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1.
Nat Commun ; 15(1): 5206, 2024 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-38897986

RESUMO

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson's disease, however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5'-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson's patient midbrain cultures accumulate glucose and uridine-5'-triphosphate, while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme, GFPT2, through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate, while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological α-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity, and its failure in Parkinson's disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement.


Assuntos
Vias Biossintéticas , Glucose , Hexosaminas , Células-Tronco Pluripotentes Induzidas , Lisossomos , Mesencéfalo , Neurônios , Doença de Parkinson , Resposta a Proteínas não Dobradas , Humanos , Hexosaminas/biossíntese , Hexosaminas/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Neurônios/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Mesencéfalo/metabolismo , Glucose/metabolismo , Glicosilação , alfa-Sinucleína/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética
2.
ACS Chem Biol ; 19(6): 1237-1242, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38723147

RESUMO

As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.


Assuntos
Bacillus subtilis , Glucosamina , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Engenharia de Proteínas , Glucosamina/biossíntese , Glucosamina/metabolismo , Glucosamina/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Simulação de Acoplamento Molecular , Frutose/metabolismo , Frutose/química , Frutose/biossíntese , Simulação de Dinâmica Molecular , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Domínio Catalítico
3.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732103

RESUMO

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.


Assuntos
Proliferação de Células , Neoplasias Colorretais , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , N-Acetilglucosaminiltransferases , Humanos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Glicosilação , Animais , Camundongos , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Regulação para Cima , Camundongos Nus , Ácido Graxo Sintase Tipo I
4.
Cell Death Dis ; 15(4): 244, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38575607

RESUMO

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.


Assuntos
Interleucina-18 , Neoplasias Pancreáticas , Humanos , Glicosilação , Interleucina-18/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas/metabolismo , Vias Biossintéticas , Hexosaminas , Microambiente Tumoral , Proteína 1 de Ligação a Y-Box/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética
5.
Nat Chem Biol ; 20(6): 770-778, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38409364

RESUMO

Metal-binding proteins (MBPs) have various and important biological roles in all living species and many human diseases are intricately linked to dysfunctional MBPs. Here, we report a chemoproteomic method named 'metal extraction-triggered agitation logged by thermal proteome profiling' (METAL-TPP) to globally profile MBPs in proteomes. The method involves the extraction of metals from MBPs using chelators and monitoring the resulting protein stability changes through thermal proteome profiling. Applying METAL-TPP to the human proteome with a broad-spectrum chelator, EDTA, revealed a group of proteins with reduced thermal stability that contained both previously known MBPs and currently unannotated MBP candidates. Biochemical characterization of one potential target, glutamine-fructose-6-phosphate transaminase 2 (GFPT2), showed that zinc bound the protein, inhibited its enzymatic activity and modulated the hexosamine biosynthesis pathway. METAL-TPP profiling with another chelator, TPEN, uncovered additional MBPs in proteomes. Collectively, this study developed a robust tool for proteomic discovery of MBPs and provides a rich resource for functional studies of metals in cell biology.


Assuntos
Proteoma , Proteômica , Humanos , Proteoma/metabolismo , Proteômica/métodos , Quelantes/química , Quelantes/farmacologia , Metais/metabolismo , Metais/química , Zinco/metabolismo , Zinco/química , Temperatura , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/antagonistas & inibidores , Estabilidade Proteica
6.
Carcinogenesis ; 45(5): 324-336, 2024 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-38267812

