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1.
Front Immunol ; 13: 926304, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36119118

RESUMO

Existing immune signatures and tumor mutational burden have only modest predictive capacity for the efficacy of immune check point inhibitors. In this study, we developed an immune-metabolic signature suitable for personalized ICI therapies. A classifier using an immune-metabolic signature (IMMETCOLS) was developed on a training set of 77 metastatic colorectal cancer (mCRC) samples and validated on 4,200 tumors from the TCGA database belonging to 11 types. Here, we reveal that the IMMETCOLS signature classifies tumors into three distinct immune-metabolic clusters. Cluster 1 displays markers of enhanced glycolisis, hexosamine byosinthesis and epithelial-to-mesenchymal transition. On multivariate analysis, cluster 1 tumors were enriched in pro-immune signature but not in immunophenoscore and were associated with the poorest median survival. Its predicted tumor metabolic features suggest an acidic-lactate-rich tumor microenvironment (TME) geared to an immunosuppressive setting, enriched in fibroblasts. Cluster 2 displays features of gluconeogenesis ability, which is needed for glucose-independent survival and preferential use of alternative carbon sources, including glutamine and lipid uptake/ß-oxidation. Its metabolic features suggest a hypoxic and hypoglycemic TME, associated with poor tumor-associated antigen presentation. Finally, cluster 3 is highly glycolytic but also has a solid mitochondrial function, with concomitant upregulation of glutamine and essential amino acid transporters and the pentose phosphate pathway leading to glucose exhaustion in the TME and immunosuppression. Together, these findings suggest that the IMMETCOLS signature provides a classifier of tumors from diverse origins, yielding three clusters with distinct immune-metabolic profiles, representing a new predictive tool for patient selection for specific immune-metabolic therapeutic approaches.


Assuntos
Glutamina , Neoplasias , Carbono , Glucose , Hexosaminas , Humanos , Hipoglicemiantes , Lactatos , Lipídeos , Microambiente Tumoral/genética
2.
Nature ; 609(7927): 590-596, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002575

RESUMO

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.


Assuntos
Acetilmuramil-Alanil-Isoglutamina , Proteína Adaptadora de Sinalização NOD2 , Fosfotransferases (Aceptor do Grupo Álcool) , Acetilmuramil-Alanil-Isoglutamina/química , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Bactérias/química , Bactérias/imunologia , Parede Celular/química , Hexosaminas/biossíntese , Imunidade Inata , Macrófagos/enzimologia , Macrófagos/imunologia , Camundongos , Proteína Adaptadora de Sinalização NOD2/agonistas , Proteína Adaptadora de Sinalização NOD2/metabolismo , Peptidoglicano/química , Peptidoglicano/imunologia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo
3.
Cells ; 11(14)2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35883652

RESUMO

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.


Assuntos
DNA Glicosilases , Hexosaminas , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Vias Biossintéticas/genética , DNA , DNA Glicosilases/genética , Epigênese Genética , Hexosaminas/metabolismo , Humanos , Integrinas , Infecções por Vírus Respiratório Sincicial/genética
4.
J Adv Res ; 37: 119-131, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35499042

