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1.
Chem Commun (Camb) ; 55(37): 5339-5342, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30973558

RESUMO

A tunable chemoenzymatic strategy provides access to the entire class of magic spot nucleotides and modified analogues. The approach combines chemoselective bisphosphorylations using phosphoramidites with regioselective ribonuclease T2 cyclo-phosphate hydrolysis, leading to flexible and simple gram-scale operations.


Assuntos
Endorribonucleases/metabolismo , Nucleotídeos/biossíntese , Biocatálise , Ciclização , Eletroforese em Gel de Poliacrilamida , Hidrólise , Nucleotídeos/química , Fosfatos/química , Fosfatos/metabolismo , Estereoisomerismo
2.
Methods Mol Biol ; 1973: 39-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016695

RESUMO

Synthesis of base-modified dNTPs through the Suzuki or Sonogashira cross-coupling reactions of halogenated dNTPs with boronic acids or alkynes is reported, as well as the use of these modified dNTPs in polymerase incorporations to oligonucleotides or DNA by primer extension or PCR.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Nucleotídeos/biossíntese , DNA/química , Halogenação , Nucleotídeos/química
3.
Nature ; 567(7747): 194-199, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787435

RESUMO

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.


Assuntos
Proteínas de Bactérias/metabolismo , Nucleotídeos/biossíntese , Nucleotídeos/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Animais , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/biossíntese , Fosfatos de Dinucleosídeos/metabolismo , Células HEK293 , Humanos , Camundongos , Nucleotídeos/química , Nucleotidiltransferases/genética , Óperon/genética , Simbiose
4.
Int J Mol Sci ; 20(4)2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30769780

RESUMO

Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a "thymidine block", but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications.


Assuntos
Terapia Genética , Células-Tronco Pluripotentes Induzidas/enzimologia , Simplexvirus/enzimologia , Timidina Quinase/genética , Apoptose/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ganciclovir/farmacologia , Edição de Genes , Regulação Enzimológica da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Genes Transgênicos Suicidas/genética , Vetores Genéticos/uso terapêutico , Humanos , Células-Tronco Pluripotentes Induzidas/transplante , Lentivirus/genética , Nucleotídeos/biossíntese , Nucleotídeos/genética , Simplexvirus/genética
5.
Nat Commun ; 10(1): 201, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643150

RESUMO

Under hypoxia, most of glucose is converted to secretory lactate, which leads to the overuse of glutamine-carbon. However, under such a condition how glutamine nitrogen is disposed to avoid over-accumulating ammonia remains to be determined. Here we identify a metabolic flux of glutamine to secretory dihydroorotate, which is indispensable to glutamine-carbon metabolism under hypoxia. We found that glutamine nitrogen is necessary to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. Dihyroorotate, not orotate, is then secreted out of cells. Furthermore, we found that the specific metabolic pathway occurs in vivo and is required for tumor growth. The identified metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis, with the rest carbon and nitrogen being safely removed. Therefore, our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism.


Assuntos
Glutamina/metabolismo , Redes e Vias Metabólicas , Metaboloma , Neoplasias/metabolismo , Ácido Orótico/análogos & derivados , Acetilcoenzima A/metabolismo , Amônia/metabolismo , Amônia/toxicidade , Animais , Carbono/química , Carbono/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Glucose/metabolismo , Glutamina/química , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Lipogênese , Metabolômica , Camundongos , Camundongos Nus , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/patologia , Nitrogênio/química , Nitrogênio/metabolismo , Nucleotídeos/biossíntese , Ácido Orótico/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Proc Natl Acad Sci U S A ; 116(3): 826-834, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598453

RESUMO

Toxin-antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654-COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.


