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BioMed Research International 2020Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis...
Reprogrammed glucose and glutamine metabolism are essential for tumor initiation and development. As a branch of glucose and metabolism, the hexosamine biosynthesis pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and contributes to the O-GlcNAcylation process. However, the spectrum of HBP-dependent tumors and the mechanisms by which the HBP promotes tumor aggressiveness remain areas of active investigation. In this study, we analyzed the activity of the HBP and its prognostic value across 33 types of human cancers. Increased HBP activity was observed in pancreatic ductal adenocarcinoma (PDAC), and higher HBP activity predicted a poor prognosis in PDAC patients. Genetic silencing or pharmacological inhibition of the first and rate-limiting enzyme of the HBP, glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), inhibited PDAC cell proliferation, invasive capacity, and triggered cell apoptosis. Notably, these effects can be restored by addition of UDP-GlcNAc. Moreover, similar antitumor effects were noticed by pharmacological inhibition of GFAT1 with 6-diazo-5-oxo-l-norleucine (DON) or Azaserine. PDAC is maintained by oncogenic Wnt/-catenin transcriptional activity. Our data showed that GFAT1 can regulate -catenin expression via modulation of the O-GlcNAcylation process. TOP/FOP-Flash and real-time qPCR analysis showed that GFAT1 knockdown inhibited -catenin activity and the transcription of its downstream target genes and . Ectopic expression of a stabilized form of -catenin restored the suppressive roles of GFAT1 knockdown on PDAC cell proliferation and invasion. Collectively, our findings indicate that higher GFAT1/HBP/O-GlcNAcylation exhibits tumor-promoting roles by maintaining -catenin activity in PDAC.
Topics: Cell Line, Tumor; Cell Proliferation; Databases, Genetic; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); Glycosylation; Hexosamines; Humans; Pancreatic Neoplasms; beta Catenin
PubMed: 32149084
DOI: 10.1155/2020/1921609 -
Cell Reports Feb 2018Sirtuin 3 (SIRT3) is a NAD-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet...
Sirtuin 3 (SIRT3) is a NAD-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen.
Topics: Amino Acid Sequence; Animals; Azaserine; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Fibroblasts; Glutamine; Mechanistic Target of Rapamycin Complex 1; Mice, Knockout; Mice, Nude; Nucleotides; Promoter Regions, Genetic; Signal Transduction; Sirtuin 3; Small Molecule Libraries; Up-Regulation
PubMed: 29466723
DOI: 10.1016/j.celrep.2018.01.076 -
Molecules (Basel, Switzerland) Apr 2020is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian...
is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to survival. Recently, we described that can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.
Topics: Animals; Azaserine; CHO Cells; Cell Cycle; Cell Proliferation; Cricetulus; Energy Metabolism; Glutamic Acid; Glutamine; Isoxazoles; Life Cycle Stages; Molecular Structure; Trypanocidal Agents; Trypanosoma cruzi
PubMed: 32252252
DOI: 10.3390/molecules25071628 -
MSystems 2017requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise...
requires the capability to accommodate to environmental changes in order to proliferate in oligotrophic oceans, in particular regarding nitrogen availability. A precise knowledge of the composition and changes in the proteome can yield fundamental insights into such a response. Here we report a detailed proteome analysis of the important model cyanobacterium SS120 after treatment with azaserine, an inhibitor of ferredoxin-dependent glutamate synthase (GOGAT), to simulate extreme nitrogen starvation. In total, 1,072 proteins, corresponding to 57% of the theoretical proteome, were identified-the maximum proteome coverage obtained for any strain thus far. Spectral intensity, calibrated quantification by the Hi3 method, was obtained for 1,007 proteins. Statistically significant changes ( value of <0.05) were observed for 408 proteins, with the majority of proteins (92.4%) downregulated after 8 h of treatment. There was a strong decrease in ribosomal proteins upon azaserine addition, while many transporters were increased. The regulatory proteins P and PipX were decreased, and the global nitrogen regulator NtcA was upregulated. Furthermore, our data for indicate that NtcA also participates in the regulation of photosynthesis. responds to the lack of nitrogen by slowing down translation, while inducing photosynthetic cyclic electron flow and biosynthesis of proteins involved in nitrogen uptake and assimilation. is the most abundant photosynthetic organism on Earth, contributing significantly to global primary production and playing a prominent role in biogeochemical cycles. Here we study the effects of extreme nitrogen limitation, a feature of the oligotrophic oceans inhabited by this organism. Quantitative proteomics allowed an accurate quantification of the proteome, finding three main responses to nitrogen limitation: upregulation of nitrogen assimilation-related proteins, including transporters; downregulation of ribosome proteins; and induction of the photosystem II cyclic electron flow. This suggests that nitrogen limitation affects a range of metabolic processes far wider than initially believed, with the ultimate goal of saving nitrogen and maximizing the nitrogen uptake and assimilation capabilities of the cell.
