RESUMEN
Acute hepatic injury is observed in response to various stressors, including trauma, ingestion of hepatic toxins, and hepatitis. Investigations to date have focused on extrinsic and intrinsic signals required for hepatocytes to proliferate and regenerate the liver in response to injury, though there is a more limited understanding of induced stress responses promoting hepatocyte survival upon acute injury. In this issue of the JCI, Sun and colleagues detail a mechanism by which local activation of the nuclear receptor liver receptor homolog-1 (LRH-1; NR5A2) directly induces de novo asparagine synthesis and expression of asparagine synthetase (ASNS) in response to injury and show that this response restrains hepatic damage. This work opens up several avenues for inquiry, including the potential for asparagine supplementation to ameliorate acute hepatic injury.
Asunto(s)
Asparagina , Hígado , Asparagina/metabolismo , Hígado/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , HepatocitosRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Moxibustion is a traditional medical intervention of traditional Chinese medicine. It refers to the direct or indirect application of ignited moxa wool made of mugwort leaves to acupuncture points or other specific parts of the body for either treating or preventing diseases. Moxibustion has been proven to be effective in treating skin lesions of psoriasis. AIM OF THE STUDY: This study was performed to elucidate molecular mechanisms underlying the effects of moxibustion treatment on imiquimod-induced psoriatic mice. MATERIALS AND METHODS: We established an imiquimod (IMQ)-induced psoriatic mice (Model) and assessed the effects of moxibustion (Moxi) treatment on skin lesions of psoriatic mice by the PASI scores and expressions of inflammation-related factors relative to normal control mice (NC). We then performed nuclear magnetic resonance (NMR)-based metabolomic analysis on the skin tissues of the NC, Model and Moxi-treated mice to address metabolic differences among the three groups. RESULTS: Moxi mice showed reduced PASI scores and decreased expressions of the pro-inflammatory cytokines IL-8, IL-17A and IL-23 relative to Model mice. Compared with the Model group, the NC and Moxi groups shared 9 characteristic metabolites and 4 significantly altered metabolic pathways except for taurine and hypotaurine metabolism uniquely identified in the NC group. To a certain extent, moxibustion treatment improved metabolic disorders of skin lesions of psoriatic mice by decreasing glucose, valine, asparagine, aspartate and alanine-mediated cell proliferation and synthesis of scaffold proteins, alleviating histidine-mediated hyperproliferation of blood vessels, and promoting triacylglycerol decomposition. CONCLUSIONS: This study reveals the molecular mechanisms underlying the effects of moxibustion treatment on the skin lesions of psoriasis, potentially improving the clinical efficacy of moxibustion.
Asunto(s)
Moxibustión , Psoriasis , Alanina/metabolismo , Alanina/farmacología , Alanina/uso terapéutico , Animales , Asparagina/metabolismo , Asparagina/farmacología , Asparagina/uso terapéutico , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Ácido Aspártico/uso terapéutico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Glucosa/metabolismo , Histidina/metabolismo , Histidina/farmacología , Histidina/uso terapéutico , Imiquimod , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Interleucina-23/farmacología , Interleucina-23/uso terapéutico , Interleucina-8/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Psoriasis/tratamiento farmacológico , Psoriasis/terapia , Piel , Taurina/metabolismo , Triglicéridos/metabolismo , Valina/metabolismo , Valina/farmacología , Valina/uso terapéuticoRESUMEN
OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.
Asunto(s)
Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Asparaginasa/metabolismo , Asparaginasa/farmacología , Asparagina/antagonistas & inhibidores , Asparagina/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Mesilato de Imatinib/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismoRESUMEN
Chinese hamster ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13 C-glucose, 13 C-glutamine, and 13 C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-acetyl-glucosamine and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13 C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.
Asunto(s)
Asparagina , Glutamina , Animales , Asparagina/metabolismo , Células CHO , Cricetinae , Cricetulus , Glucosa/metabolismo , Glutamina/metabolismo , Ácido LácticoRESUMEN
As a potential carcinogen, acrylamide (AA) widely exists in starch-rich foods during frying, triggering international health alerts. l-Asparaginase (l-ASNase, EC 3.5.1.1) could efficiently inhibit the AA by hydrolyzing its precursor l-Asparagine. Here, a novel recombinant l-ASNase from Palaeococcus ferrophilus was identified for the first time. The purified enzyme exhibited its highest activity at pH 8.5 and 95 °C and retained more than 70% relative activity after incubation at 80 °C for 2 h. Compared to untreated French fries, the AA content in the enzyme-treated (10 U/mL, 85 °C, 15 min) French fries was significantly reduced by 79%. Notably, the l-ASNase could remain over 98% of initial activity after three months of storage at 4 °C, suggesting good storage stability. These results demonstrated that P. ferrophilusl-ASNase could be a great candidate in controlling AA in the food industry, especially at high blanching temperature.