RESUMO

Tripartite Motif 14 (TRIM14) is an oncoprotein that belongs to the E3 ligase TRIM family, which is involved in the progression of various tumors except for non-small cell lung carcinoma (NSCLC). However, little is currently known regarding the function and related mechanisms of TRIM14 in NSCLC. Here, we found that the TRIM14 protein was downregulated in lung adenocarcinoma tissues compared with the adjacent tissues, which can suppress tumor cell proliferation and migration both in vitro and in vivo. Moreover, TRIM14 can directly bind to glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), which in turn results in the degradation of GFAT1 and reduced O-glycosylation levels. GFAT1 is a key enzyme in the rate-limiting step of the hexosamine biosynthetic pathway (HBP). Replenishment of N-acetyl-d-glucosamine can successfully reverse the inhibitory effect of TRIM14 on the NSCLC cell growth and migration as expected. Collectively, our data revealed that TRIM14 suppressed NSCLC cell proliferation and migration through ubiquitination and degradation of GFAT1, providing a new regulatory role for TRIM14 on HBP.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Movimento Celular , Proliferação de Células , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Hexosaminas , Neoplasias Pulmonares , Proteínas com Motivo Tripartido , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Proteínas com Motivo Tripartido/genética , Hexosaminas/biossíntese , Hexosaminas/metabolismo , Animais , Camundongos , Regulação Neoplásica da Expressão Gênica , Progressão da Doença , Ubiquitinação , Linhagem Celular Tumoral , Masculino , Camundongos Nus , Feminino , Glicosilação , Camundongos Endogâmicos BALB C , Vias Biossintéticas , Peptídeos e Proteínas de Sinalização Intracelular
7.
J Exp Clin Cancer Res ; 42(1): 338, 2023 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-38093368

RESUMO

BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. CONCLUSION: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Animais , Camundongos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Espectrometria de Massas em Tandem , Peixe-Zebra , Apoptose , Proteínas Proto-Oncogênicas c-mdm2/genética , Morte Celular , Mutação , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
8.
J Pathol Clin Res ; 9(5): 391-408, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37395335

RESUMO

Glutamine fructose-6-phosphate aminotransferase 2 (GFPT2) is a rate-limiting enzyme in hexosamine biosynthesis involved in the occurrence and progress of many cancers. What role it plays in gastric cancer (GC) is still unclear. In this study, transcriptome sequencing data from the Harbin Medical University (HMU)-GC cohort and The Cancer Genome Atlas (TCGA) dataset were combined with the HMU-TCGA training cohort to analyze the biological function and clinical significance of GFPT2. The correlation of GFPT2 with immune cells and stromal cells was analyzed in the GC immune microenvironment through transcriptome sequencing data and a public single-cell sequencing database. In cell lines, GC tissues, and the tissue microarray, GFPT2 protein expression was confirmed by western blotting and immunohistochemistry. The mRNA of GFPT2 was highly expressed in the tumor (p < 0.001), and GC cells and tumors expressed high levels of GFPT2 protein. Compared to low expression, high GFPT2 mRNA expression was associated with higher levels of tumor invasion, higher pathological stages, and poor prognosis (p = 0.02) in GC patients. In a drug susceptibility analysis, GFPT2 mRNA expression was associated with multiple chemotherapeutic drug sensitivity, including docetaxel, paclitaxel, and cisplatin. Gene enrichment analysis found that GFPT2 was mainly primarily involved in the extracellular matrix receptor interaction pathway. The ESTIMATE, CIBERSORT, and ssGSEA algorithms showed that GFPT2 was associated with immune cell infiltration. In addition, GFPT2 was more likely to be expressed within cancer-associated fibroblasts (CAFs), and high levels of GFPT2 expression were highly correlated with four CAFs scores (all p < 0.05). Finally, a prognostic model to assess the risk of death in GC patients was constructed based on GFPT2 protein expression and lymph node metastasis rate. In conclusion, GFPT2 plays an essential role in the function of CAFs in GC. It can be used as a biomarker to assess GC prognosis and immune infiltration.


Assuntos
Fibroblastos Associados a Câncer , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Prognóstico , Glutamina/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Tumoral , RNA Mensageiro/metabolismo , Microambiente Tumoral
10.
J Biomol Struct Dyn ; 41(21): 12038-12054, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36629053

