Studies on the control of hexosamine biosynthesis by glucosamine synthetase.
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ABSTRACT:
SUBMITTER: Winterburn PJ
PROVIDER: S-EPMC1176649 | biostudies-other | 1971 Feb
REPOSITORIES: biostudies-other
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Project description:Background: Endothelial cells (ECs) use glycolysis to produce energy. In pre-clinical models of peripheral arterial disease (PAD), the further activation of EC glycolysis was ineffective and/or deleterious in promoting hypoxia-dependent angiogenesis while pentose phosphate pathway (PPP) activation was effective. Hexosamine biosynthesis pathway (HBP), PPP, and glycolysis are closely linked. Glucosamine directly activates HBP.Methods:Hind-limb ischemia (HLI)ineNOS-/-and Balb/c mice was used. Glucosamine (600 ug/g/day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging (LDPI), angiogenesis was studied by CD31immunostaining.In-vitro: human umbilical vein ECs (HUVECs) and mouse microvascular EC with glucosamine, L-glucose or vascular endothelial growth factor (VEGF165a), were tested underhypoxiaand serum starvation (HSS). Cell counting kit-8 (CCK-8), tube formation, intracellular reactive oxygen species (ROS), Electric Cell-substrate Impedance Sensing (ECIS) and FITC dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time qPCR, and western blot. Human muscle biopsies from patients with PAD were assessed for EC O-GlcNAcylation before and after supervised exercise vs. standard medical care. Results: Day-3 post-HLI,glucosamine vs. control-treated eNOS-/-had less necrosis.Beginning Day-7 post-HLI, glucosamine vs. control-treated Balb/chad higherblood flowthat persisted to Day-21whereischemic musclesshowed greaterCD31 staining/muscle fiber. In-vitro,glucosamine vs. L-glucoseshowedimproved EC survival and tube formation. RNA-sequencing of glucosamine vs. L-glucose showed increased amino acid metabolism. That resulted in increased oxidative phosphorylation and serine biosynthesis pathway (SBP) without an increase in glycolysis or PPP genes. This was associated with better barrier function and less ROS compared to activating glycolysis by VEGF165a. These effects were mediated by activating transcription factor 4 (ATF4); a driver of exercise-induced angiogenesis. Finally, in muscle biopsies from humans with PAD,EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise vs. standard medical care. Conclusion: In-cells, mice, and humans activation of HBP by glucosamine in PAD inducesan “exercise-like” angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder.
2024-06-29 | GSE270552 | GEO
Project description:Aspergillus fumigatus is a human opportunistic fungal pathogen whose cell wall protects it from the extracellular environment including host defenses. Chitin, an essential component of the fungal cell wall, is synthesized from UDP-GlcNAc produced in the hexosamine biosynthetic pathway. As this pathway is critical for fungal cell wall integrity, the hexosamine biosynthesis enzymes represent potential targets of antifungal drugs. Here, we provide genetic and chemical evidence that glucosamine 6-phosphate N-acetyltransferase (Gna1), a key enzyme in this pathway, is an exploitable antifungal drug target. GNA1 deletion resulted in loss of fungal viability and disruption of the cell wall, phenotypes that could be rescued by exogenous GlcNAc, the product of the Gna1 enzyme. In a murine model of aspergillosis, the ?gna1 mutant strain exhibited attenuated virulence. Using a fragment-based approach, we discovered a small heterocyclic scaffold that binds proximal to the Gna1 active site and can be optimized to a selective submicromolar binder. Taken together, we have provided genetic, structural, and chemical evidence that Gna1 is an antifungal target in A. fumigatus.
| S-EPMC7324522 | biostudies-literature
Project description:Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) turnover. Compared with physiological glucose (5.6 mM), treatment with high glucose (25 mM) for 24 h stimulated MC proliferation, an effect that was mimicked by exposure to low concentrations of glucosamine (0.05 mM). The percentage of cells in G(0)/G(1) phase of the cell cycle was reduced with a concomitant increase of the number of cells in G(2)/M phase. Proliferating cell nuclear antigen, phosphorylated mammalian target of rapamycin [phospho-mTOR (Ser(2448))], and total regulatory-associated protein of mTOR were increased by high glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (10 muM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher glucosamine concentrations (0.25-10 mM) caused MC apoptosis after 48 h, and, in addition, GFAT overexpression also increased MC apoptosis (TdT-dUTP nick end-labeling-positive cells: 3.8 +/- 0.3 vs. 1.1 +/- 0.2% for empty vector; P < 0.05). Hence, hexosamine flux is an important determinant of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin-sensitive mTOR complex 1 (mTORC1).
| S-EPMC2822474 | biostudies-literature
Project description:The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1's essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell's energy resources are converted into structural carbohydrates in response to changing cellular needs.
| S-EPMC7340321 | biostudies-literature
Project description:Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.