Asunto(s)
Acrilamida/química , Asparaginasa/metabolismo , Asparagina/metabolismo , Manipulación de Alimentos , Calor , Solanum tuberosum/química , Asparagina/química , Proteínas Bacterianas/metabolismo , Estabilidad de EnzimasRESUMEN
The antioxidant function and metabolic profiles in mice after dietary supplementation with methionine were investigated. The results showed that methionine supplementation enhanced liver GSH-Px activity and upregulated Gpx1 expression in the liver and SOD1 and Gpx4 expressions in the jejunum. Nrf2/Keap1 is involved in oxidative stress, and the western blotting data exhibited that dietary methionine markedly increased Keap1 abundance, while failed to influence the Nrf2 signal. Metabolomics investigation showed that methionine administration increased 2-hydroxypyridine, salicin, and asparagine and reduced D-Talose, maltose, aminoisobutyric acid, and inosine 5'-monophosphate in the liver, which are widely reported to involve in oxidative stress, lipid metabolism, and nucleotides generation. In conclusion, our study provides insights into antioxidant function and liver metabolic profiles in response to dietary supplementation with methionine.
Asunto(s)
Suplementos Dietéticos , Regulación de la Expresión Génica/efectos de los fármacos , Yeyuno/efectos de los fármacos , Hígado/efectos de los fármacos , Metaboloma/efectos de los fármacos , Metionina/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animales , Antioxidantes/metabolismo , Asparagina/metabolismo , Alcoholes Bencílicos/metabolismo , Dieta/métodos , Femenino , Glucósidos/metabolismo , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Inosina Monofosfato/metabolismo , Yeyuno/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Lactonas/metabolismo , Hígado/metabolismo , Maltosa/metabolismo , Metaboloma/fisiología , Metionina/administración & dosificación , Ratones , Ratones Endogámicos ICR , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Piridonas/metabolismo , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
l-Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l-asparaginase was screened. Type I l-asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l-asparaginase had an activity of 42.0 IU mL-1 and showed no activity toward l-glutamine and d-asparagine. The recombinant l-asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and ß-mercaptoethanol. The Km and Vmax values of the l-asparaginase for the l-asparagine were 3.22 mmol L-1 and 1.55 IU µg-1, respectively. Moreover, the recombinant l-asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l-asparaginase has the potential for application in the food processing industry.
Asunto(s)
Acinetobacter/enzimología , Acrilamida/metabolismo , Asparaginasa/metabolismo , Solanum tuberosum/metabolismo , Acinetobacter/genética , Asparaginasa/genética , Asparagina/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Glutamina/metabolismo , BocadillosRESUMEN
Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.
Asunto(s)
Asparagina/metabolismo , Glutamina/metabolismo , Virus Vaccinia/metabolismo , Vaccinia/virología , Animales , Antivirales , Línea Celular , Humanos , Metaboloma , Poxviridae , Biosíntesis de Proteínas , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Peptidoglycan synthesis is an important target for antibiotics and relies on intermediates derived from central metabolism. As a result, alterations of metabolism may affect antibiotic sensitivity. An aspB mutant is auxotrophic for aspartate (Asp) and asparagine (Asn) and lyses when grown in Difco sporulation medium (DSM), but not in LB medium. Genetic and physiological studies, supported by amino acid analysis, reveal that cell lysis in DSM results from Asp limitation due to a relatively low Asp and high glutamate (Glu) concentrations, with Glu functioning as a competitive inhibitor of Asp uptake by the major Glu/Asp transporter GltT. Lysis can be specifically suppressed by supplementation with 2,6-diaminopimelate (DAP), which is imported by two different cystine uptake systems. These studies suggest that aspartate limitation depletes the peptidoglycan precursor meso-2,6-diaminopimelate (mDAP), inhibits peptidoglycan synthesis, upregulates the cell envelope stress response mediated by σM and eventually leads to cell lysis. Aspartate limitation sensitizes cells to antibiotics targeting late steps of PG synthesis, but not steps prior to the addition of mDAP into the pentapeptide sidechain. This work highlights the ability of perturbations of central metabolism to sensitize cells to peptidoglycan synthesis inhibitors.
