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1.
Biochim Biophys Acta Proteins Proteom ; 1872(4): 141015, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38615986

RESUMEN

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.


Asunto(s)
Aminoimidazol Carboxamida , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/química , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/farmacología , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/química , Simulación del Acoplamiento Molecular , Ribonucleótidos/metabolismo , Ribonucleótidos/química , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/antagonistas & inhibidores , Nucleótido Desaminasas/metabolismo , Nucleótido Desaminasas/química , Nucleótido Desaminasas/genética , Especificidad por Sustrato , Proliferación Celular/efectos de los fármacos , Transferasas de Hidroximetilo y Formilo/metabolismo , Transferasas de Hidroximetilo y Formilo/química , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Complejos Multienzimáticos
2.
Int J Biol Sci ; 17(15): 4442-4458, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34803509

RESUMEN

Background: Autophagy regulates many cell functions related to cancer, ranging from cell proliferation and angiogenesis to metabolism. Due to the close relationship between autophagy and tumors, we investigated the predictive value of autophagy-related genes. Methods: Data from patients with hepatocellular carcinoma were obtained from The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC) databases. A regression analysis of differentially expressed genes was performed. Based on a prognostic model, patients were divided into a high-risk or low-risk group. Kaplan-Meier survival analyses of patients were conducted. The immune landscapes, as determined using single-sample gene set enrichment analysis (ssGSEA), exhibited different patterns in the two groups. The prognostic model was verified using the ICGC database and clinical data from patients collected at Zhongnan Hospital. Based on the results of multivariate Cox regression analysis, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC) had the largest hazard ratio, and thus we studied the effect of ATIC on autophagy and tumor progression by performing in vitro and in vivo experiments. Results: Fifty-eight autophagy-related genes were differentially expressed (false discovery rate (FDR)<0.05, log2 fold change (logFC)>1); 23 genes were related to the prognosis of patients. A prognostic model based on 12 genes (ATG10, ATIC, BIRC5, CAPN10, FKBP1A, GAPDH, HDAC1, PRKCD, RHEB, SPNS1, SQSTM1 and TMEM74) was constructed. A significant difference in survival rate was observed between the high-risk group and low-risk group distinguished by the model (P<0.001). The model had good predictive power (area under the curve (AUC)>0.7). Risk-related genes were related to the terms type II IFN response, MHC class I (P<0.001) and HLA (P<0.05). ATIC was confirmed to inhibit autophagy and promote the proliferation, invasion and metastasis of liver cancer cells through the AKT/Forkhead box subgroup O3 (FOXO3) signaling pathway in vitro and in vivo. Conclusions: The prediction model effectively predicts the survival time of patients with liver cancer. The risk score reflects the immune cell features and immune status of patients. ATIC inhibits autophagy and promotes the progression of liver cancer through the AKT/FOXO3 signaling pathway.


Asunto(s)
Carcinoma Hepatocelular/metabolismo , Cromonas/farmacología , Proteína Forkhead Box O3/metabolismo , Transferasas de Hidroximetilo y Formilo/metabolismo , Neoplasias Hepáticas/metabolismo , Morfolinas/farmacología , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetatos/farmacología , Benzopiranos/farmacología , Biomarcadores de Tumor , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Supervivencia Celular , Proteína Forkhead Box O3/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Neoplasias Hepáticas/genética , Modelos Biológicos , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Sobrevida
3.
Int J Rheum Dis ; 24(5): 654-662, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33780152