| S-EPMC8093268 | biostudies-literature
Project description:1. The binding of substrates and effectors to glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was studied by using the ligand to alter the denaturation rate of the enzyme. The free enzyme bound fructose 6-phosphate, glucose 6-phosphate and UDP-N-acetylglucosamine, but not glutamine, AMP or UTP. Glucose 6-phosphate and AMP increased the binding of UDP-N-acetylglucosamine whereas UTP decreased the interaction between the enzyme and the feedback inhibitor. UDP-N-acetylglucosamine induced a glutamine-binding site on the enzyme. 2. Selective thermal or chemical denaturation revealed that the UDP-N-acetylglucosamine-binding site was not located at the catalytic site. The UTP site could not be distinguished from that for the nucleotide sugar. The AMP- and glucose 6-phosphate-binding sites were distinct from the catalytic and feedback-inhibitor-binding sites. 3. The specificity of the glutamine-binding site was investigated by using a series of potential analogues. 4. A model is proposed for the action of the effectors and the mechanism of the reaction discussed in kinetic and chemical terms.
| S-EPMC1176650 | biostudies-other
Project description:Pancreatic ductal adenocarcinoma (PDAC) is considered to be a highly immunosuppressive and heterogenous neoplasm. Despite improved knowledge regarding the genetic background of the tumor and better understanding of the tumor microenvironment, immune checkpoint inhibitor therapy (targeting CTLA4, PD1, PDL1) has not been very successful against PDAC. The robust desmoplastic stroma, along with an extensive extracellular matrix (ECM) that is rich in hyaluronan, plays an integral role in this immune evasion. Hexosamine biosynthesis pathway (HBP), a shunt pathway of glycolysis, is a metabolic node in cancer cells that can promote survival pathways on the one hand and influence the hyaluronan synthesis in the ECM on the other. The rate-limiting enzyme of the pathway, glutamine-fructose amidotransferase 1 (GFAT1), uses glutamine and fructose 6-phosphate to eventually synthesize uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). In the current manuscript, we targeted this glutamine-utilizing enzyme by a small molecule glutamine analog (6-diazo-5-oxo-l-norleucine [DON]). Our results showed that DON decreased the self-renewal potential and metastatic ability of tumor cells. Further, treatment with DON decreased hyaluronan and collagen in the tumor microenvironment, leading to an extensive remodeling of the ECM and an increased infiltration of CD8+ T cells. Additionally, treatment with DON sensitized pancreatic tumors to anti-PD1 therapy, resulting in tumor regression and prolonged survival.
| S-EPMC6934212 | biostudies-literature
Project description:The hexosamine biosynthetic pathway (HBP) plays critical roles in nutrient sensing, stress response, and cell growth. However, its contribution to cardiac hypertrophic growth and heart failure remains incompletely understood. Here, we show that the HBP is induced in cardiomyocytes during hypertrophic growth. Overexpression of Gfat1 (glutamine:fructose-6-phosphate amidotransferase 1), the rate-limiting enzyme of HBP, promotes cardiomyocyte growth. On the other hand, Gfat1 inhibition significantly blunts phenylephrine-induced hypertrophic growth in cultured cardiomyocytes. Moreover, cardiac-specific overexpression of Gfat1 exacerbates pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Conversely, deletion of Gfat1 in cardiomyocytes attenuates pathological cardiac remodeling in response to pressure overload. Mechanistically, persistent upregulation of the HBP triggers decompensated hypertrophy through activation of mTOR while Gfat1 deficiency shows cardioprotection and a concomitant decrease in mTOR activity. Taken together, our results reveal that chronic upregulation of the HBP under hemodynamic stress induces pathological cardiac hypertrophy and heart failure through persistent activation of mTOR.
| S-EPMC7156663 | biostudies-literature
Project description:Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly ?-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis.
| S-EPMC5006067 | biostudies-literature
Project description:Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of an acetyl group from acetyl CoA to the primary amino group of glucosamine. The enzyme from Clostridium acetobutylicum is thought to be involved in cell wall rescue. In addition to glucosamine, GlmA has been shown to function on di- and trisaccharides of glucosamine as well. Here we present a structural and kinetic analysis of the enzyme. For this investigation, eight structures were determined to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA places it into the tandem GNAT superfamily. Each subunit of the dimer folds into two distinct domains which exhibit high three-dimensional structural similarity. Whereas both domains bind acetyl CoA, it is the C-terminal domain that is catalytically competent. On the basis of the various structures determined in this investigation, two amino acid residues were targeted for further study: Asp 287 and Tyr 297. Although their positions in the active site suggested that they may play key roles in catalysis by functioning as active site bases and acids, respectively, this was not borne out by characterization of the D287N and Y297F variants. The kinetic properties revealed that both residues were important for substrate binding but had no critical roles as acid/base catalysts. Kinetic analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA binding first followed by glucosamine. The product N-acetylglucosamine is then released prior to CoA. The investigation described herein provides significantly new information on enzymes belonging to the tandem GNAT superfamily.
| S-EPMC5095700 | biostudies-literature