Asunto(s)
Antibacterianos/farmacología , Aspartato Aminotransferasas/genética , Ácido Aspártico/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/metabolismo , Peptidoglicano/biosíntesis , Asparagina/metabolismo , Bacillus subtilis/genética , Pared Celular/metabolismo , Ácido Diaminopimélico/metabolismo , Ácido Glutámico/metabolismoRESUMEN
Alterations of the cellular proteome over time due to spontaneous or toxin-mediated enzymatic deamidation of glutamine (Gln) and asparagine (Asn) residues contribute to bacterial infection and might represent a source of aging-related diseases. Here, we put into perspective what is known about the mode of action of the CNF1 toxin from pathogenic Escherichia coli, a paradigm of bacterial deamidases that activate Rho GTPases, to illustrate the importance of determining whether exposure to these factors are risk factors in the etiology age-related diseases, such as cancer. In particular, through in silico analysis of the distribution of the CNF1-like deamidase active site Gly-Cys-(Xaa)n-His sequence motif in bacterial genomes, we unveil the wide distribution of the super-family of CNF-like toxins and CNF-like deamidase domains among members of the Enterobacteriacae and in association with a large variety of toxin delivery systems. We extent our discussion with recent findings concerning cellular systems that control activated Rac1 GTPase stability and provide protection against cancer. These findings point to the urgency for developing holistic approaches toward personalized medicine that include monitoring for asymptomatic carriage of pathogenic toxin-producing bacteria and that ultimately might lead to improved public health and increased lifespans.
Asunto(s)
Amidohidrolasas/metabolismo , Toxinas Bacterianas/metabolismo , Enterobacteriaceae/enzimología , Proteínas de Escherichia coli/metabolismo , Factores Inmunológicos/metabolismo , Factores de Virulencia/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Amidohidrolasas/genética , Asparagina/metabolismo , Toxinas Bacterianas/genética , Biología Computacional , Infecciones por Enterobacteriaceae/complicaciones , Infecciones por Enterobacteriaceae/patología , Proteínas de Escherichia coli/genética , Glutamina/metabolismo , Neoplasias/etiología , Neoplasias/fisiopatología , Factores de Virulencia/genéticaRESUMEN
Proliferation of acute lymphoblastic leukemic cells is nutritionally dependent on the external supply of asparagine. l-asparaginase, an enzyme hydrolyzing l-asparagine in blood, is used for treatment of acute lymphoblastic leukemic and other related blood cancers. Although previous studies demonstrated that l-asparaginase suppresses the proliferation of cultured solid tumor cells, it remains unclear whether this enzyme prevents the growth of solid tumors in vivo. In this study, we demonstrated the importance of optimizing dosing schedules for the anti-tumor activity of l-asparaginase in 4T1 breast tumor-bearing mice. Cultures of several types of murine solid tumor cells were dependent on the external supply of asparagine. Among them, we selected murine 4T1 breast cancer cells and implanted them into BALB/c female mice kept under standardized light/dark cycle conditions. The growth of 4T1 tumor cells implanted in mice was significantly suppressed by intravenous administration of l-asparaginase during the light phase, whereas its administration during the dark phase failed to show significant anti-tumor activity. Decreases in plasma asparagine levels due to the administration of l-asparaginase were closely related to the dosing time-dependency of its anti-tumor effects. These results suggest that the anti-tumor efficacy of l-asparaginase in breast tumor-bearing mice is improved by optimizing the dosing schedule.