PubMed: 28593196
DOI: 10.1128/mSystems.00008-17 -
American Journal of Physiology.... Mar 2020The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin...
The extent of glucose metabolism during oocyte maturation is closely related to oocyte developmental potential. Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that negatively regulates glucose uptake into cells. However, little information is available regarding the function of TXNIP in bovine oocytes. Accordingly, the present study was performed to investigate the influence of TXNIP on glucose metabolism in bovine oocytes during in vitro maturation. Pharmacological inhibition of TXNIP by azaserine enhanced glucose uptake and imparted a specific metabolic effect on glycolysis and pentose phosphate pathway (PPP). RNA interference (RNAi) was adopted to further determine the biological significance of TXNIP in regulating glucose metabolism. The maturation rate and the developmental competence of TXNIP siRNA-treated oocytes were significantly improved. Knockdown of TXNIP in bovine oocytes significantly increased glycolysis by increasing the activities of phosphofructokinase (PFK), pyruvate kinase, and lactate dehydrogenase; pyruvate and lactate production; and intracellular ATP level, as well as mitochondrial activity. Furthermore, glucose metabolism through PPP was also enhanced by TXNIP depletion, as TXNIP siRNA treatment promoted glucose-6-phosphate dehydrogenase (G6PDH) activity and NADPH content, and helped maintain a high level of glutathione and a low level of reactive oxygen species within the oocytes. Further studies revealed that inhibition of TXNIP resulted increases in glucose transporter 1 (GLUT1) expression, as well as PFK1 platelet isoform () and mRNA levels. These results reveal that TXNIP depletion promotes oocyte maturation by enhancing both glycolysis and the PPP. During in vitro maturation of bovine oocytes, TXNIP serves as a key regulator of glucose uptake by controlling GLUT1 expression.
Topics: Adenosine Triphosphate; Animals; Azaserine; Carrier Proteins; Cattle; Female; Gene Knockdown Techniques; Glucose; Glycolysis; In Vitro Oocyte Maturation Techniques; Infertility, Female; Intracellular Space; Mitochondria; Oocytes; Oxidation-Reduction; RNA Interference; RNA, Small Interfering
PubMed: 31935112
DOI: 10.1152/ajpendo.00057.2019 -
Bioscience Reports Jul 2021High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate...
Glucose regulates expression of pro-inflammatory genes, IL-1β and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin.
High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1β and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.
Topics: Animals; Glucose; Hexosamines; Inflammation; Inflammation Mediators; Interleukin-12; Interleukin-1beta; Lipopolysaccharides; Macrophages; Mice; Phenotype; RAW 264.7 Cells; Up-Regulation; alpha Catenin
PubMed: 34139004
DOI: 10.1042/BSR20211066 -
Oxidative Medicine and Cellular... 2018Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues...
Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues or organs. O-linked -N-acetylglucosamine (O-GlcNAc) glycosylation of intracellular proteins denotes a type of posttranslational modification that influences multiple cytoplasmic and nuclear protein functions. Growing evidence indicates that stress can induce an acute increase in O-GlcNAc levels, which can be cytoprotective. The current study aimed to determine whether RIPC can provide renoprotection against contrast-induced acute kidney injury (CI-AKI) by augmenting O-GlcNAc signaling. We established a stable model of CI-AKI using 5/6 nephrectomized rats exposed to dehydration followed by iohexol injection via the tail vein. We found that RIPC increased UDP-GlcNAc levels through the hexosamine biosynthetic pathway as well as global renal O-GlcNAcylation. RIPC-induced elevation of O-GlcNAc signaling ameliorated CI-AKI based on the presence of less tubular damage and apoptosis and the amount of reactive oxygen species. In addition, the use of alloxan, an O-GlcNAc transferase inhibitor, and azaserine, a glutamine fructose-6-phosphate amidotransferase inhibitor, neutralized the protective effect of RIPC against oxidative stress and tubular apoptosis. In conclusion, RIPC attenuates local oxidative stress and tubular apoptosis induced by contrast exposure by enhancing O-GlcNAc glycosylation levels; this can be a potentially useful approach for lowering the risk of CI-AKI.