RESUMO

Introduction: O-linked ß-D-N-acetylglucosamine (O-GlcNAc) modification is a post-translational modification in which a single O-GlcNAc is added to serine or threonine residues in nuclear, cytoplasmic, and mitochondrial proteins, and is involved in a variety of physiological processes. Objectives: In the present study, the role of O-GlcNAcylation in embryo implantation was evaluated. Furthermore, whether O-GlcNAcylation is involved in orchestrating glucose metabolism to influence endometrial cell physiological functions was investigated. Methods: Different endometrial tissues were detected using immunohistochemistry. Pregnant mouse models were established to verify molecular expression. O-GlcNAc transferase and aquaporin 3 (AQP3) knockdown were used to detect embryo implantation efficiency in vitro and in vivo. Western blotting and immunofluorescence were used to detect protein expression and stability. Dual luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to verify the binding transcription factor. Glycolysis was detected using bioenergy analyzer, and metabolites were analyzed using isotope 13C-labeled LC-MS. Metabolic-related genes were determined using RNA sequencing. Results: Activation of endometrial hexosamine biosynthetic pathway (HBP) caused elevated O-GlcNAcylation during the window of implantation, affecting endometrial cell function and embryo implantation. Specifically, elevated O-GlcNAcylation increased glucose uptake via glucose transporter 1 (GLUT1) leading to glucose metabolic flow into the pentose phosphate pathways and HBP, which regulate the metabolic reprogramming of endometrial cells. Furthermore, O-GlcNAcylation mediated the intracellular transport of glycerol to support and compensate for glycolysis through regulation of AQP3. Unexpectedly, elevated AQP3 also increased glucose uptake via GLUT1. These processes maintained higher metabolic requirements for endometrial physiology. Furthermore, the transcription factor SP1 specifically bound to the AQP3 promoter region, and O-GlcNAcylation of SP1 increased its stability and transcriptional regulation of AQP3 which is associated with O-GlcNAcylation of SP1. Conclusion: Overall, O-GlcNAcylation regulated glucose metabolism in endometrial cells, and AQP3-mediated compensation provides new insights into the communication between glycolysis and O-GlcNAcylation.


Assuntos
Aquaporina 3 , Glicólise , Animais , Aquaporina 3/metabolismo , Implantação do Embrião , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Hexosaminas , Camundongos
5.
Biomater Adv ; 134: 112675, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35599100

RESUMO

This study sets the stage for the therapeutic use of Ac5ManNTProp, an N-acetylmannosamine (ManNAc) analog that installs thiol-modified sialoglycans onto the surfaces of human neural stem cells (hNSC). First, we compared hNSC adhesion to the extracellular matrix (ECM) proteins laminin, fibronectin, and collagen and found preferential adhesion and concomitant changes to cell morphology and cell spreading for Ac5ManNTProp-treated cells to laminin, compared to fibronectin where there was a modest response, and collagen where there was no observable increase. PCR array transcript analysis identified several classes of cell adhesion molecules that responded to combined Ac5ManNTProp treatment and hNSC adhesion to laminin. Of these, we focused on integrin α6ß1 expression, which was most strongly upregulated in analog-treated cells incubated on laminin. We also characterized downstream responses including vinculin display as well as the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). In these experiments, Ac5ManNTProp more strongly induced all tested biological endpoints compared to Ac5ManNTGc, showing that the single methylene unit that structurally separates the two analogs finely tunes biological responses. Together, the concerted modulation of multiple pro-regenerative activities through Ac5ManNTProp treatment, in concert with crosstalk with ECM components, lays a foundation for using our metabolic glycoengineering approach to treat neurological disorders by favorably modulating endpoints that contribute to the viability of transplanted NSCs.


Assuntos
Laminina , Células-Tronco Neurais , Colágeno , Fibronectinas , Hexosaminas , Humanos , Laminina/farmacologia , Células-Tronco Neurais/metabolismo , Compostos de Sulfidrila
6.
Carbohydr Res ; 518: 108585, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35597209

RESUMO

N-Trichloroacetyl analogs of N-acetylmannosamine 1-phosphate repeating units, which are found in capsular polysaccharides of Neisseria meningitidis serotype A, were successfully obtained via solid-phase synthesis using an oxazaphospholidine monomer. The introduction of the trichloroacetyl group into the amino group of mannosamine resulted in the stabilization of the reaction intermediates. Monosaccharide, disaccharide, and tetrasaccharide derivatives were obtained and isolated. This is the first example to demonstrate the synthesis of the N-acylated mannosamine 1-phosphate structure having no less than four phosphate linkages.


