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1.
Artigo em Inglês | MEDLINE | ID: mdl-38359644

RESUMO

Adenine phosphoribosyltransferase (APRT) deficiency is a rare , hereditary disorder characterized by renal excretion of 2,8-dihydroxyadenine (DHA), leading to kidney stone formation and chronic kidney disease (CKD). Treatment with a xanthine oxidoreductase inhibitor, allopurinol or febuxostat, reduces urinary DHA excretion and slows the progression of CKD. The method currently used for therapeutic monitoring of APRT deficiency lacks specificity and thus, a more reliable measurement technique is needed. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma was optimized and validated. Plasma samples were prepared with protein precipitation using acetonitrile followed by evaporation. The chemometric approach design of experiments was implemented to optimize gradient steepness, amount of organic solvent, flow rate, column temperature, cone voltage, desolvation temperature and desolvation flow rate. Experimental screening was conducted using fractional factorial design with addition of complementary experiments at the axial points for optimization of peak area, peak resolution and peak width. The assay was validated according to the US Food and Drug Administration guidelines for bioanalytical method validation over the concentration range of 50 to 5000 ng/mL for DHA, allopurinol and febuxostat, 100 to 5000 ng/mL for adenine and 50 to 12,000 ng/mL for oxypurinol, with r2 ≥ 0.99. The analytical assay achieved acceptable performance of accuracy (-10.8 to 8.3 %) and precision (CV < 15 %). DHA, adenine, allopurinol, oxypurinol and febuxostat were stable in plasma samples after five freeze-thaw cycles at -80 °C and after storage at -80 °C for 12 months. The assay was evaluated for quantification of the five analytes in clinical plasma samples from six APRT deficiency patients and proved to be both efficient and accurate. The proposed assay will be valuable for guiding pharmacotherapy and thereby contribute to improved and more personalized care for patients with APRT deficiency.


Assuntos
Adenina Fosforribosiltransferase/deficiência , Adenina/análogos & derivados , Alopurinol , Erros Inatos do Metabolismo , Insuficiência Renal Crônica , Urolitíase , Humanos , Alopurinol/uso terapêutico , Oxipurinol , Febuxostat , Cromatografia Líquida , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massa com Cromatografia Líquida , Adenina/metabolismo , Adenina Fosforribosiltransferase/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico
2.
PLoS Negl Trop Dis ; 16(2): e0009926, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35104286

RESUMO

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based pro-drug activation.


Assuntos
Adenina Fosforribosiltransferase/genética , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Adenina Fosforribosiltransferase/metabolismo , Escherichia coli , Isoformas de Proteínas , Proteínas de Protozoários/metabolismo , Purinas/metabolismo , Saccharomycetales , Trypanosoma brucei brucei/metabolismo
3.
Chembiochem ; 23(3): e202100596, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-34859954

RESUMO

AICA (5'-aminoimidazole-4-carboxamide) ribonucleotides with different phosphorylation levels are the pharmaceutically active metabolites of AICA nucleoside-based drugs. The chemical synthesis of AICA ribonucleotides with defined phosphorylation is challenging and expensive. In this study, we describe two enzymatic cascades to synthesize AICA derivatives with defined phosphorylation levels from the corresponding nucleobase and the co-substrate phosphoribosyl pyrophosphate. The cascades are composed of an adenine phosphoribosyltransferase from Escherichia coli (EcAPT) and different polyphosphate kinases: polyphosphate kinase from Acinetobacter johnsonii (AjPPK), and polyphosphate kinase from Meiothermus ruber (MrPPK). The role of the EcAPT is to bind the nucleobase to the sugar moiety, while the kinases are responsible for further phosphorylation of the nucleotide to produce the desired phosphorylated AICA ribonucleotide. The selected enzymes were characterized, and conditions were established for two enzymatic cascades. The diphosphorylated AICA ribonucleotide derivative ZDP, synthesized from the cascade EcAPT/AjPPK, was produced with a conversion up to 91 %. The EcAPT/MrPPK cascade yielded ZTP with conversion up to 65 % with ZDP as a side product.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polifosfatos/metabolismo , Ribonucleotídeos/biossíntese , Acinetobacter/enzimologia , Aminoimidazol Carboxamida/química , Bactérias/enzimologia , Escherichia coli/enzimologia , Concentração de Íons de Hidrogênio , Polifosfatos/química , Ribonucleotídeos/química , Temperatura
4.
Eur J Med Chem ; 225: 113798, 2021 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-34482272

RESUMO

Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered. Herein, we designed and synthesized two series of new ANPs branched at the C1' position as mimics of adenosine monophosphate. The novel ANPs efficaciously inhibited Trypanosoma brucei adenine PRT (TbrAPRT1) activity in vitro and it was shown that the configuration on the C1' chiral centre strongly influenced their activity: the (R)-enantiomers proved to be more potent compared to the (S)-enantiomers. Two ANPs, with Ki values of 0.39 µM and 0.57 µM, represent the most potent TbrAPRT1 inhibitors reported to date and they are an important tool to further study purine metabolism in various parasites.