RESUMEN

INTRODUCTION: The adenosine pathway is one of the ways through which methotrexate (MTX) ameliorates inflammation. We therefore explored an association of polymorphism of genes involved in adenosine and MTX metabolic pathways with response to MTX. METHODS: Association of polymorphism in 7 genes (rs2236225 [MTHFD1 1958G>A], rs17602729 [AMPD1 G>A], rs1127354 [ITPA C>A], rs1431131 [TGFBR2 A>T], rs2372536 [ATIC C>G], rs11188513 [ENTPD1 C>T] and rs5751876 [ADORA2A T>C]) with efficacy of MTX was studied in Indian rheumatoid arthritis (RA) patients. The patients, classified by European League Against Rheumatology (EULAR)/American College of Rheumatology (ACR) 2010 criteria, were DMARD (disease-modifying antirheumatic drug)-naïve, with Disease Activity Score (DAS28) >3.2. After 4 months of MTX monotherapy, patients were classified as responders (R) or non-responders (NR) based on EULAR response criteria. Genotyping was done by TaqMan 5' nuclease assay and association of gene polymorphisms with response to MTX was determined by Chi-squared test. RESULTS: Two hundred and twenty-six patients (86% female, median age 40 [interquartile range, IQR = 17.25] years), with disease duration of 24 (IQR = 38.25) months and DAS28-C-reactive protein score of 4.61 (IQR = 1.34) were enrolled. After therapy, 186 patients were classified as R and 40 as NR. GG genotype of ATIC (P = .01, odds ratio [OR] 2.56, 95% CI, 1.04-6.30) and CC genotype of ITPA (P = .009, OR 1.34, 95% CI 1.02-1.76) genes were found to be associated with the response. On binary logistic regression analysis, GG genotype of ATIC and CC of ITPA genes were independent predictors of the response. CONCLUSION: Polymorphisms of ATIC and ITPA genes alone or with clinical variables were associated with response to MTX therapy in Indian RA patients.


Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Reumatoide/tratamiento farmacológico , Transferasas de Hidroximetilo y Formilo/metabolismo , Metotrexato/uso terapéutico , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Pirofosfatasas/metabolismo , Adulto , Artritis Reumatoide/epidemiología , Genotipo , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Inmunosupresores , India/epidemiología , Metotrexato/efectos adversos , Persona de Mediana Edad , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/genética , Polimorfismo de Nucleótido Simple , Pirofosfatasas/genética
4.
Nat Protoc ; 16(2): 1170-1192, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33462439

RESUMEN

Digested genome sequencing (Digenome-seq) is a highly sensitive, easy-to-carry-out, cell-free method for experimentally identifying genome-wide off-target sites of programmable nucleases and deaminases (also known as base editors). Genomic DNA is digested in vitro using clustered regularly interspaced short palindromic repeats ribonucleoproteins (RNPs; plus DNA-modifying enzymes to cleave both strands of DNA at sites containing deaminated base products, in the case of base editors) and subjected to whole-genome sequencing (WGS) with a typical sequencing depth of 30×. A web-based program is available to map in vitro cleavage sites corresponding to on- and off-target sites. Chromatin DNA, in parallel with histone-free genomic DNA, can also be used to account for the effects of chromatin structure on off-target nuclease activity. Digenome-seq is more sensitive and comprehensive than cell-based methods for identifying off-target sites. Unlike other cell-free methods, Digenome-seq does not involve enrichment of DNA ends through PCR amplification. The entire process other than WGS, which takes ~1-2 weeks, including purification and preparation of RNPs, digestion of genomic DNA and bioinformatic analysis after WGS, takes about several weeks.


Asunto(s)
Edición Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Cromatina , Mapeo Cromosómico/métodos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN , Endonucleasas/metabolismo , Genoma Humano , Humanos , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , ARN Guía de Kinetoplastida/genética , Ribonucleasas/genética , Ribonucleasas/metabolismo , Secuenciación Completa del Genoma/métodos
5.
Mol Cell ; 79(5): 758-767.e6, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32755596

RESUMEN

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.