RESUMO

Candida albicans is one of the most common species of fungus with life-threatening systemic infections and a high mortality rate. The outer cell wall layer of C. albicans is packed with mannoproteins and glycosylated polysaccharide moieties that play an essential role in the interaction with host cells and tissues. The glucosamine-6-phosphate synthase enzyme produces N-acetylglucosamine, which is a crucial chemical component of the cell wall of Candida albicans. Collectively, these components are essential to maintain the cell shape and for infection. So, its disruption can have serious effects on cell growth and morphology, resulting in cell death. Hence, it is considered a good antifungal target. In this study, we have performed an in silico approach to analyze the inhibitory potential of some polyphenols obtained from plants. Those can be considered important in targeting against the enzyme glucosamine-6-phosphate synthase (PDB-2VF5). The results of the study revealed that the binding affinity of complexes theaflavin and 3-o-malonylglucoside have significant docking scores and binding free energy followed by significant ADMET parameters that predict the drug-likeness property and toxicity of polyphenols as potential ligands. A molecular dynamic simulation was used to test the validity of the docking scores, and it showed that the complex remained stable during the period of the simulation, which ranged from 0 to 100 ns. Theaflavins and 3-o-malonylglucoside may be effective against Candida albicans using a computer-aided drug design methodology that will further enable researchers for future in vitro and in vivo studies, according to our in silico study.Communicated by Ramaswamy H. Sarma.


Assuntos
Candida albicans , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Polifenóis/farmacologia , Antifúngicos/farmacologia , Antifúngicos/química , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular
11.
Bone ; 166: 116575, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36195245

RESUMO

INTRODUCTION: Dental pulp stem cells (DPSCs) have high proliferative and multilineage differentiation potential in mesenchymal stem cells. However, several studies have indicated that there are individual differences in the potential for osteogenic differentiation of DPSCs, and the factors determining these differences are unknown. OBJECTIVE: To identify the genes responsible for the individual differences in the osteogenic differentiation ability of DPSCs. METHODS: We divided DPSCs into high and low osteogenic differentiation ability groups (HG or LG) with ALP and von Kossa stain, and compared the gene expression patterns using RNA-seq. In addition, genes that may affect osteogenic differentiation were knocked down using small interfering RNA (siRNA) and their effects were investigated. RESULTS: The RNA-seq patterns revealed that VCAM1 and GFPT2 were significantly expressed at higher levels in the HG than in the LG. The results of siRNA analysis showed that VCAM1 and GFPT2 knockdown significantly reduced the expression of osteogenic markers. Furthermore, we analyzed the involvement of these two genes in cell signaling in DPSC differentiation. The results indicated that the VCAM1-mediated Ras-MEK-Erk and PI3K/Akt pathways are involved in the osteogenic differentiation of DPSCs, and that GFPT2-mediated HBP signaling influences the osteogenic differentiation of DPSCs. CONCLUSIONS: These findings indicate that DPSCs that highly express VCAM1 and GFPT2 have a high capacity for osteogenic differentiation. Evaluation of VCAM1 and GFPT2 expression in undifferentiated DPSCs may predict the outcome of bone regenerative therapy using DPSCs. Moreover, the expression levels of VCAM1 and GFPT2 in DPSCs may be useful in setting criteria for selecting donors for allogeneic cell transplantation for bone regeneration.


Assuntos
Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Osteogênese , Molécula 1 de Adesão de Célula Vascular , Humanos , Biomarcadores/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Polpa Dentária , Osteoblastos , Osteogênese/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Células-Tronco/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
12.
Front Cell Infect Microbiol ; 12: 976924, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36211971

RESUMO

The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, ß-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White.


Assuntos
Sporothrix , beta-Glucanas , Animais , Antifúngicos , Benzenossulfonatos , Parede Celular/metabolismo , Celulose , Quitina , Vermelho Congo , Glutamina , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hexosaminas/análise , Hexosaminas/metabolismo , Humanos , Mamíferos/metabolismo , Polímeros/análise , Sporothrix/metabolismo , Açúcares , Difosfato de Uridina , beta-Glucanas/análise
13.
Adv Sci (Weinh) ; 9(30): e2202993, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36045101