Assuntos
Adenina Fosforribosiltransferase/antagonistas & inibidores , Monofosfato de Adenosina/farmacologia , Antiprotozoários/farmacologia , Inibidores Enzimáticos/farmacologia , Nucleosídeos/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Adenina Fosforribosiltransferase/metabolismo , Monofosfato de Adenosina/síntese química , Monofosfato de Adenosina/química , Antiprotozoários/síntese química , Antiprotozoários/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estrutura Molecular , Nucleosídeos/síntese química , Nucleosídeos/química , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade , Trypanosoma brucei brucei/enzimologia
5.
Sci Rep ; 11(1): 13317, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172767

RESUMO

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).


Assuntos
Nucleotídeos de Adenina/metabolismo , Adenina Fosforribosiltransferase/metabolismo , Nucleosídeos/metabolismo , Organofosfonatos/metabolismo , Trypanosoma brucei brucei/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Purinas/metabolismo
6.
Int J Mol Sci ; 22(2)2021 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-33418923

RESUMO

To date, Chlorella vulgaris is the most used species of microalgae in the food and feed additive industries, and also considered as a feasible cell factory for bioproducts. However, the lack of an efficient genetic engineering tool makes it difficult to improve the physiological characteristics of this species. Therefore, the development of new strategic approaches such as genome editing is trying to overcome this hurdle in many research groups. In this study, the possibility of editing the genome of C. vulgaris UTEX395 using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been proven to target nitrate reductase (NR) and adenine phosphoribosyltransferase (APT). Genome-edited mutants, nr and apt, were generated by a DNA-mediated and/or ribonucleoprotein (RNP)-mediated CRISPR-Cas9 system, and isolated based on the negative selection against potassium chlorate or 2-fluoroadenine in place of antibiotics. The null mutation of edited genes was demonstrated by the expression level of the correspondent proteins or the mutation of transcripts, and through growth analysis under specific nutrient conditions. In conclusion, this study offers relevant empirical evidence of the possibility of genome editing in C. vulgaris UTEX395 by CRISPR-Cas9 and the practical methods. Additionally, among the generated mutants, nr can provide an easier screening strategy during DNA transformation than the use of antibiotics owing to their auxotrophic characteristics. These results will be a cornerstone for further advancement of the genetics of C. vulgaris.


Assuntos
Sistemas CRISPR-Cas/genética , Chlorella vulgaris/genética , Edição de Genes/métodos , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo
7.
Sci Rep ; 10(1): 13330, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770053

RESUMO

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.


Assuntos
Cinetina/metabolismo , Mamíferos/metabolismo , Estresse Oxidativo/fisiologia , Adenina Fosforribosiltransferase/metabolismo , Animais , Linhagem Celular Tumoral , Células HL-60 , Humanos , Oxirredução , Receptores Purinérgicos P1/metabolismo
8.
J Am Soc Nephrol ; 31(4): 799-816, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32086278

RESUMO

BACKGROUND: Hereditary deficiency of adenine phosphoribosyltransferase causes 2,8-dihydroxyadenine (2,8-DHA) nephropathy, a rare condition characterized by formation of 2,8-DHA crystals within renal tubules. Clinical relevance of rodent models of 2,8-DHA crystal nephropathy induced by excessive adenine intake is unknown. METHODS: Using animal models and patient kidney biopsies, we assessed the pathogenic sequelae of 2,8-DHA crystal-induced kidney damage. We also used knockout mice to investigate the role of TNF receptors 1 and 2 (TNFR1 and TNFR2), CD44, or alpha2-HS glycoprotein (AHSG), all of which are involved in the pathogenesis of other types of crystal-induced nephropathies. RESULTS: Adenine-enriched diet in mice induced 2,8-DHA nephropathy, leading to progressive kidney disease, characterized by crystal deposits, tubular injury, inflammation, and fibrosis. Kidney injury depended on crystal size. The smallest crystals were endocytosed by tubular epithelial cells. Crystals of variable size were excreted in urine. Large crystals obstructed whole tubules. Medium-sized crystals induced a particular reparative process that we term extratubulation. In this process, tubular cells, in coordination with macrophages, overgrew and translocated crystals into the interstitium, restoring the tubular luminal patency; this was followed by degradation of interstitial crystals by granulomatous inflammation. Patients with adenine phosphoribosyltransferase deficiency showed similar histopathological findings regarding crystal morphology, crystal clearance, and renal injury. In mice, deletion of Tnfr1 significantly reduced tubular CD44 and annexin two expression, as well as inflammation, thereby ameliorating the disease course. In contrast, genetic deletion of Tnfr2, Cd44, or Ahsg had no effect on the manifestations of 2,8-DHA nephropathy. CONCLUSIONS: Rodent models of the cellular and molecular mechanisms of 2,8-DHA nephropathy and crystal clearance have clinical relevance and offer insight into potential future targets for therapeutic interventions.