Asunto(s)
Caulobacter crescentus/metabolismo , Nucleótidos/metabolismo , Proteasa La/metabolismo , Pliegue de Proteína , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/enzimología , Elementos Transponibles de ADN , Didesoxinucleósidos/metabolismo , Regulación Bacteriana de la Expresión Génica , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Ribonucleótido Reductasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Regulación hacia Arriba
6.
J Inherit Metab Dis ; 43(6): 1254-1264, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32557644

RESUMEN

5-Amino-4-imidazolecarboxamide-ribosiduria (AICA)-ribosiduria is an exceedingly rare autosomal recessive condition resulting from the disruption of the bifunctional purine biosynthesis protein PURH (ATIC), which catalyzes the last two steps of de novo purine synthesis. It is characterized biochemically by the accumulation of AICA-riboside in urine. AICA-ribosiduria had been reported in only one individual, 15 years ago. In this article, we report three novel cases of AICA-ribosiduria from two independent families, with two novel pathogenic variants in ATIC. We also provide a clinical update on the first patient. Based on the phenotypic features shared by these four patients, we define AICA-ribosiduria as the syndromic association of severe-to-profound global neurodevelopmental impairment, severe visual impairment due to chorioretinal atrophy, ante-postnatal growth impairment, and severe scoliosis. Dysmorphic features were observed in all four cases, especially neonatal/infancy coarse facies with upturned nose. Early-onset epilepsy is frequent and can be pharmacoresistant. Less frequently observed features are aortic coarctation, chronic hepatic cytolysis, minor genital malformations, and nephrocalcinosis. Alteration of the transformylase activity of ATIC might result in a more severe impairment than the alteration of the cyclohydrolase activity. Data from literature points toward a cytotoxic mechanism of the accumulated AICA-riboside.


Asunto(s)
Anomalías Congénitas/genética , Epilepsia/genética , Transferasas de Hidroximetilo y Formilo/deficiencia , Discapacidad Intelectual/genética , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/deficiencia , Aminoimidazol Carboxamida/metabolismo , Niño , Preescolar , Femenino , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/metabolismo , Lactante , Recién Nacido , Masculino , Complejos Multienzimáticos/metabolismo , Mutación , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Fenotipo , Ribonucleósidos/metabolismo
7.
J Biol Chem ; 295(28): 9551-9566, 2020 07 10.
Artículo en Inglés | MEDLINE | ID: mdl-32439803

RESUMEN

The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand.


Asunto(s)
Transferasas de Hidroximetilo y Formilo/metabolismo , Factor 1 Inducible por Hipoxia/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Purinas/biosíntesis , Hipoxia de la Célula , Células HeLa , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Factor 1 Inducible por Hipoxia/genética , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/genética
8.
Comb Chem High Throughput Screen ; 23(8): 723-739, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32321396

RESUMEN

OBJECTIVE: The study aims at the derivatization of "Phthalides" and synthesizes 3- arylaminophthalides & 3-indolyl-phthalides compounds, and evaluates their anti-tubercular and antioxidant activities. The study has also intended to employ the in silico methods for the identification of possible drug targets in Mycobacterium and evaluate the binding affinities of synthesized compounds. METHODS: This report briefly explains the synthesis of phthalide derivatives using ammonium chloride. The synthesized compounds were characterized using spectral analysis. Resazurin Microtiter Assay (REMA) plate method was used to demonstrate the anti-mycobacterial activity of the synthesized compounds. An in-silico pharmacophore probing approach was used for target identification in Mycobacterium. The structural level interaction between the identified putative drug target and synthesized phthalides was studied using Lamarckian genetic algorithm-based software. RESULTS AND DISCUSSION: In the present study, we report an effective, environmentally benign scheme for the synthesis of phthalide derivatives. Compounds 5c and 5d from the current series appear to possess good anti-mycobacterial activity. dCTP: deaminasedUTPase was identified as a putative drug target in Mycobacterium. The docking results clearly showed the interactive involvement of conserved residues of dCTP with the synthesized phthalide compounds. CONCLUSION: On the eve of evolving anti-TB drug resistance, the data on anti-tubercular and allied activities of the compounds in the present study demonstrates the enormous significance of these newly synthesized derivatives as possible candidate leads in the development of novel anti-tubercular agents. The docking results from the current report provide a structural rationale for the promising anti-tubercular activity demonstrated by 3-arylaminophthalides and 3-indolyl-phthalides compounds.