RESUMO

A malformed tumour vascular network provokes the nutrient-deprived tumour microenvironment (TME), which conversely activates endothelial cell (EC) functions and stimulates neovascularization. Emerging evidence suggests that the flexible metabolic adaptability of tumour cells helps to establish a metabolic symbiosis among various cell subpopulations in the fluctuating TME. In this study, the authors propose a novel metabolic link between bladder cancer (BCa) cells and ECs in the nutrient-scarce TME, in which BCa-secreted glutamine-fructose-6-phosphate aminotransferase 1 (GFAT1) via small extracellular vesicles (sEVs) reprograms glucose metabolism by increasing hexosamine biosynthesis pathway flux in ECs and thus enhances O-GlcNAcylation. Moreover, seryl-tRNA synthetase (SerRS) O-GlcNAcylation at serine 101 in ECs promotes its degradation by ubiquitination and impeded importin α5-mediated nuclear translocation. Intranuclear SerRS attenuates vascular endothelial growth factor transcription by competitively binding to the GC-rich region of the proximal promotor. Additionally, GFAT1 knockout in tumour cells blocks SerRS O-GlcNAcylation in ECs and attenuates angiogenesis both in vitro and in vivo. However, administration of GFAT1-overexpressing BCa cells-derived sEVs increase the angiogenetic activity in the ECs of GFAT1-knockout mice. In summary, this study suggests that inhibiting sEV-mediated GFAT1 secretion from BCa cells and targeting SerRS O-GlcNAcylation in ECs may serve as novel strategies for BCa antiangiogenetic therapy.


Assuntos
Vesículas Extracelulares , Serina-tRNA Ligase , Neoplasias da Bexiga Urinária , Camundongos , Animais , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Células Endoteliais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Serina-tRNA Ligase/metabolismo , Hexosaminas/metabolismo , Serina/metabolismo , Glucose/metabolismo , Vesículas Extracelulares/metabolismo , Carioferinas , Microambiente Tumoral
14.
Carcinogenesis ; 43(10): 969-979, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36040914

RESUMO

Cervical cancer demonstrates the fourth incidence and death rate in females worldwide. Glutamine--fructose-6-phosphate transaminase 1 (GFPT1), the first rate-limited enzyme of the hexosamine biosynthesis pathway, has been reported to promote the progression of cancers. However, the prognostic value and roles of GFPT1 in cervical cancer are largely unknown. Transcription expression data for cervical cancer were downloaded from public databases. GFPT1 overexpressed and knockdown cell lines were constructed. Colony formation assays, Edu assays and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to measure the proliferation capabilities of cervical cancer cells. Western blot, Immunofluorescence and co-immunoprecipitation assays were performed to verify the interaction between GFPT1and Phosphatase and tensin homolog (PTEN). Animal assays were applied to verify the results in vivo. GFPT1 expression was higher in cervical cancer cell lines. The proliferation capabilities of cervical cancer cells were suppressed in GFPT1 knockdown cells and GFPT1 inhibitor L-DON treated cells. And overexpression of GFPT1 promoted cell proliferation. PTEN was up-regulated in GFPT1 knockdown cells and downregulated in GFPT1 overexpression cells. Immunofluorescence and co-immunoprecipitation results showed that GFPT1 was co-localized and interacted with PTEN. GFPT1 promoted the ubiquitination and degradation of PTEN. Silence of PTEN offsets the growth inhibition of cervical cancer caused by GFPT1 knockdown. Animal assays showed that GFPT1 promoted the proliferation of cervical cancer in vivo. Our study revealed that GFPT1 could promote the progression of cervical cancer by regulating PTEN expression. Our study highlights the GFPT1-PTEN regulation as a potential therapy target for cervical cancer. .


Assuntos
Neoplasias do Colo do Útero , Humanos , Feminino , Animais , Neoplasias do Colo do Útero/genética , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Proliferação de Células , Ubiquitinação , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
15.
J Biol Chem ; 298(10): 102437, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36041631

