Interplay between mTOR and Purine Metabolism Enzymes and Its Relevant Role in Cancer.

Tumor cells reprogram their metabolism to meet the increased demand for nucleotides and other molecules necessary for growth and proliferation. In fact, cancer cells are characterized by an increased "de novo" synthesis of purine nucleotides. Therefore, it is not surprising that specific enzymes of purine metabolism are the targets of drugs as antineoplastic agents, and a better knowledge of the mechanisms underlying their regulation would be of great help in finding new therapeutic approaches. The mammalian target of the rapamycin (mTOR) signaling pathway, which is often activated in cancer cells, promotes anabolic processes and is a major regulator of cell growth and division. Among the numerous effects exerted by mTOR, noteworthy is its empowerment of the "de novo" synthesis of nucleotides, accomplished by supporting the formation of purinosomes, and by increasing the availability of necessary precursors, such as one-carbon formyl group, bicarbonate and 5-phosphoribosyl-1-pyrophosphate. In this review, we highlight the connection between purine and mitochondrial metabolism, and the bidirectional relation between mTOR signaling and purine synthesis pathways.

Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Inborn Errors of Purine Salvage and Catabolism.

Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all.

The Good, the Bad and the New about Uric Acid in Cancer.

Uric acid is the final product of purine catabolism in man and apes. The serum concentration of uric acid is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant, while at high intracellular concentrations, it is a pro-oxidant molecule. In this review, we describe the possible causes of uric acid accumulation or depletion and some of the metabolic and regulatory pathways it may impact. Particular attention has been given to fructose, which, because of the complex correlation between carbohydrate and nucleotide metabolism, causes uric acid accumulation. We also present recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in a variety of signaling pathways, which can play a role in the pathogenesis of diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. The loss of uricase in Homo sapiens and great apes, although exposing these species to the potentially adverse effects of uric acid, appears to be associated with evolutionary advantages.

Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.

Cytosolic 5'-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.

Metabolic Aspects of Adenosine Functions in the Brain.

Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5'-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington's disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator.

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

Purine-Metabolising Enzymes and Apoptosis in Cancer.

The enzymes of both de novo and salvage pathways for purine nucleotide synthesis are regulated to meet the demand of nucleic acid precursors during proliferation. Among them, the salvage pathway enzymes seem to play the key role in replenishing the purine pool in dividing and tumour cells that require a greater amount of nucleotides. An imbalance in the purine pools is fundamental not only for preventing cell proliferation, but also, in many cases, to promote apoptosis. It is known that tumour cells harbour several mutations that might lead to defective apoptosis-inducing pathways, and this is probably at the basis of the initial expansion of the population of neoplastic cells. Therefore, knowledge of the molecular mechanisms that lead to apoptosis of tumoural cells is key to predicting the possible success of a drug treatment and planning more effective and focused therapies. In this review, we describe how the modulation of enzymes involved in purine metabolism in tumour cells may affect the apoptotic programme. The enzymes discussed are: ectosolic and cytosolic 5'-nucleotidases, purine nucleoside phosphorylase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, and inosine-5'-monophosphate dehydrogenase, as well as recently described enzymes particularly expressed in tumour cells, such as deoxynucleoside triphosphate triphosphohydrolase and 7,8-dihydro-8-oxoguanine triphosphatase.

Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors.

The growing evidence of the involvement of purine compounds in signaling, of nucleotide imbalance in tumorigenesis, the discovery of purinosome and its regulation, cast new light on purine metabolism, indicating that well known biochemical pathways may still surprise. Adenosine deaminase is important not only to preserve functionality of immune system but also to ensure a correct development and function of central nervous system, probably because its activity regulates the extracellular concentration of adenosine and therefore its function in brain. A lot of work has been done on extracellular 5'-nucleotidase and its involvement in the purinergic signaling, but also intracellular nucleotidases, which regulate the purine nucleotide homeostasis, play unexpected roles, not only in tumorigenesis but also in brain function. Hypoxanthine guanine phosphoribosyl transferase (HPRT) appears to have a role in the purinosome formation and, therefore, in the regulation of purine synthesis rate during cell cycle with implications in brain development and tumors. The final product of purine catabolism, uric acid, also plays a recently highlighted novel role. In this review, we discuss the molecular mechanisms underlying the pathological manifestations of purine dysmetabolisms, focusing on the newly described/hypothesized roles of cytosolic 5'-nucleotidase II, adenosine kinase, adenosine deaminase, HPRT, and xanthine oxidase.

Cell-specific pattern of berberine pleiotropic effects on different human cell lines.

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.

Cytosolic 5'-Nucleotidase II Silencing in a Human Lung Carcinoma Cell Line Opposes Cancer Phenotype with a Concomitant Increase in p53 Phosphorylation.

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5'-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology.

Metabolic interaction between urea cycle and citric acid cycle shunt: A guided approach.

This article is a guided pedagogical approach, devoted to postgraduate students specializing in biochemistry, aimed at presenting all single reactions and overall equations leading to the metabolic interaction between ureagenesis and citric acid cycle to be incorporated into a two-three lecture series about the interaction of urea cycle with other metabolic pathways. We emphasize that citrate synthetase, aconitase, and isocitrate dehydrogenase, three enzymes of the citric acid cycle are not involved, thus creating a shunt in citric acid cycle. In contrast, the glutamic-oxaloacetate transaminase, which does not belong to citric acid cycle, has a paramount importance in the metabolic interaction of the two cycles, because it generates aspartate, one of the two fuel molecules of urea cycle, and a-ketoglutarate, an intermediate of the citric acid cycle. Finally, students should appreciate that balancing equations for all atoms and charges is not only a stoichiometric task, but strongly facilitates the discussion of the physiological roles of metabolic pathways. Indeed, this exercise has been used in the classroom, to encourage a deeper level of understanding of an important biochemical issue. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(2):182-185, 2018.