Assuntos
Adenina Fosforribosiltransferase/deficiência , Adenina/análogos & derivados , Nefropatias/etiologia , Nefropatias/patologia , Erros Inatos do Metabolismo/etiologia , Erros Inatos do Metabolismo/patologia , Urolitíase/etiologia , Urolitíase/patologia , Adenina/fisiologia , Adenina Fosforribosiltransferase/metabolismo , Adulto , Animais , Estudos de Coortes , Dieta , Modelos Animais de Doenças , Feminino , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/metabolismo , Camundongos , Pessoa de Meia-Idade , Urolitíase/metabolismo
9.
J Biol Chem ; 294(32): 11992-11993, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399535

RESUMO

Two phosphoribosyltransferases in the purine salvage pathway exhibit exquisite substrate specificity despite the chemical similarity of their distinct substrates, but the basis for this discrimination was not fully understood. Ozeir et al. now employ a complementary biochemical, structural, and computational approach to deduce the chemical constraints governing binding and propose a distinct mechanism for catalysis in one of these enzymes, adenine phosphoribosyltransferase. These insights, built on data from an unexpected finding, finally provide direct answers to key questions regarding these enzymes and substrate recognition more generally.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Hipoxantina Fosforribosiltransferase/metabolismo , Adenina Fosforribosiltransferase/química , Biocatálise , Humanos , Hipoxantina Fosforribosiltransferase/química , Purinas/química , Purinas/metabolismo , Especificidade por Substrato
10.
J Biol Chem ; 294(32): 11980-11991, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31160323

RESUMO

The reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore, a quantum mechanics/molecular mechanics (QM/MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (SN2) with a mix of SN1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted SN2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Adenina/química , Adenina/metabolismo , Adenina Fosforribosiltransferase/química , Biocatálise , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Estrutura Terciária de Proteína , Teoria Quântica , Especificidade por Substrato
11.
J Bacteriol ; 201(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036724

RESUMO

Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells.IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Monofosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Glutamato Desidrogenase/metabolismo , Leucina/metabolismo , Thermus thermophilus/enzimologia , Adenina Fosforribosiltransferase/genética , Proteínas de Bactérias/genética , Catálise , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Glutamato Desidrogenase/genética , Ácido Glutâmico/metabolismo , Thermus thermophilus/genética
12.
Pediatr Nephrol ; 34(3): 435-442, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30443743

RESUMO

BACKGROUND: Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary purine metabolism disorder that causes kidney stones and chronic kidney disease (CKD). The purpose of this study was to examine the course of APRT deficiency in patients who presented in childhood. METHODS: The disease course of 21 (35%) patients in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, who presented with manifestations of APRT deficiency and/or were diagnosed with the disorder before the age of 18 years, was studied. The effect of pharmacotherapy on renal manifestations and outcomes was thoroughly assessed. RESULTS: Fourteen children were placed on allopurinol, 100 (25-200) mg/day, at the age of 2.6 (0.6-16.5) years. Six of these patients had experienced kidney stone events and three had developed acute kidney injury (AKI) prior to allopurinol treatment. During 18.9 (1.7-31.5) years of pharmacotherapy, stones occurred in two patients and AKI in three. Six adult patients started allopurinol treatment, 200 (100-300) mg/day, at age 29.8 (20.5-42.4) years. Five of these patients had experienced 28 stone episodes and AKI had occurred in two. Stone recurrence occurred in four patients and AKI in two during 11.2 (4.2-19.6) years of allopurinol therapy. Lack of adherence and insufficient dosing contributed to stone recurrence and AKI during pharmacotherapy. At latest follow-up, estimated glomerular filtration rate (eGFR) was 114 (70-163) and 62 (10-103) mL/min/1.73 m2 in those who initiated treatment as children and adults, respectively. All three patients with CKD stages 3-5 at the last follow-up were adults when pharmacotherapy was initiated. CONCLUSION: Timely diagnosis and treatment of APRT deficiency decreases renal complications and preserves kidney function.