Asunto(s)
Cloruro de Amonio/química , Antituberculosos/síntesis química , Benzofuranos/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , Algoritmos , Antioxidantes/química , Antituberculosos/farmacología , Benzofuranos/farmacología , Diseño de Fármacos , Humanos , Radical Hidroxilo/química , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Nucleótido Desaminasas/metabolismo , Relación Estructura-Actividad
9.
Annu Rev Immunol ; 37: 349-375, 2019 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-30673536

RESUMEN

Detection of double-stranded RNAs (dsRNAs) is a central mechanism of innate immune defense in many organisms. We here discuss several families of dsRNA-binding proteins involved in mammalian antiviral innate immunity. These include RIG-I-like receptors, protein kinase R, oligoadenylate synthases, adenosine deaminases acting on RNA, RNA interference systems, and other proteins containing dsRNA-binding domains and helicase domains. Studies suggest that their functions are highly interdependent and that their interdependence could offer keys to understanding the complex regulatory mechanisms for cellular dsRNA homeostasis and antiviral immunity. This review aims to highlight their interconnectivity, as well as their commonalities and differences in their dsRNA recognition mechanisms.


Asunto(s)
Inmunidad Innata/genética , ARN Bicatenario/genética , Virosis/inmunología , 2',5'-Oligoadenilato Sintetasa/metabolismo , Animales , Proteína 58 DEAD Box/metabolismo , Humanos , Inmunomodulación , Mamíferos , Nucleótido Desaminasas/metabolismo , Interferencia de ARN , eIF-2 Quinasa/metabolismo
10.
Artículo en Inglés | MEDLINE | ID: mdl-30587076

RESUMEN

Huntington's disease (HD) is a neurodegenerative disorder that is caused by expanded CAG repeats within the exon-1 of the huntingtin (HTT) gene. It has been shown that HTT interacts with the proteins involved in the gene transcription, endocytosis and metabolism, nevertheless the biochemical pathways by which mutant HTT causes a cellular dysfunction remain unclear. Thus, this study aimed to establish the role of mutant HTT expansion in energy and nucleotide metabolism deteriorations. We examined HEK 293 T cell line transfected with plasmids expressing wild-type (control) or mutant exon 1 of the HTT gene (HD). Analysis of intracellular concentration of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD+), as well as activities of intra- and extracellular enzymes of nucleotide catabolism (such as adenine monophosphate deaminase (AMPD), adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and ectonucleoside triphosphate diphosphohydrolase (eNTPD), ecto-5'-nucleotidase (e5NT), ecto-adenosine deaminase (eADA) were performed with high pressure liquid chromatography. Protein concentration was measured with Bradford method. We found diminished intracellular ATP concentration (22.5 ± 1.7 in HD; 29.3 ± 1.4 nmol/mg protein in control), increased ADA activity (27.9 ± 1.0 in HD; 21.1 ± 1.6 nmol/min/mg protein in control) and reduced activities of eNTPD (2.4 ± 0.5 in HD; 5.8 ± 0.7 nmol/min/mg protein in control), e5NT (0.1 ± 0.01 in HD; 0.2 ± 0.01 nmol/min/mg protein in control) and eADA (0.3 ± 0.03 in HD; 0.4 ± 0.04 nmol/min/mg protein in control) while NAD+ concentration, AMPD and PNP activities remained unchanged. This study highlights that the mutant HTT expansion resulted in depletion of cellular ATP concentration and reduced rates of extracellular nucleotide breakdown. In conclusion, such changes may contribute to the pathology of HD.


Asunto(s)
Adenina/metabolismo , Enfermedad de Huntington/fisiopatología , Nucleótidos/metabolismo , 5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/metabolismo , Exones/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Proteína Huntingtina/genética , Mutación/genética , NAD/metabolismo , Nucleótido Desaminasas/metabolismo , Purina-Nucleósido Fosforilasa/metabolismo , Transfección/métodos
11.
Sci Rep ; 8(1): 15458, 2018 10 18.
Artículo en Inglés | MEDLINE | ID: mdl-30337562

RESUMEN

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.


Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Antineoplásicos , Inhibidores Enzimáticos/farmacología , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Neoplasias Pulmonares , Complejos Multienzimáticos/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Nucleótido Desaminasas/antagonistas & inhibidores , Ribonucleótidos , Aminoimidazol Carboxamida/farmacocinética , Aminoimidazol Carboxamida/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Transferasas de Hidroximetilo y Formilo/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Complejos Multienzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleótido Desaminasas/metabolismo , Ribonucleótidos/farmacocinética , Ribonucleótidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Pharmacol Res ; 138: 37-42, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30267763

RESUMEN

Mitochondrial myopathy (MM) is characterised by muscle weakness, exercise intolerance and various histopathological changes. Recently, a subset of MM has also been associated with aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle. This aberrant mTORC1 activation promotes increased de novo nucleotide synthesis, which contributes to abnormal expansion and imbalance of skeletal muscle deoxyribonucleoside triphosphates (dNTP) pools. However, the exact mechanism via which mTORC1-stimulated de novo nucleotide biosynthesis ultimately disturbs muscle dNTP pools remains unclear. In this article, it is proposed that mTORC1-stimulated de novo nucleotide synthesis in skeletal muscle cells with respiratory chain dysfunction promotes an asymmetric increase of purine nucleotides, probably due to NAD+ deficiency. This in turn could disrupt purine nucleotide-dependent allosteric feedback regulatory mechanisms, ultimately leading to dNTP pools aberration. Pharmacological down-modulation of aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) activity is also proposed as a potential therapeutic strategy in MM exhibiting mTORC1-driven abnormal metabolic reprogramming, including aberrant dNTPs pools.


Asunto(s)
Miopatías Mitocondriales/metabolismo , Nucleótidos de Purina/metabolismo , Animales , Humanos , Transferasas de Hidroximetilo y Formilo/antagonistas & inhibidores , Transferasas de Hidroximetilo y Formilo/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Miopatías Mitocondriales/tratamiento farmacológico , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/antagonistas & inhibidores , Nucleótido Desaminasas/metabolismo
13.
Mol Oncol ; 12(10): 1778-1796, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30099851

RESUMEN

Although metabolomics has attracted considerable attention in the field of lung cancer (LC) detection and management, only a very limited number of works have applied it to tissues. As such, the aim of this study was the thorough analysis of metabolic profiles of relevant LC tissues, including the most important histological subtypes (adenocarcinoma and squamous cell lung carcinoma). Mass spectrometry-based metabolomics, along with genetic expression and histological analyses, were performed as part of this study, the widest to date, to identify metabolic alterations in tumors of the most relevant histological subtypes in lung. A total of 136 lung tissue samples were analyzed and 851 metabolites were identified through metabolomic analysis. Our data show the existence of a clear metabolic alteration not only between tumor vs. nonmalignant tissue in each patient, but also inherently intrinsic changes in both AC and SCC. Significant changes were observed in the most relevant biochemical pathways, and nucleotide metabolism showed an important number of metabolites with high predictive capability values. The present study provides a detailed analysis of the metabolomic changes taking place in relevant biochemical pathways of the most important histological subtypes of LC, which can be used as biomarkers and also to identify novel targets.


Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Metabolómica/métodos , Nucleótidos/metabolismo , Anciano , Femenino , Regulación Neoplásica de la Expresión Génica , Glucosa/metabolismo , Glutatión/metabolismo , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/metabolismo , Neoplasias Pulmonares/genética , Masculino , Metaboloma , Persona de Mediana Edad , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Estrés Oxidativo , Poliaminas/metabolismo , Purinas/metabolismo , Curva ROC
14.
Biochem Biophys Res Commun ; 503(1): 195-201, 2018 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-29864427