Assuntos
ADP Ribose Transferases/metabolismo , Nucleotídeos/biossíntese , Ribose-Fosfato Pirofosfoquinase/metabolismo , Sphingomonadaceae/enzimologia , Sistemas Toxina-Antitoxina , Sequência de Aminoácidos , Cristalografia , Escherichia coli
7.
Biotechnol J ; 14(4)2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30367549

RESUMO

The availability of nucleotide sugars is considered as bottleneck for Leloir-glycosyltransferases mediated glycan synthesis. A breakthrough for the synthesis of nucleotide sugars is the development of salvage pathway like enzyme cascades with high product yields from affordable monosaccharide substrates. In this regard, the authors aim at high enzyme productivities of these cascades by a repetitive batch approach. The authors report here for the first time that the exceptional high enzyme cascade stability facilitates the synthesis of Uridine-5'-diphospho-α-d-galactose (UDP-Gal), Uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and Uridine-5'-diphospho-N-acetylgalactosamine (UDP-GalNAc) in a multi-gram scale by repetitive batch mode. The authors obtained 12.8 g UDP-Gal through a high mass based total turnover number (TTNmass ) of 494 [gproduct /genzyme ] and space-time-yield (STY) of 10.7 [g/L*h]. Synthesis of UDP-GlcNAc in repetitive batch mode gave 11.9 g product with a TTNmass of 522 [gproduct /genzyme ] and a STY of 9.9 [g/L*h]. Furthermore, the scale-up to a 200 mL scale using a pressure operated concentrator was demonstrated for a UDP-GalNAc producing enzyme cascade resulting in an exceptional high STY of 19.4 [g/L*h] and 23.3 g product. In conclusion, the authors demonstrate that repetitive batch mode is a versatile strategy for the multi-gram scale synthesis of nucleotide sugars by stable enzyme cascades.


Assuntos
Polissacarídeos/química , Uridina Difosfato Galactose/biossíntese , Uridina Difosfato N-Acetilglicosamina/biossíntese , Açúcares de Uridina Difosfato/biossíntese , Glicosiltransferases/química , Nucleotídeos/biossíntese , Nucleotídeos/química , Transferases (Outros Grupos de Fosfato Substituídos)/química , Uridina Difosfato Galactose/química , Uridina Difosfato N-Acetilglicosamina/química , Açúcares de Uridina Difosfato/química
8.
New Phytol ; 221(3): 1289-1302, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30368816

RESUMO

Viruses that infect photoautotrophs have a fundamental relationship with light, given the need for host resources. We investigated the role of light on Coccolithovirus (EhV) infection of the globally distributed coccolithophore, Emiliania huxleyi. Light was required for EhV adsorption, and viral production was highest when host cultures were maintained in continuous light or at irradiance levels of 150-300 µmol m-2  s-1 . During the early stages of infection, photosynthetic electron transport remained high, while RuBisCO expression decreased concomitant with an induction of the pentose phosphate pathway, the primary source of de novo nucleotides. A mathematical model developed and fitted to the laboratory data supported the hypothesis that EhV replication was controlled by a trade-off between host nucleotide recycling and de novo synthesis, and that photoperiod and photon flux could toggle this switch. Laboratory results supported field observations that light was the most robust driver of EhV replication within E. huxleyi populations collected across a 2000 nautical mile transect in the North Atlantic. Collectively, these findings demonstrate that light can drive host-virus interactions through a mechanistic interplay between host metabolic processes, which serve to structure infection and phytoplankton mortality in the upper ocean.


Assuntos
Haptófitas/efeitos da radiação , Haptófitas/virologia , Interações Hospedeiro-Patógeno/efeitos da radiação , Luz , Phycodnaviridae/fisiologia , Adsorção , Haptófitas/crescimento & desenvolvimento , NADP/metabolismo , Nucleotídeos/biossíntese , Via de Pentose Fosfato/efeitos da radiação , Fotoperíodo , Fotossíntese/efeitos da radiação
9.
Protein Expr Purif ; 156: 36-43, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30557611

RESUMO

Nuclease P1 gene (nuc P1) which was cloned from Penicillium citrinum and expressed in A. niger Bdel4 with the low-background extracellular protein. The expression strategy of multi-copy nuc P1 in the A. niger with the linker of 2A peptide was applied to improve the enzyme activity of nuclease P1, the highest activity up to 77.6 U/mL. After Ni-chelate purification, the specific enzyme activity, the optimum temperature and pH were 32.4 U/mg, 65 °C and 5.3 respectively. The recombination nuclease P1 was activated by addition of Mg2+, Zn2+ and Cu2+, and inhibited by addition of Ca2+, Fe2+, Mn2+, Ni2+, Co2+, Mg2+, K+ and EDTA. Furthermore, the enzyme hydrolyses yeast RNA efficiently into 5'- nucleotides. Through enzymolysis, the highest concentration of nucleotides achieved 15.12 mg/mL, and 75U nuclease P1 is suitable amount should be added to the enzymolysis system.