RESUMO

Mammalian target of rapamycin (mTOR), which is part of mTOR complex 1 (mTORC1) and mTORC2, controls cellular metabolism in response to levels of nutrients and other growth signals. A hallmark of mTORC2 activation is the phosphorylation of Akt, which becomes upregulated in cancer. How mTORC2 modulates Akt phosphorylation remains poorly understood. Here, we found that the RNA-binding protein, AUF1 (ARE/poly(U)-binding/degradation factor 1), modulates mTORC2/Akt signaling. We determined that AUF1 is required for phosphorylation of Akt at Thr308, Thr450, and Ser473 and that AUF1 also mediates phosphorylation of the mTORC2-modulated metabolic enzyme glutamine fructose-6-phosphate amidotransferase 1 at Ser243. In addition, AUF1 immunoprecipitation followed by quantitative RT-PCR revealed that the mRNAs of Akt, glutamine fructose-6-phosphate amidotransferase 1, and the mTORC2 component SIN1 associate with AUF1. Furthermore, expression of the p40 and p45, but not the p37 or p42, isoforms of AUF1 specifically mediate Akt phosphorylation. In the absence of AUF1, subcellular fractionation indicated that Akt fails to localize to the membrane. However, ectopic expression of a membrane-targeted allele of Akt is sufficient to allow Akt-Ser473 phosphorylation despite AUF1 depletion. Finally, conditions that enhance mTORC2 signaling, such as acute glutamine withdrawal, augment AUF1 phosphorylation, whereas mTOR inhibition abolishes AUF1 phosphorylation. Our findings unravel a role for AUF1 in promoting membrane localization of Akt to facilitate its phosphorylation on this cellular compartment. Targeting AUF1 could have therapeutic benefit for cancers with upregulated mTORC2/Akt signaling.


Assuntos
Ribonucleoproteína Nuclear Heterogênea D0 , Proteínas Proto-Oncogênicas c-akt , Proteínas de Ligação a RNA , Proliferação de Células , Glutamina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Humanos , Ribonucleoproteína Nuclear Heterogênea D0/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , Membrana Celular/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo
16.
J Enzyme Inhib Med Chem ; 37(1): 1928-1956, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-35801410

RESUMO

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is known as a promising target for antimicrobial agents and antidiabetics. Several compounds of natural or synthetic origin have been identified as inhibitors of this enzyme. This set comprises highly selective l-glutamine, amino sugar phosphate or transition state intermediate cis-enolamine analogues. Relatively low antimicrobial activity of these inhibitors, poorly penetrating microbial cell membranes, has been improved using the pro-drug approach. On the other hand, a number of heterocyclic and polycyclic compounds demonstrating antimicrobial activity have been presented as putative inhibitors of the enzyme, based on the results of molecular docking to GlcN-6-P synthase matrix. The most active compounds of this group could be considered promising leads for development of novel antimicrobial drugs or antidiabetics, provided their selective toxicity is confirmed.


Assuntos
Anti-Infecciosos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Hipoglicemiantes , Simulação de Acoplamento Molecular
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 38(7): 651-656, 2022 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-35786458

RESUMO

Objective To prepare rabbit polyclonal antibody specifically against human glutamine fructose-6-phosphate amidotransferase 1(GFPT1). Methods The protein sequences of GFPT1 and its highly homologous isozyme GFPT2 were compared. Two peptides for the specific sequence of GFPT1 were designed and synthesized. New Zealand rabbits were immunized by peptide coupled with Keyhole Limpet hemocyanin (KLH) as antigen. Antiserum was obtained after 3 booster immunizations. The titer of the antiserum against GFPT1 were detected by ELISA. The E.coli expression vectors of GFPT1 and GFPT2 were constructed, and the recombinant proteins of GFPT1 and GFPT2 were obtained by induced expression. GFPT1 and GFPT2 recombinant proteins were analyzed by Western blot to verify the specificity of the antiserum. Immuno-fluorescence cytochemical staining for GFPT1 expression in 786-O cells was verified as for whether the obtained antiserum could recognize the endogenously expressed GFPT1. Results Polyclonal antibody specifically recognizing GFPT1 was obtained and the titer of polyclonal antibody reached 1:1 458 000. Conclusion The experiment successfully prepared the specific rabbit polyclonal antibody against GFPT1.


Assuntos
Anticorpos , Glutamina , Animais , Escherichia coli/genética , Frutosefosfatos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante) , Humanos , Soros Imunes , Coelhos , Proteínas Recombinantes
18.
J Neuromuscul Dis ; 9(4): 533-541, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35694932

RESUMO

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.


Assuntos
Doença de Depósito de Glicogênio Tipo II , Síndromes Miastênicas Congênitas , Adulto , Diagnóstico Diferencial , Testes Genéticos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Glicogênio , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Debilidade Muscular/genética , Síndromes Miastênicas Congênitas/diagnóstico , Síndromes Miastênicas Congênitas/genética
19.
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
20.
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
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