RESUMEN

Archaeal/fungal Rib7 and eubacterial RibG possess a reductase domain for ribosyl reduction in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for an amino and a carbonyl group of the pyrimidine ring, respectively. Here, several crystal structures of Methanosarcina mazei Rib7 are reported at 2.27-1.95 Šresolution, which are the first archaeal dimeric Rib7 structures. Mutational analysis displayed that no detectable activity was observed for the Bacillus subtilis RibG K151A, K151D, and K151E mutants, and the M. mazei Rib7 D33N, D33K, and E156Q variants, while 0.1-0.6% of the activity was detected for the M. mazei Rib7 N9A, S29A, D33A, and D57N variants. Our results suggest that Lys151 in B. subtilis RibG, while Asp33 together with Arg36 in M. mazei Rib7, ensure the specific substrate recognition. Unexpectedly, an endogenous NADPH cofactor is observed in M. mazei Rib7, in which the 2'-phosphate group interacts with Ser88, and Arg91. Replacement of Ser88 with glutamate eliminates the endogenous NADPH binding and switches preference to NADH. The lower melting temperature of ∼10 °C for the S88E and R91A mutants suggests that nature had evolved a tightly bound NADPH to greatly enhance the structural stability of archaeal Rib7.


Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Nucleótido Desaminasas/metabolismo , Oxidorreductasas/metabolismo , Riboflavina/biosíntesis , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Arqueales/química , Proteínas Arqueales/genética , Bacillus subtilis/enzimología , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Dominio Catalítico/genética , Cristalografía por Rayos X , Estabilidad de Enzimas , Evolución Molecular , Methanosarcina/enzimología , Methanosarcina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , NAD/metabolismo , NADP/metabolismo , Nucleótido Desaminasas/química , Nucleótido Desaminasas/genética , Oxidorreductasas/química , Oxidorreductasas/genética , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Electricidad Estática , Especificidad por Sustrato , Deshidrogenasas del Alcohol de Azúcar/química , Deshidrogenasas del Alcohol de Azúcar/genética
15.
Microbiology (Reading) ; 164(7): 982-991, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29799386

RESUMEN

Dihydrofolate reductase (DHFR) and 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/IMP cyclohydrolase (PurH) play key roles in maintaining folate pools in cells, and are targets of antimicrobial and anticancer drugs. While the activities of bacterial DHFR and PurH on their classical substrates (DHF and 10-CHO-THF, respectively) are known, their activities and kinetic properties of utilisation of 10-CHO-DHF are unknown. We have determined the kinetic properties (kcat/Km) of conversion of 10-CHO-DHF to 10-CHO-THF by DHFR, and to DHF by PurH. We show that DHFR utilises 10-CHO-DHF about one third as efficiently as it utilises DHF. The 10-CHO-DHF is also utilised (as a formyl group donor) by PurH albeit slightly less efficiently than 10-CHO-THF. The utilisation of 10-CHO-DHF by DHFR is ~50 fold more efficient than its utilisation by PurH. A folate deficient Escherichia coli (∆pabA) grows well when supplemented with adenine, glycine, thymine and methionine, the metabolites that arise from the one-carbon metabolic pathway. Notably, when the ∆pabA strain harboured a folate transporter, it grew in the presence of 10-CHO-DHF alone, suggesting that it (10-CHO-DHF) can enter one-carbon metabolic pathway to provide the required metabolites. Thus, our studies reveal that both DHFR and PurH could utilise 10-CHO-DHF for folate homeostasis in E. coli.


Asunto(s)
Escherichia coli/metabolismo , Ácido Fólico/análogos & derivados , Nucleótido Desaminasas/metabolismo , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Ácido 4-Aminobenzoico , Clonación Molecular , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/genética , Homeostasis , Cinética , Redes y Vías Metabólicas , Nucleótido Desaminasas/genética , Fosforribosilaminoimidazolcarboxamida-Formiltransferasa/genética , Tetrahidrofolato Deshidrogenasa/genética
16.
J Biol Chem ; 293(13): 4845-4859, 2018 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-29414769