Assuntos
Neisseria meningitidis , Hexosaminas , Fosfatos , Polissacarídeos , Polissacarídeos Bacterianos/química , Sorogrupo , Técnicas de Síntese em Fase Sólida
7.
Acta Crystallogr C Struct Chem ; 78(Pt 4): 223-230, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-35380125

RESUMO

D-Mannosamine hydrochloride (2-amino-2-deoxy-D-mannose hydrochloride), C6H14NO5+·Cl-, (I), crystallized from a methanol/ethyl acetate/n-hexane solvent mixture at room temperature in a 4C1 chair conformation that is slightly distorted towards the C3,O5B form. A comparison of the structural parameters of (I) with the corresponding parameters in α-D-glucosamine hydrochloride, (II), and ß-D-galactosamine hydrochloride, (III)/(III'), was undertaken to evaluate the effects of ionic hydrogen bonding on structural properties. Three types of ionic hydrogen bonds are present in the crystals of (I)-(III)/(III'), i.e. N+-H...O, N+-H...Cl-, and O-H...Cl-. The exocyclic structural parameters in (I), (II), and (III)/(III') appear to be most influenced by this bonding, especially the exocyclic hydroxy groups, which adopt eclipsed conformations enabled by ionic hydrogen bonding to the chloride anion. Anomeric disorder was observed in crystals of (I), with an α:ß ratio of 37:63. However, anomeric configuration appears to exert minimal structural effects; that is, bond lengths, bond angles, and torsion angles are essentially identical in both anomers. The observed disorder at the anomeric C atom of (I) appears to be caused by the presence of the chloride anion and atom O3 or O4 in proximal voids, which provide opportunities for hydrogen bonding to atom O1 in both axial and equatorial orientations.


Assuntos
Cloretos , Manose , Cristalografia por Raios X , Hexosaminas , Hidrogênio , Ligação de Hidrogênio
8.
Life Sci Alliance ; 5(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35396334

RESUMO

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N-glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment.


Assuntos
Glucose , Hexosaminas , Receptores ErbB/genética , Glucose/metabolismo , Glicosilação , Hexosaminas/metabolismo , Humanos , Nutrientes
9.
Int Immunopharmacol ; 107: 108711, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35338958

RESUMO

Podocyte pyroptosis is an inflammatory form of cell death associated with Diabetic nephropathy (DN). It is reported that hyposialylated Angiopoietin-like-4 (Angptl4) secreted by glomerular podocytes plays an important role in the formation of proteinuria. Previous study indicated that supplementation of sialic acid precursor N-acetylmannosamine (ManNAc) could inhibit podocyte apoptosis and actin cytoskeleton rearrangement. Nevertheless, whether ManNAc could improve diabetic kidney damage by inhibiting podocyte pyroptosis remains unclear. This study aimed to explore the effect of ManNAc therapy on alleviating diabetic renal injury and podocyte pyroptosis, and its possible mechanism was also figured out. The male 8-week-old C57BL/6 mice were divided into three groups: control group, Streptozocin (STZ)-induced DN group, and ManNAc treated diabetic group. Then, the changes in renal function, renal histopathology, podocyte pyroptosis, reactive oxygen species (ROS), and mitochondrial dysfunction were measured. Herein, we observed that the upregulated expression of Angptl4 was involved in podocyte injury. ManNAc treatment ameliorated podocyte ultrastructure, renal function, and renal histopathology in STZ-induced DN mice. In addition, ManNAc administration attenuated podocyte cell death and suppressed the activation of Nucleotide leukin-rich polypeptide 3 (NLRP3), caspase-1, and interleukin-1ß (IL-1ß), and the cleavage of gasdermin-D (GSDMD). Moreover, ManNAc inhibited ROS production and restored mitochondrial morphology in vivo and vitro. Further, ManNAc administration significantly alleviated podocyte pyroptosis through inhibiting ROS/NLRP3 signaling pathway. Therefore, these results elucidated that the upregulated expression of Angptl4 was involved in podocyte injury and ManNAc treatment protected against podocyte pyroptosis via inhibiting mitochondrial injury and ROS/NLRP3 signaling pathway in DN mice.


Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Podócitos , Animais , Diabetes Mellitus/patologia , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Feminino , Hexosaminas , Humanos , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotídeos/metabolismo , Nucleotídeos/farmacologia , Piroptose , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estreptozocina
10.
Elife ; 112022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35229715

RESUMO

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Hexosaminas , Animais , Vias Biossintéticas , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glicosilação , Hexosaminas/metabolismo , Camundongos
11.
Cells ; 11(3)2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-35159258

RESUMO

The zeppelin (zep) locus is known for its essential role in the development of the embryonic cuticle of Drosophila melanogaster. We show here that zep encodes Gfat1 (Glutamine: Fructose-6-Phosphate Aminotransferase 1; CG12449), the enzyme that catalyzes the rate-limiting step in the hexosamine biosynthesis pathway (HBP). This conserved pathway diverts 2%-5% of cellular glucose from glycolysis and is a nexus of sugar (fructose-6-phosphate), amino acid (glutamine), fatty acid [acetyl-coenzymeA (CoA)], and nucleotide/energy (UDP) metabolism. We also describe the isolation and characterization of lethal mutants in the euchromatic paralog, Gfat2 (CG1345), and demonstrate that ubiquitous expression of Gfat1+ or Gfat2+ transgenes can rescue lethal mutations in either gene. Gfat1 and Gfat2 show differences in mRNA and protein expression during embryogenesis and in essential tissue-specific requirements for Gfat1 and Gfat2, suggesting a degree of functional evolutionary divergence. An evolutionary, cytogenetic analysis of the two genes in six Drosophila species revealed Gfat2 to be located within euchromatin in all six species. Gfat1 localizes to heterochromatin in three melanogaster-group species, and to euchromatin in the more distantly related species. We have also found that the pattern of flanking-gene microsynteny is highly conserved for Gfat1 and somewhat less conserved for Gfat2.


Assuntos
Drosophila melanogaster , Hexosaminas , Animais , Vias Biossintéticas/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Eucromatina , Glutamina/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
12.
Cells ; 11(4)2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35203353

RESUMO

Unresolved hyperglycaemia, a hallmark of type 2 diabetes mellitus (T2DM), is a well characterised manifestation of altered fuel homeostasis and our understanding of its role in the pathologic activation of the inflammatory system continues to grow. Metabolic disorders like T2DM trigger changes in the regulation of key cellular processes such as cell trafficking and proliferation, and manifest as chronic inflammatory disorders with severe long-term consequences. Activation of inflammatory pathways has recently emerged as a critical link between T2DM and inflammation. A substantial body of evidence has suggested that this is due in part to increased flux through the hexosamine biosynthetic pathway (HBP). The HBP, a unique nutrient-sensing metabolic pathway, produces the activated amino sugar UDP-GlcNAc which is a critical substrate for protein O-GlcNAcylation, a dynamic, reversible post-translational glycosylation of serine and threonine residues in target proteins. Protein O-GlcNAcylation impacts a range of cellular processes, including inflammation, metabolism, trafficking, and cytoskeletal organisation. As increased HBP flux culminates in increased protein O-GlcNAcylation, we propose that targeting O-GlcNAcylation may be a viable therapeutic strategy for the prevention and management of glucose-dependent pathologies with inflammatory components.


Assuntos
Diabetes Mellitus Tipo 2 , Glicosilação , Hexosaminas/metabolismo , Humanos , Inflamação , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo
13.
Eur Surg Res ; 63(2): 64-76, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34986481