Assuntos
Aspergillus niger , Proteínas Fúngicas/biossíntese , Nucleotídeos/biossíntese , Penicillium/enzimologia , Proteínas Recombinantes/biossíntese , Endonucleases Específicas para DNA e RNA de Cadeia Simples/biossíntese , Aspergillus niger/enzimologia , Proteínas Fúngicas/genética , Hidrólise , Proteínas Recombinantes/genética , Endonucleases Específicas para DNA e RNA de Cadeia Simples/genética
10.
Nat Commun ; 9(1): 5442, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30575741

RESUMO

Phosphoglycerate dehydrogenase (PHGDH) catalyzes the committed step in de novo serine biosynthesis. Paradoxically, PHGDH and serine synthesis are required in the presence of abundant environmental serine even when serine uptake exceeds the requirements for nucleotide synthesis. Here, we establish a mechanism for how PHGDH maintains nucleotide metabolism. We show that inhibition of PHGDH induces alterations in nucleotide metabolism independent of serine utilization. These changes are not attributable to defects in serine-derived nucleotide synthesis and redox maintenance, another key aspect of serine metabolism, but result from disruption of mass balance within central carbon metabolism. Mechanistically, this leads to simultaneous alterations in both the pentose phosphate pathway and the tri-carboxylic acid cycle, as we demonstrate based on a quantitative model. These findings define a mechanism whereby disruption of one metabolic pathway induces toxicity by simultaneously affecting the activity of multiple related pathways.


Assuntos
Ciclo do Ácido Cítrico , Nucleotídeos/biossíntese , Via de Pentose Fosfato , Fosfoglicerato Desidrogenase/metabolismo , Células HCT116 , Humanos , Células MCF-7 , Análise do Fluxo Metabólico , Serina/biossíntese
11.
EMBO J ; 37(22)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30348863

RESUMO

The Hippo pathway and its nuclear effector Yap regulate organ size and cancer formation. While many modulators of Hippo activity have been identified, little is known about the Yap target genes that mediate these growth effects. Here, we show that yap -/- mutant zebrafish exhibit defects in hepatic progenitor potential and liver growth due to impaired glucose transport and nucleotide biosynthesis. Transcriptomic and metabolomic analyses reveal that Yap regulates expression of glucose transporter glut1, causing decreased glucose uptake and use for nucleotide biosynthesis in yap -/- mutants, and impaired glucose tolerance in adults. Nucleotide supplementation improves Yap deficiency phenotypes, indicating functional importance of glucose-fueled nucleotide biosynthesis. Yap-regulated glut1 expression and glucose uptake are conserved in mammals, suggesting that stimulation of anabolic glucose metabolism is an evolutionarily conserved mechanism by which the Hippo pathway controls organ growth. Together, our results reveal a central role for Hippo signaling in glucose metabolic homeostasis.


Assuntos
Glucose/metabolismo , Fígado/embriologia , Nucleotídeos/biossíntese , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Glucose/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Camundongos , Nucleotídeos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
13.
Int J Med Microbiol ; 308(8): 990-999, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30145133

RESUMO

Growth in urinary tract depends on the ability of uropathogenic E. coli to adjust metabolism in response to available nutrients, especially to synthesize metabolites that are present in urinary tract with limited concentrations. In this study, a genome-wide assay was applied and identified five nucleotide biosynthetic genes purA, guaAB and carAB that are required for optimal growth of UPEC in human urine and colonization in vivo. Subsequent functional analyses revealed that either interruption of de novo nucleotide biosynthesis or blocking of salvage pathways alone could not decrease UPEC's growth, while only simultaneous interruption of both two pathways significantly reduced UPEC's growth in urine. Evidences showed that uracil, xanthine, and hypoxanthine in human urine could support nucleotide biosynthesis through salvage pathways when the de novo pathways were interrupted. Moreover, the expression of genes involved in salvage pathways of nucleotide biosynthesis were significantly upregulated when UPEC are cultured in human urine and artificial urine medium with uracil, xanthine or hypoxanthine. Finally, animal tests showed that further deletion of genes involved in salvage nucleotide biosynthesis from mutants with defects in de novo pathways significantly reduced UPEC's colonization in host bladders and kidneys. These results indicated that UPEC preferentially utilize abundant metabolites in urine for nucleotide biosynthesis through salvage pathways, which is not like in serum, where the limiting amounts of substrates for salvage biosynthesis force invading pathogens to rely on de novo nucleotide biosynthesis. Taken together, our study implied the importance of salvage pathways of nucleotides biosynthesis for UPEC's fitness during urinary tract infection.