RESUMEN

The enzyme AICAR-transformylase/IMP cyclohydrolase (ATIC) catalyzes the last two steps of purine de novo synthesis. It metabolizes 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which is an AMP analogue, leading to activation of AMP-activated kinase (AMPK). We investigated whether the AICAR-ATIC pathway plays a role in the high glucose (HG)-mediated DNA damage response and AICAR-mediated AMPK activation, explaining the detrimental effects of glucose on neuronal damage and shortening of the lifespan. HG up-regulated the expression and activity of the Caenorhabditis elegans homologue of ATIC, C55F2.1 (atic-1), and increased the levels of reactive oxygen species and methylglyoxal-derived advanced glycation end products. Overexpression of atic-1 decreased the lifespan and head motility and increased neuronal damage under both standard and HG conditions. Inhibition of atic-1 expression, by RNAi, under HG was associated with increased lifespan and head motility and reduced neuronal damage, reactive oxygen species, and methylglyoxal-derived advanced glycation end product accumulation. This effect was independent of an effect on DNA damage or antioxidant defense pathways, such as superoxide dismutase (sod-3) or glyoxalase-1 (glod-4), but was dependent on AMPK and accumulation of AICAR. Through AMPK, AICAR treatment also reduced the negative effects of HG. The mitochondrial inhibitor rotenone abolished the AICAR/AMPK-induced amelioration of HG effects, pointing to mitochondria as a prime target of the glucotoxic effects in C. elegans We conclude that atic-1 is involved in glucotoxic effects under HG conditions, either by blocked atic-1 expression or via AICAR and AMPK induction.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Glucosa/metabolismo , Transferasas de Hidroximetilo y Formilo/metabolismo , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Transferasas de Hidroximetilo y Formilo/genética , Complejos Multienzimáticos/genética , Neuronas/metabolismo , Nucleótido Desaminasas/genética , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo
17.
Biochim Biophys Acta Proteins Proteom ; 1866(2): 254-263, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29042184

RESUMEN

Aminoimidazolecarboxamide ribonucleotide formyl transferase (AICARFT): Inosine monophosphate cyclohydrolase (IMPCH, collectively called ATIC) is a bifunctional enzyme that catalyses the penultimate and final steps in the purine de novo biosynthesis pathway. The bifunctional protein is dimeric and each monomer contains two different active sites both of which are capable of binding nucleotide substrates, this means to a potential total of four distinct binding events might be observed. Within this work we used a combination of site-directed and truncation mutants of ATIC to independently investigate the binding at these two sites using calorimetry. A single S10W mutation is sufficient to block the IMPCH active site allowing investigation of the effects of mutation on ligand binding in the AICARFT active site. The majority of nucleotide ligands bind selectively at one of the two active sites with the exception of xanthosine monophosphate, XMP, which, in addition to binding in both AICARFT and IMPCH active sites, shows evidence for cooperative binding with communication between symmetrically-related active sites in the two IMPCH domains. The AICARFT site is capable of independently binding both nucleotide and folate substrates with high affinity however no evidence for positive cooperativity in binding could be detected using the model ligands employed in this study.


Asunto(s)
Transferasas de Hidroximetilo y Formilo/química , Modelos Moleculares , Complejos Multienzimáticos/química , Nucleótido Desaminasas/química , Nucleótidos/química , Dominio Catalítico , Humanos , Transferasas de Hidroximetilo y Formilo/genética , Transferasas de Hidroximetilo y Formilo/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Nucleótidos/genética , Nucleótidos/metabolismo , Unión Proteica , Especificidad por Sustrato/fisiología
18.
Cell Commun Signal ; 15(1): 52, 2017 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-29246230