Topics: Acute Kidney Injury; Animals; Contrast Media; Humans; Male; N-Acetylglucosaminyltransferases; Rats; Rats, Sprague-Dawley
PubMed: 30186544
DOI: 10.1155/2018/4895913 -
Chemical Science Aug 2023Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative...
characterization of nonribosomal peptide synthetase-dependent -(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.
Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene () cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the genome. Azaserine was produced by the heterologous expression of the cluster in . enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes -(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.
PubMed: 37621439
DOI: 10.1039/d3sc01906c -
PloS One 2014The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the...
The enzyme isocitrate dehydrogenase (ICDH; EC 1.1.1.42) catalyzes the oxidative decarboxylation of isocitrate, to produce 2-oxoglutarate. The incompleteness of the tricarboxylic acids cycle in marine cyanobacteria confers a special importance to isocitrate dehydrogenase in the C/N balance, since 2-oxoglutarate can only be metabolized through the glutamine synthetase/glutamate synthase pathway. The physiological regulation of isocitrate dehydrogenase was studied in cultures of Prochlorococcus sp. strain PCC 9511, by measuring enzyme activity and concentration using the NADPH production assay and Western blotting, respectively. The enzyme activity showed little changes under nitrogen or phosphorus starvation, or upon addition of the inhibitors DCMU, DBMIB and MSX. Azaserine, an inhibitor of glutamate synthase, induced clear increases in the isocitrate dehydrogenase activity and icd gene expression after 24 h, and also in the 2-oxoglutarate concentration. Iron starvation had the most significant effect, inducing a complete loss of isocitrate dehydrogenase activity, possibly mediated by a process of oxidative inactivation, while its concentration was unaffected. Our results suggest that isocitrate dehydrogenase responds to changes in the intracellular concentration of 2-oxoglutarate and to the redox status of the cells in Prochlorococcus.
Topics: Bacterial Proteins; Isocitrate Dehydrogenase; Ketoglutaric Acids; NADP; Oxidation-Reduction; Prochlorococcus
PubMed: 25061751
DOI: 10.1371/journal.pone.0103380 -
Analytical Chemistry Aug 2016Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield...
Quantification of Azaserine-Induced Carboxymethylated and Methylated DNA Lesions in Cells by Nanoflow Liquid Chromatography-Nanoelectrospray Ionization Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.
Humans are exposed to N-nitroso compounds through environmental exposure and endogenous metabolism. Some N-nitroso compounds can be metabolically activated to yield diazoacetate, which is known to induce DNA carboxymethylation. DNA lesion measurement remains one of the core tasks in toxicology and in evaluating human health risks associated with carcinogen exposure. In this study, we developed a highly sensitive nanoflow liquid chromatography-nanoelectrospray ionization-multistage tandem mass spectrometry (nLC-nESI-MS(3)) method for the simultaneous quantification of O(6)-carboxymethyl-2'-deoxyguanosine (O(6)-CMdG), O(6)-methyl-2'-deoxyguanosine (O(6)-MedG), and N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA). We were able to measure the levels of these three lesions with the use of low-microgram quantities of DNA from cultured human skin fibroblasts and human colorectal carcinoma cells treated with azaserine, a DNA carboxymethylating agent. Our results revealed that the levels of O(6)-CMdG and O(6)-MedG increased when the dose of azaserine was increased from 0 to 450 μM. We, however, did not observe an apparent dose-dependent induction of N(6)-CMdA, suggesting the presence of repair mechanism(s) for the rapid clearance of this lesion in cells. This is the first report about the application of nLC-nESI-MS(3) technique for the simultaneous quantification of O(6)-CMdG, O(6)-MedG, and N(6)-CMdA. The method reported here will be useful for future investigations about the repair of the carboxymethylated DNA lesions and about the implications of these lesions in carcinogenesis.
Topics: Azaserine; Cells, Cultured; Chromatography, Liquid; DNA Damage; DNA Methylation; Dose-Response Relationship, Drug; Humans; Indicator Dilution Techniques; Methylation; Molecular Structure; Nanotechnology; Spectrometry, Mass, Electrospray Ionization; Structure-Activity Relationship; Tandem Mass Spectrometry
PubMed: 27441891
DOI: 10.1021/acs.analchem.6b01349