Assuntos
Infecções por Escherichia coli/microbiologia , Nucleotídeos/biossíntese , Infecções Urinárias/microbiologia , Sistema Urinário/metabolismo , Escherichia coli Uropatogênica/fisiologia , Animais , Infecções por Escherichia coli/urina , Proteínas de Escherichia coli/genética , Feminino , Humanos , Hipoxantina/urina , Rim/microbiologia , Camundongos , Camundongos Endogâmicos CBA , Mutagênese , Uracila/urina , Bexiga Urinária/microbiologia , Sistema Urinário/microbiologia , Infecções Urinárias/urina , Escherichia coli Uropatogênica/genética , Xantina/urina
14.
Nature ; 559(7715): 632-636, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29995852

RESUMO

The chemotherapeutic drug methotrexate inhibits the enzyme dihydrofolate reductase1, which generates tetrahydrofolate, an essential cofactor in nucleotide synthesis2. Depletion of tetrahydrofolate causes cell death by suppressing DNA and RNA production3. Although methotrexate is widely used as an anticancer agent and is the subject of over a thousand ongoing clinical trials4, its high toxicity often leads to the premature termination of its use, which reduces its potential efficacy5. To identify genes that modulate the response of cancer cells to methotrexate, we performed a CRISPR-Cas9-based screen6,7. This screen yielded FTCD, which encodes an enzyme-formimidoyltransferase cyclodeaminase-that is required for the catabolism of the amino acid histidine8, a process that has not previously been linked to methotrexate sensitivity. In cultured cancer cells, depletion of several genes in the histidine degradation pathway markedly decreased sensitivity to methotrexate. Mechanistically, histidine catabolism drains the cellular pool of tetrahydrofolate, which is particularly detrimental to methotrexate-treated cells. Moreover, expression of the rate-limiting enzyme in histidine catabolism is associated with methotrexate sensitivity in cancer cell lines and with survival rate in patients. In vivo dietary supplementation of histidine increased flux through the histidine degradation pathway and enhanced the sensitivity of leukaemia xenografts to methotrexate. The histidine degradation pathway markedly influences the sensitivity of cancer cells to methotrexate and may be exploited to improve methotrexate efficacy through a simple dietary intervention.


Assuntos
Histidina/metabolismo , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Amônia-Liases/deficiência , Amônia-Liases/genética , Amônia-Liases/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Feminino , Antagonistas do Ácido Fólico/farmacologia , Antagonistas do Ácido Fólico/uso terapêutico , Glutamato Formimidoiltransferase/deficiência , Glutamato Formimidoiltransferase/genética , Glutamato Formimidoiltransferase/metabolismo , Histidina/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Nucleotídeos/biossíntese , Proteína Carregadora de Folato Reduzido/genética , Proteína Carregadora de Folato Reduzido/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Tetra-Hidrofolatos/deficiência , Tetra-Hidrofolatos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Proc Natl Acad Sci U S A ; 115(30): E7158-E7165, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987044

RESUMO

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.


Assuntos
Infecções por Picornaviridae/metabolismo , Rhinovirus/fisiologia , Replicação Viral/fisiologia , Animais , Desoxiglucose/farmacologia , Feminino , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Camundongos , Nucleotídeos/biossíntese , Nucleotídeos/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Infecções por Picornaviridae/tratamento farmacológico , Infecções por Picornaviridae/genética , Infecções por Picornaviridae/patologia , Replicação Viral/efeitos dos fármacos
16.
Nat Med ; 24(7): 1036-1046, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29892070

RESUMO

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.