Metabolic interaction between purine nucleotide cycle and oxypurine cycle during skeletal muscle contraction of different intensities: a biochemical reappraisal.

BACKGROUND: A substrate cycle is a metabolic transformation in which a substrate A is phosphorylated to A-P at the expense of ATP (or another "high energy" compound), and A-P is converted back to A by a nucleotidase or a phosphatase. Many biochemists resisted the idea of such an ATP waste. Why a non-phosphorylated metabolite should be converted into a phosphorylated form, and converted back to its non-phosphorylated form through a "futile cycle"? AIM OF REVIEW: In this Review we aim at presenting our present knowledge on the biochemical features underlying the interrelation between the muscle purine nucleotide cycle and the oxypurine cycle, and on the metabolic responses of the two cycles to increasing intensities of muscle contraction. KEY SCIENTIFIC CONCEPTS OF REVIEW: Nowadays it is widely accepted that the substrate cycles regulate many vital functions depending on the expense of large amounts of ATP, including skeletal muscle contraction, so that the expense of some extra ATP and "high energy" compounds, such as GTP and PRPP via substrate cycles, is not surprising. The Review emphasizes the strict metabolic interrelationship between the purine nucleotide cycle and the oxipurine cycle.

Understanding the interrelationship between the synthesis of urea and gluconeogenesis by formulating an overall balanced equation.

It is well known that a strong metabolic interrelationship exists between ureagenesis and gluconeogenesis. In this paper, we present a detailed, overall equation, describing a possible metabolic link between ureagenesis and gluconeogenesis. We adopted a guided approach in which we strongly suggest that students, when faced with the problem of obtaining the overall equation of a metabolic pathway, carefully account for all atoms and charges of the single reactions, as well as the cellular localizations of the substrates, and the related transport systems. If this suggestion is always taken into account, a balanced, overall equation of a metabolic pathway will be obtained, which strongly facilitates the discussion of its physiological role. Unfortunately, textbooks often report unbalanced overall equations of metabolic pathways, including ureagenesis and gluconeogenesis. Most likely the reason is that metabolism and enzymology have been neglected for about three decades, owing to the remarkable advances of molecular biology and molecular genetics. In this paper, we strongly suggest that students, when faced with the problem of obtaining the overall reaction of a metabolic pathway, carefully control if the single reactions are properly balanced for atoms and charges. Following this suggestion, we were able to obtain an overall equation describing the metabolic interrelationship between ureagenesis and gluconeogenesis, in which urea and glucose are the final products. The aim is to better rationalize this topic and to convince students and teachers that metabolism is an important and rewarding chapter of human physiology.

IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism.

BACKGROUND: Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS: HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS: An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE: Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE: Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.

Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

What is the true nitrogenase reaction? A guided approach.

Only diazotrophic bacteria, called Rizhobia, living as symbionts in the root nodules of leguminous plants and certain free-living prokaryotic cells can fix atmospheric N2 . In these microorganisms, nitrogen fixation is carried out by the nitrogenase protein complex. However, the reduction of nitrogen to ammonia has an extremely high activation energy due to the stable (unreactive) N≡N triple bond. The structural and functional features of the nitrogenase protein complex, based on the stepwise transfer of eight electrons from reduced ferredoxin to the nitrogenase, coupled to the hydrolysis of 16 ATP molecules, to fix one N2 molecule into two NH3 molecules, is well understood. Yet, a number of different nitrogenase-catalyzed reactions are present in biochemistry textbooks, which might cause misinterpretation. In this article, we show that when trying to balance the reaction catalyzed by the nitrogenase protein complex, it is important to show explicitly the 16 H(+) released by the hydrolysis of the 16 ATP molecules needed to fix the atmospheric N2.

A native electrophoretic technique to study oligomerization and activity of cytosolic 5'-nucleotidase II.

The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5'-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

Expression of bovine cytosolic 5'-nucleotidase (cN-II) in yeast: nucleotide pools disturbance and its consequences on growth and homologous recombination.

Cytosolic 5'-nucleotidase II is a widespread IMP hydrolyzing enzyme, essential for cell vitality, whose role in nucleotide metabolism and cell function is still to be exactly determined. Cytosolic 5'-nucleotidase overexpression and silencing have both been demonstrated to be toxic for mammalian cultured cells. In order to ascertain the effect of enzyme expression on a well-known eukaryote simple model, we expressed cytosolic 5'-nucleotidase II in Saccharomyces cerevisiae, which normally hydrolyzes IMP through the action of a nucleotidase with distinct functional and structural features. Heterologous expression was successful. The yeast cells harbouring cytosolic 5'-nucleotidase II displayed a shorter duplication time and a significant modification of purine and pyrimidine derivatives concentration as compared with the control strain. Furthermore the capacity of homologous recombination in the presence of mutagenic compounds of yeast expressing cytosolic 5'-nucleotidase II was markedly impaired.