeletons and remobilize the N assimilated. The glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle represents a crucial metabolic step of N assimilation, regulating crop yield. In this review, the physiological and genetic studies on GS and GOGAT of the main cereal crops will be examined, giving emphasis on their implications in NUE.

Ammonia scavenger and glutamine synthetase inhibitors cocktail in targeting mTOR/ß-catenin and MMP-14 for nitrogen homeostasis and liver cancer.

The glutamine synthetase (GS) facilitates cancer cell growth by catalyzing de novo glutamine synthesis. This enzyme removes ammonia waste from the liver following the urea cycle. Since cancer development is associated with dysregulated urea cycles, there has been no investigation of GS's role in ammonia clearance. Here, we demonstrate that, although GS expression is increased in the setting of ß-catenin oncogenic activation, it is insufficient to clear the ammonia waste burden due to the dysregulated urea cycle and may thus be unable to prevent cancer formation. In vivo study, a total of 165 male Swiss albino mice allocated in 11 groups were used, and liver cancer was induced by p-DAB. The activity of GS was evaluated along with the relative expression of mTOR, ß-catenin, MMP-14, and GS genes in liver samples and HepG2 cells using qRT-PCR. Moreover, the cytotoxicity of the NH3 scavenger phenyl acetate (PA) and/or GS-inhibitor L-methionine sulfoximine (MSO) and the migratory potential of cells was assessed by MTT and wound healing assays, respectively. The Swiss target prediction algorithm was used to screen the mentioned compounds for probable targets. The treatment of the HepG2 cell line with PA plus MSO demonstrated strong cytotoxicity. The post-scratch remaining wound area (%) in the untreated HepG2 cells was 2.0%. In contrast, the remaining wound area (%) in the cells treated with PA, MSO, and PA + MSO for 48 h was 61.1, 55.8, and 78.5%, respectively. The combination of the two drugs had the greatest effect, resulting in the greatest decrease in the GS activity, ß-catenin, and mTOR expression. MSO and PA are both capable of suppressing mTOR, a key player in the development of HCC, and MMP-14, a key player in the development of HCC. PA inhibited the MMP-14 enzyme more effectively than MSO, implying that PA might be a better way to target HCC as it inhibited MMP-14 more effectively than MSO. A large number of abnormal hepatocytes (5%) were found to be present in the HCC mice compared to mice in the control group as determined by the histopathological lesions scores. In contrast, PA, MSO, and PA + MSO showed a significant reduction in the hepatic lesions score either when protecting the liver or when treating the liver. The molecular docking study indicated that PA and MSO form a three-dimensional structure with NF-κB and COX-II, blocking their ability to promote cancer and cause gene mutations. PA and MSO could be used to manipulate GS activities to modulate ammonia levels, thus providing a potential treatment for ammonia homeostasis.

A carbon-nitrogen negative feedback loop underlies the repeated evolution of cnidarian-Symbiodiniaceae symbioses.

Symbiotic associations with Symbiodiniaceae have evolved independently across a diverse range of cnidarian taxa including reef-building corals, sea anemones, and jellyfish, yet the molecular mechanisms underlying their regulation and repeated evolution are still elusive. Here, we show that despite their independent evolution, cnidarian hosts use the same carbon-nitrogen negative feedback loop to control symbiont proliferation. Symbiont-derived photosynthates are used to assimilate nitrogenous waste via glutamine synthetase-glutamate synthase-mediated amino acid biosynthesis in a carbon-dependent manner, which regulates the availability of nitrogen to the symbionts. Using nutrient supplementation experiments, we show that the provision of additional carbohydrates significantly reduces symbiont density while ammonium promotes symbiont proliferation. High-resolution metabolic analysis confirmed that all hosts co-incorporated glucose-derived 13C and ammonium-derived 15N via glutamine synthetase-glutamate synthase-mediated amino acid biosynthesis. Our results reveal a general carbon-nitrogen negative feedback loop underlying these symbioses and provide a parsimonious explanation for their repeated evolution.

M. mazei glutamine synthetase and glutamine synthetase-GlnK1 structures reveal enzyme regulation by oligomer modulation.

Glutamine synthetases (GS) play central roles in cellular nitrogen assimilation. Although GS active-site formation requires the oligomerization of just two GS subunits, all GS form large, multi-oligomeric machines. Here we describe a structural dissection of the archaeal Methanosarcina mazei (Mm) GS and its regulation. We show that Mm GS forms unstable dodecamers. Strikingly, we show this Mm GS oligomerization property is leveraged for a unique mode of regulation whereby labile Mm GS hexamers are stabilized by binding the nitrogen regulatory protein, GlnK1. Our GS-GlnK1 structure shows that GlnK1 functions as molecular glue to affix GS hexamers together, stabilizing formation of GS active-sites. These data, therefore, reveal the structural basis for a unique form of enzyme regulation by oligomer modulation.

Glutamine synthetase and hepatocellular carcinoma.

Glutamine synthetase (GS) is an enzyme that converts ammonia and glutamate to glutamine using adenosine triphosphate. GS is expressed in the brain, kidney, and liver tissues under normal physiological conditions. GS is involved in abnormal lipid metabolism of the liver by catalyzing de novo synthesis of glutamine, thereby inducing liver inflammation. Metabolic dysfunction-associated steatotic liver diseases (MASLD), such as Metabolic Associated Fatty Liver Disease and Metabolic Associated Steato Hepatitis, are considered risk factors for HCC. GS may also be involved in the development and progression of hepatocellular carcinoma (HCC) through other signaling pathways, including the rapamycin (mTOR) and Wnt/ß-catenin signaling pathways. Furthermore, the correct combination of HSP70, GPC3, and GS can improve the accuracy and precision of HCC diagnosis. However, the prognostic value of GS in different HCC populations remains controversial. The expression of GS affects the sensitivity of HCC cells to radiotherapy and chemotherapy. In addition, immunotherapy has been approved for the treatment of advanced HCC. This article delves into the development and application of GS in HCC, laying a theoretical foundation for the subsequent exploration of GS as a potential target for treating HCC.