Assuntos
Injúria Renal Aguda/epidemiologia , Adenina Fosforribosiltransferase/deficiência , Alopurinol/uso terapêutico , Cálculos Renais/epidemiologia , Erros Inatos do Metabolismo/complicações , Insuficiência Renal Crônica/epidemiologia , Urolitíase/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Progressão da Doença , Feminino , Seguimentos , Taxa de Filtração Glomerular/fisiologia , Humanos , Lactente , Rim/fisiopatologia , Cálculos Renais/química , Cálculos Renais/diagnóstico , Cálculos Renais/etiologia , Masculino , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Recidiva , Sistema de Registros/estatística & dados numéricos , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/fisiopatologia , Urolitíase/tratamento farmacológico , Urolitíase/genética , Urolitíase/metabolismo , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/metabolismo , Adulto Jovem
14.
Nat Commun ; 9(1): 3924, 2018 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-30254261

RESUMO

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest.


Assuntos
Diatomáceas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Transfecção/métodos , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sistemas CRISPR-Cas , Diatomáceas/metabolismo , Microalgas/genética , Microalgas/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Orotato Fosforribosiltransferase/genética , Orotato Fosforribosiltransferase/metabolismo , Orotidina-5'-Fosfato Descarboxilase/genética , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Reprodutibilidade dos Testes , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Homologia de Sequência de Aminoácidos
15.
Biosci Rep ; 38(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30104401

RESUMO

Uric acid (UA) is the end product of the catabolism of purines, and its serum levels are commonly increased in cancer patients. We aimed to explore the transcriptional regulation of tumour uricogenesis in human tumours, and relate uricogenesis with tumour pathological and pharmacological findings. Using data from The Cancer Genome Atlas (TCGA), we analysed the expression levels of xanthine dehydrogenase (XDH) and adenine phosphoribosyltransferase (APRT), two key enzymes in UA production and the purine salvage pathway, respectively. We found large differences between tumour types and individual tumours in their expression of XDH and APRT Variations in locus-specific DNA methylation and gene copy number correlated with the expression levels of XDH and APRT in human tumours respectively. We explored the consequences of this differential regulation of uricogenesis. Tumours with high levels of XDH mRNA were characterised by higher expression of several genes encoding pro-inflammatory and immune cytokines, and increased levels of tumour infiltration with immune cells. Finally, we studied cancer drug sensitivity using data from the National Cancer Institute-60 (NCI-60) database. A specific correlation was found between the expression levels of APRT and cell sensitivity to the chemotherapeutic agent 5-fluorouracil (5-FU). Our findings underline the existence of great differences in uricogenesis between different types of human tumours. The study of uricogenesis offers promising perspectives for the identification of clinically relevant molecular biomarkers and for tumour stratification in the therapeutic context.


Assuntos
Adenina Fosforribosiltransferase/genética , Neoplasias/genética , Ácido Úrico/sangue , Xantina Desidrogenase/genética , Adenina Fosforribosiltransferase/metabolismo , Variações do Número de Cópias de DNA/genética , Metilação de DNA/genética , Bases de Dados Factuais , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Masculino , Neoplasias/sangue , Neoplasias/classificação , Xantina Desidrogenase/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(30): E7081-E7090, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987005

RESUMO

The huntingtin N17 domain is a modulator of mutant huntingtin toxicity and is hypophosphorylated in Huntington's disease (HD). We conducted high-content analysis to find compounds that could restore N17 phosphorylation. One lead compound from this screen was N6-furfuryladenine (N6FFA). N6FFA was protective in HD model neurons, and N6FFA treatment of an HD mouse model corrects HD phenotypes and eliminates cortical mutant huntingtin inclusions. We show that N6FFA restores N17 phosphorylation levels by being salvaged to a triphosphate form by adenine phosphoribosyltransferase (APRT) and used as a phosphate donor by casein kinase 2 (CK2). N6FFA is a naturally occurring product of oxidative DNA damage. Phosphorylated huntingtin functionally redistributes and colocalizes with CK2, APRT, and N6FFA DNA adducts at sites of induced DNA damage. We present a model in which this natural product compound is salvaged to provide a triphosphate substrate to signal huntingtin phosphorylation via CK2 during low-ATP stress under conditions of DNA damage, with protective effects in HD model systems.