RESUMO

INTRODUCTION: Diabetes mellitus (DM)-induced testicular damage is characterized by abnormal apoptosis of spermatogenic cells. Here, we clarified the roles and the molecular mechanism of microRNA (miR)-27b-3p in high glucose (HG)-induced spermatogenic cell damage. METHODS: GC-1 spg cells were treated with 30 mmol/L glucose for 24 h. Cell viability was assessed by 2.3 3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay. And, levels of O-linked N-acetylglucosamine (OGT), apoptosis-related proteins, and autophagy-related proteins were evaluated using Western blot. Levels of tumor necrosis factor-α (TNF-α), IL-1ß, IL-6, and UDP-N-acetylglucosamine (UDP-GlcNAc) were assessed by enzyme linked immunosorbent (ELISA) assay. Levels of reactive oxygen species (ROS), malonic dialdehyde (MDA) and activity of superoxide dismutase (SOD) in cells were determined using kits. Cell apoptosis was determined using flow cytometry assay. Besides, dual luciferase reporter assay was employed to verify the binding relationship between miR-27b-3p and glutamine-fructose-6-phosphate transaminase 1 (Gfpt1). RESULTS: miR-27b-3p was markedly downregulated in HG-treated GC-1 spg cells. HG treatment caused decreased cell viability, increased oxidative stress and inflammation, and induced autophagy and apoptosis, which were abolished by miR-27b-3p overexpression. miR-27b-3p suppressed the activation of hexosamine biosynthetic pathway (HBP) signaling in HG-treated spermatogenic cells. miR-27b-3p directly bound to Gfpt1 and negatively regulated its expression. CONCLUSION: miR-27b-3p could improve HG-induced spermatogenic cell damage via regulating Gfpt1/HBP signaling, providing a new treatment strategy for the treatment of DM-induced testicular damage.


Assuntos
MicroRNAs , Acetilglucosamina , Apoptose , Vias Biossintéticas , Glucose , Hexosaminas , MicroRNAs/genética , Difosfato de Uridina/metabolismo
14.
Cells ; 11(1)2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-35011738

RESUMO

In non-small-cell lung cancer (NSCLC), concurrent mutations in the oncogene KRAS and tumor suppressor STK11 (also known as LKB1) confer an aggressive malignant phenotype, an unfavourability towards immunotherapy, and overall poor prognoses in patients. In a previous study, we showed that murine KRAS/LKB1 co-mutant tumors and human co-mutant cancer cells have an enhanced dependence on glutamine-fructose-6-phosphate transaminase 2 (GFPT2), a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP), which could be targeted to reduce survival of KRAS/LKB1 co-mutants. Here, we found that KRAS/LKB1 co-mutant cells also exhibit an increased dependence on N-acetylglucosamine-phosphate mutase 3 (PGM3), an enzyme downstream of GFPT2. Genetic or pharmacologic suppression of PGM3 reduced KRAS/LKB1 co-mutant tumor growth in both in vitro and in vivo settings. Our results define an additional metabolic vulnerability in KRAS/LKB1 co-mutant tumors to the HBP and provide a rationale for targeting PGM3 in this aggressive subtype of NSCLC.


Assuntos
/genética , Neoplasias Pulmonares/genética , Terapia de Alvo Molecular , Fosfoglucomutase/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Vias Biossintéticas/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glicosilação/efeitos dos fármacos , Hexosaminas/biossíntese , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Fosfoglucomutase/antagonistas & inibidores , Fosfoglucomutase/genética
15.
Curr Diabetes Rev ; 18(2): e011121190177, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-33430751

RESUMO

It is well established that diabetes and its associated hyperglycemia negatively impact retinal function, yet we know little about the role played by augmented flux through the Hexosamine Biosynthetic Pathway (HBP). This offshoot of the glycolytic pathway produces UDP-Nacetyl- glucosamine, which serves as the substrate for post-translational O-linked modification of proteins in a process referred to as O-GlcNAcylation. HBP flux and subsequent protein O-GlcNAcylation serve as nutrient sensors, enabling cells to integrate metabolic information to appropriately modulate fundamental cellular processes including gene expression. Here we summarize the impact of diabetes on retinal physiology, highlighting recent studies that explore the role of O-GlcNAcylation- induced variation in mRNA translation in retinal dysfunction and the pathogenesis of Diabetic Retinopathy (DR). Augmented O-GlcNAcylation results in wide variation in the selection of mRNAs for translation, in part, due to O-GlcNAcylation of the translational repressor 4E-BP1. Recent studies demonstrate that 4E-BP1 plays a critical role in regulating O-GlcNAcylation-induced changes in the translation of the mRNAs encoding Vascular Endothelial Growth Factor (VEGF), a number of important mitochondrial proteins, and CD40, a key costimulatory molecule involved in diabetes-induced retinal inflammation. Remarkably, 4E-BP1/2 ablation delays the onset of diabetes- induced visual dysfunction in mice. Thus, pharmacological interventions to prevent the impact of O-GlcNAcylation on 4E-BP1 may represent promising therapeutics to address the development and progression of DR. In this regard, we discuss the potential interplay between retinal O-GlcNAcylation and the ocular renin-angiotensin system as a potential therapeutic target of future interventions.