Asunto(s)
Antineoplásicos/administración & dosificación , Asparaginasa/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Cronoterapia de Medicamentos , Animales , Asparagina/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Esquema de Medicación , Femenino , Humanos , Infusiones Intravenosas , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
Streptococcus mutans, the primary aetiological agent of dental caries, is one of the major bacteria of the human oral cavity. The pathogenicity of this bacterium is attributed not only to the expression of virulence factors, but also to its ability to respond and adapt rapidly to the ever-changing conditions of the oral cavity. The two-component signal transduction system (TCS) CovR/S plays a crucial role in virulence and stress response in many streptococci. Surprisingly, in S. mutans the response regulator CovR appears to be an orphan, as the cognate sensor kinase, CovS, is absent in all the strains. We found that acetyl phosphate, an intracellular phosphodonor molecule known to act in signalling, might play a role in CovR phosphorylation in vivo. We also found that in vitro, upon phosphorylation by potassium phosphoramide (a high-energy phophodonor) CovR formed a dimer and showed altered electrophoretic mobility. As expected, we found that the conserved aspartic acid residue at position 53 (D53) was the site of phosphorylation, since neither phosphorylation nor dimerization was seen when an alanine-substituted CovR mutant (D53A) was used. Surprisingly, we found that the ability of CovR to act as a transcriptional regulator does not depend upon its phosphorylation status, since the D53A mutant behaved similarly to the wild-type protein in both in vivo and in vitro DNA-binding assays. This unique phosphorylation-mediated inhibition of CovR function in S. mutans sheds light on an unconventional mechanism of the signal transduction pathway.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Streptococcus mutans/metabolismo , Factores de Transcripción/metabolismo , Asparagina/genética , Asparagina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Caries Dental/microbiología , Mutación , Organofosfatos/metabolismo , Fosforilación , Ftalimidas/farmacología , Regiones Promotoras Genéticas , Unión Proteica , Multimerización de Proteína/efectos de los fármacos , Streptococcus mutans/genética , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Insulin-like growth factor I (IGF-I) is a key regulator of chondrogenesis, but its therapeutic application to articular cartilage damage is limited by rapid elimination from the repair site. The human IGF-I gene gives rise to three IGF-I propeptides (proIGF-IA, proIGF-IB and proIGF-IC) that are cleaved to create mature IGF-I. In this study, we elucidate the processing of IGF-I precursors by articular chondrocytes, and test the hypotheses that proIGF-I isoforms bind to heparin and regulate articular chondrocyte biosynthesis. METHODS: Human IGF-I propeptides and mutants were overexpressed in bovine articular chondrocytes. IGF-I products were characterized by ELISA, western blot and FPLC using a heparin column. The biosynthetic activity of IGF-I products on articular chondrocytes was assayed for DNA and glycosaminoglycan that the cells produced. RESULTS: Secreted IGF-I propeptides stimulated articular chondrocyte biosynthetic activity to the same degree as mature IGF-I. Of the three IGF-I propeptides, only one, proIGF-IA, strongly bound to heparin. Interestingly, heparin binding of proIGF-IA depended on N-glycosylation at Asn92 in the EA peptide. To our knowledge, this is the first demonstration that N-glycosylation determines the binding of a heparin-binding protein to heparin. CONCLUSION: The biosynthetic and heparin binding abilities of proIGF-IA, coupled with its generation of IGF-I, suggest that proIGF-IA may have therapeutic value for articular cartilage repair. GENERAL SIGNIFICANCE: These data identify human pro-insulin-like growth factor IA as a bifunctional protein. Its combined ability to bind heparin and augment chondrocyte biosynthesis makes it a promising therapeutic agent for cartilage damage due to trauma and osteoarthritis.