Assuntos
Adenina , Adutos de DNA/metabolismo , Dano ao DNA , Doença de Huntington/tratamento farmacológico , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacocinética , Adenina/farmacologia , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Linhagem Celular Transformada , Adutos de DNA/genética , Modelos Animais de Doenças , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/genética
17.
FEBS J ; 285(12): 2306-2318, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29694705

RESUMO

Francisella tularensisis, the causative agent of tularemia has been classified as a category A bioterrorism agent. Here, we present the crystal structure of apo and adenine bound form of the adenine phosphoribosyltransferase (APRT) from Francisella tularensis. APRT is an enzyme involved in the salvage of adenine (a 6-aminopurine), converting it to AMP. The purine salvage pathway relies on two essential and distinct enzymes to convert 6-aminopurine and 6-oxopurines into corresponding nucleotides. The mechanism by which these enzymes differentiate different purines is not clearly understood. Analysis of the structures of apo and adenine-bound APRT from F. tularensis, together with all other available structures of APRTs, suggests that (a) the base-binding loop is stabilized by a cluster of aromatic and conformation-restricting proline residues, and (b) an N-H···N hydrogen bond between the base-binding loop and the N1 atom of adenine is the key interaction that differentiates adenine from 6-oxopurines. These observations were corroborated by bioinformatics analysis of ~ 4000 sequences of APRTs (with 80% identity cutoff), which confirmed that the residues conferring rigidity to the base-binding loop are highly conserved. Furthermore, an F23A mutation on the base-binding loop severely affects the efficiency of the enzyme. We extended our analysis to the structure and sequences of APRTs from the Trypanosomatidae family with a destabilizing insertion on the base-binding loop and propose the mechanism by which these evolutionarily divergent enzymes achieve base specificity. Our results suggest that the base-binding loop not only confers appropriate affinity but also provides defined specificity for adenine. ENZYME: EC 2.4.2.7 DATABASE: Structural data are available in Protein Data Bank (PDB) under the accession numbers 5YW2 and 5YW5.


Assuntos
Adenina Fosforribosiltransferase/química , Adenina/química , Monofosfato de Adenosina/química , Apoproteínas/química , Proteínas de Bactérias/química , Francisella tularensis/enzimologia , Adenina/metabolismo , Adenina Fosforribosiltransferase/genética , Adenina Fosforribosiltransferase/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Francisella tularensis/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Cinética , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Termodinâmica
18.
Cell Chem Biol ; 25(6): 666-676.e4, 2018 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-29576532

RESUMO

Phosphoribosyltransferases catalyze the displacement of a PRPP α-1'-pyrophosphate to a nitrogen-containing nucleobase. How they control the balance of substrates/products binding and activities is poorly understood. Here, we investigated the human adenine phosphoribosyltransferase (hAPRT) that produces AMP in the purine salvage pathway. We show that a single oxygen atom from the Tyr105 side chain is responsible for selecting the active conformation of the 12 amino acid long catalytic loop. Using in vitro, cellular, and in crystallo approaches, we demonstrated that Tyr105 is key for the fine-tuning of the kinetic activity efficiencies of the forward and reverse reactions. Together, our results reveal an evolutionary pressure on the strictly conserved Tyr105 and on the dynamic motion of the flexible loop in phosphoribosyltransferases that is essential for purine biosynthesis in cells. These data also provide the framework for designing novel adenine derivatives that could modulate, through hAPRT, diseases-involved cellular pathways.


Assuntos
Adenina Fosforribosiltransferase/metabolismo , Adenina Fosforribosiltransferase/química , Adenina Fosforribosiltransferase/isolamento & purificação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação Proteica
20.
ACS Chem Biol ; 13(1): 152-160, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29178779

RESUMO

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.


Assuntos
Adenina Fosforribosiltransferase/química , Adenina Fosforribosiltransferase/metabolismo , Inibidores Enzimáticos/metabolismo , Compostos Organofosforados/química , Adenina/química , Adenina/metabolismo , Adenina Fosforribosiltransferase/antagonistas & inibidores , Domínio Catalítico , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Modelos Moleculares , Compostos Organofosforados/síntese química , Orotato Fosforribosiltransferase/antagonistas & inibidores , Plasmodium falciparum/enzimologia , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Estereoisomerismo
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