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the cancer types with poor prognosis. To effectively treat HCC, new molecular targets and therapeutic approaches must be identified. 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate (IMP) cyclohydrolase (ATIC), a bifunctional protein enzyme, catalyzes the last two steps of the de novo purine biosynthetic pathway. Whether ATIC contributes to cancer development remains unclear. METHODS: ATIC mRNA levels in different types of human HCC samples or normal tissues were determined from Gene Expression across Normal and Tumor tissue (GENT) database. The expression level of ATIC in human HCC samples or cell lines were examined by RT-PCR and western blot. Overall survival and disease-free survival of HCC patients in the ATIC low and ATIC high groups were determined by Kaplan-Meier analysis. Effects of ATIC knockdown by lentivirus infection were evaluated on cell-proliferation, cell-apoptosis, colony formation and migration. The mechanisms involved in HCC cells growth, apoptosis and migration were analyzed by western blot and Compound C (C-C) rescue assays. RESULTS: Here, we first demonstrated that expression of ATIC is aberrantly up-regulated in HCC tissues and high level of ATIC is correlated with poor survival in HCC patients. Knockdown of ATIC expression resulted in a dramatic decrease in proliferation, colony formation and migration of HCC cells. We also identified ATIC as a novel regulator of adenosine monophosphate-activated protein kinase (AMPK) and its downstream signaling mammalian target of rapamycin (mTOR). ATIC suppresses AMPK activation, thus activates mTOR-S6 K1-S6 signaling and supports growth and motility activity of HCC cells. CONCLUSION: Taken together, our results indicate that ATIC acts as an oncogenic gene that promotes survival, proliferation and migration by targeting AMPK-mTOR-S6 K1 signaling.


Asunto(s)
Adenilato Quinasa/metabolismo , Carcinoma Hepatocelular/patología , Transferasas de Hidroximetilo y Formilo/metabolismo , Neoplasias Hepáticas/patología , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Activación Enzimática , Técnicas de Silenciamiento del Gen , Humanos , Transferasas de Hidroximetilo y Formilo/deficiencia , Transferasas de Hidroximetilo y Formilo/genética , Terapia Molecular Dirigida , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Nucleótido Desaminasas/deficiencia , Nucleótido Desaminasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba
19.
Sci Rep ; 6: 24219, 2016 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-27063406

RESUMEN

Members of the dUTPase superfamily play an important role in the maintenance of the pyrimidine nucleotide balance and of genome integrity. dCTP deaminases and the bifunctional dCTP deaminase-dUTPases are cooperatively regulated by dTTP. However, the manifestation of allosteric behavior within the same trimeric protein architecture of dUTPases, the third member of the superfamily, has been a question of debate for decades. Therefore, we designed hybrid dUTPase trimers to access conformational states potentially mimicking the ones observed in the cooperative relatives. We studied how the interruption of different steps of the enzyme cycle affects the active site cross talk. We found that subunits work independently in dUTPase. The experimental results combined with a comparative structural analysis of dUTPase superfamily enzymes revealed that subtile structural differences within the allosteric loop and the central channel in these enzymes give rise to their dramatically different cooperative behavior. We demonstrate that the lack of allosteric regulation in dUTPase is related to the functional adaptation to more efficient dUTP hydrolysis which is advantageous in uracil-DNA prevention.


Asunto(s)
ADN/metabolismo , Pirofosfatasas/metabolismo , Uracilo/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Dicroismo Circular , Humanos , Cinética , Magnesio/química , Magnesio/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Pirofosfatasas/química , Pirofosfatasas/genética , Alineación de Secuencia , Espectrometría de Fluorescencia , Nucleótidos de Timina/biosíntesis
20.
Biochemistry ; 55(7): 1107-19, 2016 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-26848874

RESUMEN

Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP(+) and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis.


Asunto(s)
Proteínas Bacterianas/química , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , NADP/química , Nucleótido Desaminasas/química , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolatos/química , Ácido Aminosalicílico/farmacología , Antituberculosos/química , Antituberculosos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Farmacorresistencia Bacteriana , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Cinética , Ligandos , Conformación Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , NADP/metabolismo , Nucleótido Desaminasas/antagonistas & inhibidores , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolatos/metabolismo , Trimetoprim/química , Trimetoprim/metabolismo , Trimetoprim/farmacología , Trimetrexato/química , Trimetrexato/metabolismo , Trimetrexato/farmacología
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