Asunto(s)
Cartílago Articular/citología , Condrocitos/efectos de los fármacos , Heparina/metabolismo , Factor I del Crecimiento Similar a la Insulina/fisiología , Precursores de Proteínas/fisiología , Procesamiento Proteico-Postraduccional , Empalme Alternativo , Animales , Asparagina/metabolismo , Secuencia de Bases , Bovinos , Células Cultivadas , Condrocitos/metabolismo , Evaluación Preclínica de Medicamentos , Glicosilación , Humanos , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/farmacología , Unión Proteica , Isoformas de Proteínas/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/farmacología , Proteínas Recombinantes/metabolismoRESUMEN
While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.IMPORTANCE We have previously found that Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
Asunto(s)
Asparagina/metabolismo , Proliferación Celular , Glutamina/metabolismo , Herpesvirus Humano 8/fisiología , Neoplasias/patología , Neoplasias/virología , Nucleótidos/biosíntesis , Asparagina/farmacología , Aspartato Aminotransferasas/genética , Ácido Aspártico/farmacología , Proliferación Celular/efectos de los fármacos , Glutamato Deshidrogenasa/genética , Ácido Glutámico/farmacología , Glutaminasa/genética , Glutamina/deficiencia , Humanos , Redes y Vías Metabólicas , Neoplasias/fisiopatología , Nitrógeno/metabolismoRESUMEN
Endothelial cell (EC) metabolism is emerging as a regulator of angiogenesis, but the precise role of glutamine metabolism in ECs is unknown. Here, we show that depriving ECs of glutamine or inhibiting glutaminase 1 (GLS1) caused vessel sprouting defects due to impaired proliferation and migration, and reduced pathological ocular angiogenesis. Inhibition of glutamine metabolism in ECs did not cause energy distress, but impaired tricarboxylic acid (TCA) cycle anaplerosis, macromolecule production, and redox homeostasis. Only the combination of TCA cycle replenishment plus asparagine supplementation restored the metabolic aberrations and proliferation defect caused by glutamine deprivation. Mechanistically, glutamine provided nitrogen for asparagine synthesis to sustain cellular homeostasis. While ECs can take up asparagine, silencing asparagine synthetase (ASNS, which converts glutamine-derived nitrogen and aspartate to asparagine) impaired EC sprouting even in the presence of glutamine and asparagine. Asparagine further proved crucial in glutamine-deprived ECs to restore protein synthesis, suppress ER stress, and reactivate mTOR signaling. These findings reveal a novel link between endothelial glutamine and asparagine metabolism in vessel sprouting.
Asunto(s)
Asparagina/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Glutamina/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Medios de Cultivo/química , Células Endoteliales/metabolismo , Glutaminasa/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Redes y Vías Metabólicas , Neovascularización PatológicaRESUMEN
BACKGROUND: Coffee substitutes made of roasted chicory are affected by the formation of acrylamide whose main precursor is asparagine. One strategy for limiting the formation of acrylamide is to reduce free asparagine in the chicory roots by lessening the supply of nitrogen in the field. However, decreasing nitrogen fertilizer could affect the formation of the volatile compounds and, consequently, the sensory characteristics of the roasted chicory. The present study aimed to investigate the impact of the nitrogen supply in five commercial varieties on their aroma profile. RESULTS: The addition of 120 kg ha-1 of nitrogen fertilizer in the field resulted in a greater amount of pyrazines in the roasted chicory. Triangle tests were performed to determine the effect of the nitrogen level on the sensory quality of the five varieties. The results revealed that the chicory aroma was modified in two out of five varieties. CONCLUSION: The results of the present study suggest that a strategy aiming to limit the amount of acrylamide could affect the sensory quality of some varieties of chicory. Further acceptance tests need to be conducted to assess the effect (whether favourable or otherwise) on the sensory quality of the coffee substitutes. © 2016 Society of Chemical Industry.
Asunto(s)
Bebidas/análisis , Cichorium intybus/metabolismo , Fertilizantes , Contaminación de Alimentos/prevención & control , Nitrógeno/metabolismo , Odorantes , Pirazinas/metabolismo , Acrilamida/metabolismo , Adulto , Agricultura/métodos , Asparagina/metabolismo , Cichorium intybus/clasificación , Café , Comportamiento del Consumidor , Culinaria , Femenino , Preferencias Alimentarias , Humanos , Masculino , Odorantes/análisis , Especificidad de la Especie , Compuestos Orgánicos Volátiles/metabolismo , Adulto JovenRESUMEN
Finafloxacin is a novel fluoroquinolone with improved antimicrobial efficacy, especially in an acidic environment. The efficacy of finafloxacin for the inhibition of Helicobacter pylori infection was compared with the efficacies of levofloxacin and moxifloxacin at neutral and acidic pH. The impacts of gyrA point mutation on the efficacy of those three fluoroquinolones were also investigated. A total of 128 clinical H. pylori strains were utilized. MICs of levofloxacin, moxifloxacin, and finafloxacin were determined at pH 5.0 and pH 7.0 by the agar dilution method. The impact of gyrA point mutations that are responsible for fluoroquinolone resistance was analyzed; the results showed 50 strains with an Asn-87 point mutation, 48 strains with an Asp-91 point mutation, and the remaining 30 strains with no gyrA mutations. The use of finafloxacin led to MIC values at pH 5.0 that were lower than the values seen at pH 7.0 for 112 strains (112/128, 87.5%), and this proportion was higher than that seen with moxifloxacin (21/128, 16.4%, P < 0.001). Finafloxacin also demonstrated a rate of susceptibility (MIC, <1 µg/ml) (37.5%, 48/128) at pH 5.0 that was higher than that seen with moxifloxacin (2.3%, 3/128) (P < 0.001). The trends were similar regardless of which of the Asn-87, Asp-91, and A2143 point mutations were present. In conclusion, the superior antimicrobial efficacy of finafloxacin against H. pylori in an acidic environment suggests the possible use of finafloxacin for treatment of H. pylori infection, as has been proposed by its developer, Merlion Pharma.