Assuntos
Diabetes Mellitus , Retinopatia Diabética , Animais , Retinopatia Diabética/metabolismo , Hexosaminas/metabolismo , Humanos , Camundongos , Processamento de Proteína Pós-Traducional , Sistema Renina-Angiotensina , Retina , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo
16.
Elife ; 102021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-34951587

RESUMO

Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Ácido Hialurônico/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Técnicas de Inativação de Genes , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Hexosaminas/biossíntese , Humanos , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante Heterólogo
17.
Biomolecules ; 11(11)2021 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-34827730

RESUMO

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a ß-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s-1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s-1 were revealed. P. alvei CsaB was inactive on synthetic pNP-ß-d-ManNAc and ß-d-ManNAc-ß-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.


Assuntos
Paenibacillus , Catálise , Hexosaminas , Fosfatos , Fosfoenolpiruvato
18.
Elife ; 102021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34844667

RESUMO

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth.


Inside tumors, cancer cells often have to compete with each other for food and other resources they need to survive. This is a key factor driving the growth and progression of cancer. One of the resources cells need is a molecule called UDP-GlcNAc, which they use to modify many proteins so they can work properly. Because cancer cells grow quickly, they likely need much more UDP-GlcNAc than healthy cells. Many tumors, including those derived from pancreatic cancers, have very poor blood supplies, so their cells cannot get the nutrients and other resources they need to grow from the bloodstream. This means that tumor cells have to find new ways to use what they already have. One example of this is developing alternative ways to obtain UDP-GlcNAc. Cells require a nutrient called glutamine to produce UDP-GlcNAc. Limiting the supply of glutamine to cells allows researchers to study how cells are producing UDP-GlcNAc in the lab. Campbell et al. used this approach to study how pancreatic cancer cells obtain UDP-GlcNAc when their access to glutamine is limited. They used a technique called isotope tracing, which allows researchers to track how a specific chemical is processed inside the cell, and what it turns into. The results showed that the pancreatic cancer cells do not make new UDP-GlcNAc but use a protein called NAGK to salvage GlcNAc (another precursor of UDP-GlcNAc), which may be obtained from cellular proteins. Cancer cells that lacked NAGK formed smaller tumors, suggesting that the cells grow more slowly because they cannot recycle UDP-GlcNAc fast enough. Pancreatic cancer is one of the most common causes of cancer deaths and is notable for being difficult to detect and treat. Campbell et al. have identified one of the changes that allows pancreatic cancers to survive and grow quickly. Next steps will include examining the role of NAGK in healthy cells and testing whether it could be targeted for cancer treatment.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Glutamina/deficiência , Hexosaminas/metabolismo , Neoplasias Pancreáticas/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Nus
19.
Biochem Biophys Res Commun ; 583: 121-127, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34735873

RESUMO

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Fibroblastos/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Hexosaminas/biossíntese , Hexosaminas/metabolismo , Células-Tronco Pluripotentes Induzidas , Camundongos , Miocárdio/citologia , Isoformas de Proteínas , Ratos Sprague-Dawley
20.
Sci Rep ; 11(1): 20705, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34667181

RESUMO

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing.


Assuntos
Acilação/fisiologia , Diabetes Mellitus/metabolismo , Endocitose/fisiologia , N-Acetilglucosaminiltransferases/metabolismo , Placenta/metabolismo , Trofoblastos/metabolismo , Acetilglucosamina/metabolismo , Adulto , Clatrina/metabolismo , Feminino , Glicosilação , Hexosaminas/metabolismo , Humanos , Mães , Gravidez , Processamento de Proteína Pós-Traducional/fisiologia , Adulto Jovem
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