Assuntos
Neoplasias/patologia , Fosforilação Oxidativa , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Metabolismo Energético , Glicólise/efeitos dos fármacos , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Mitocôndrias/metabolismo , Nucleotídeos/biossíntese , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
17.
EMBO J ; 37(14)2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-29875130

RESUMO

Cisplatin is the most widely used chemotherapeutic agent, and resistance of neoplastic cells against this cytoxicant poses a major problem in clinical oncology. Here, we explored potential metabolic vulnerabilities of cisplatin-resistant non-small human cell lung cancer and ovarian cancer cell lines. Cisplatin-resistant clones were more sensitive to killing by nutrient deprivation in vitro and in vivo than their parental cisplatin-sensitive controls. The susceptibility of cisplatin-resistant cells to starvation could be explained by a particularly strong dependence on glutamine. Glutamine depletion was sufficient to restore cisplatin responses of initially cisplatin-resistant clones, and glutamine supplementation rescued cisplatin-resistant clones from starvation-induced death. Mass spectrometric metabolomics and specific interventions on glutamine metabolism revealed that, in cisplatin-resistant cells, glutamine is mostly required for nucleotide biosynthesis rather than for anaplerotic, bioenergetic or redox reactions. As a result, cisplatin-resistant cancers became exquisitely sensitive to treatment with antimetabolites that target nucleoside metabolism.


Assuntos
Antimetabólitos/farmacologia , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glutamina/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Morte Celular , Linhagem Celular Tumoral , Metabolismo Energético , Feminino , Humanos , Espectrometria de Massas , Metaboloma , Modelos Biológicos , Nucleotídeos/biossíntese
18.
J Virol ; 92(17)2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950419

RESUMO

The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on the oxygen consumption rate) and glycolytic (based on the extracellular acidification rate) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of RVs to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population.IMPORTANCE RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data add viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. In addition, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations.


Assuntos
Metabolismo Energético , Glutamina/metabolismo , Glicólise/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Vírus da Rubéola/metabolismo , Células A549 , Células Endoteliais/metabolismo , Células Endoteliais/virologia , Glucose/metabolismo , Glucose/farmacologia , Glutamina/farmacologia , Homeostase , Humanos , Cinurenina/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Mitocôndrias/metabolismo , Nucleotídeos/biossíntese , Oxirredução , Estresse Oxidativo , Consumo de Oxigênio/efeitos dos fármacos , Fenótipo , Ácido Pirúvico/metabolismo , Ácido Pirúvico/farmacologia , Replicação Viral/efeitos dos fármacos
19.
Artigo em Inglês | MEDLINE | ID: mdl-29735559

RESUMO

Muraymycins are antibacterial natural products from Streptomyces spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'-C-glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned in vitro as a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.


Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Nucleosídeos/biossíntese , Nucleosídeos/química , Nucleotidiltransferases/química , Peptídeos/química , Fosfotransferases/química , Streptomyces/metabolismo , Transferases/antagonistas & inibidores , Antibacterianos/biossíntese , Nucleotídeos/biossíntese , Nucleotidiltransferases/genética , Fosforilação , Fosfotransferases/genética
20.
EMBO J ; 37(11)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29650681

RESUMO

YAP/TAZ, downstream transducers of the Hippo pathway, are powerful regulators of cancer growth. How these factors control proliferation remains poorly defined. Here, we found that YAP/TAZ directly regulate expression of key enzymes involved in deoxynucleotide biosynthesis and maintain dNTP precursor pools in human cancer cells. Regulation of deoxynucleotide metabolism is required for YAP-induced cell growth and underlies the resistance of YAP-addicted cells to chemotherapeutics targeting dNTP synthesis. During RAS-induced senescence, YAP/TAZ bypass RAS-mediated inhibition of nucleotide metabolism and control senescence. Endogenous YAP/TAZ targets and signatures are inhibited by RAS/MEK1 during senescence, and depletion of YAP/TAZ is sufficient to cause senescence-associated phenotypes, suggesting a role for YAP/TAZ in suppression of senescence. Finally, mechanical cues, such as ECM stiffness and cell geometry, regulate senescence in a YAP-dependent manner. This study indicates that YAP/TAZ couples cell proliferation with a metabolism suited for DNA replication and facilitates escape from oncogene-induced senescence. We speculate that this activity might be relevant during the initial phases of tumour progression or during experimental stem cell reprogramming induced by YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/genética , Nucleotídeos/biossíntese , Fosfoproteínas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Reprogramação Celular/genética , Senescência Celular/genética , Humanos , Neoplasias/patologia , Nucleotídeos/genética , Transdução de Sinais/genética , Células-Tronco/metabolismo , Fatores de Transcrição
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