Asunto(s)
Antibacterianos/uso terapéutico , Girasa de ADN/genética , Fluoroquinolonas/uso terapéutico , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori/efectos de los fármacos , Mutación Puntual , Sustitución de Aminoácidos , Asparagina/metabolismo , Ácido Aspártico/metabolismo , Girasa de ADN/metabolismo , Femenino , Expresión Génica , Infecciones por Helicobacter/microbiología , Helicobacter pylori/enzimología , Helicobacter pylori/genética , Helicobacter pylori/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Levofloxacino/uso terapéutico , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Moxifloxacino , Resultado del TratamientoRESUMEN
The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed the Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by the generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine, and proline in a macrophage cell line (RAW264.7) and an epithelial cell line (HeLa) and examined access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine, or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine, or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine- or aspartate-auxotrophic strains support distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline, or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by the extracellular addition of succinate. Complementation of the intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in the induction of Salmonella-induced filaments (SIFs) but failed in a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains and, more generally, is essential to continuously supply bacteria within the SCV with nutrients.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Glicoproteínas/genética , Salmonella enterica/metabolismo , Vacuolas/metabolismo , Alanina/metabolismo , Alanina/farmacología , Animales , Asparagina/metabolismo , Asparagina/farmacología , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacología , Proteínas Bacterianas/metabolismo , Glutamina/metabolismo , Glutamina/farmacología , Glicoproteínas/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Prolina/metabolismo , Prolina/farmacología , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética , Succinato Deshidrogenasa/deficiencia , Succinato Deshidrogenasa/genética , Ácido Succínico/metabolismo , Ácido Succínico/farmacología , Vacuolas/efectos de los fármacos , Vacuolas/microbiologíaRESUMEN
This study investigated the fate of acrylamide in thermally processed foods after ingestion. An in vitro multistep enzymatic digestion system simulating gastric, duodenal and colon phases was used to understand the fate of acrylamide in bakery and fried potato products. Acrylamide levels gradually decreased through gastric, duodenal and colon phases during in vitro digestion of biscuits. At the end of digestion, acrylamide reduction was between 49.2% and 73.4% in biscuits. Binary model systems composed of acrylamide and amino acids were used to understand the mechanism of acrylamide reduction. High-resolution mass spectrometry analyses confirmed Michael addition of amino acids to acrylamide during digestion. In contrast to bakery products, acrylamide levels increased significantly during gastric digestion of fried potatoes. The Schiff base formed between reducing sugars and asparagine disappeared rapidly, whereas the acrylamide level increased during the gastric phase. This suggests that intermediates like the Schiff base that accumulate in potatoes during frying are potential precursors of acrylamide under gastric conditions.
Asunto(s)
Acrilamida/química , Pan/análisis , Culinaria , Digestión , Modelos Moleculares , Raíces de Plantas/química , Solanum tuberosum/química , Acrilamida/análisis , Acrilamida/metabolismo , Asparagina/análisis , Asparagina/química , Asparagina/metabolismo , Carcinógenos/análisis , Carcinógenos/química , Carcinógenos/metabolismo , Cistina/análisis , Cistina/química , Cistina/metabolismo , Carbohidratos de la Dieta/análisis , Carbohidratos de la Dieta/metabolismo , Contaminación de Alimentos , Jugo Gástrico/química , Jugo Gástrico/enzimología , Jugo Gástrico/metabolismo , Calor/efectos adversos , Humanos , Secreciones Intestinales/química , Secreciones Intestinales/enzimología , Secreciones Intestinales/metabolismo , Lisina/análisis , Lisina/química , Lisina/metabolismo , Estructura Molecular , Bases de Schiff/análisis , Bases de Schiff/química , Bases de Schiff/metabolismoRESUMEN
A unique and nontraditional approach using glutamine and asparagine supplements for CHO-glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench-top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines.