74 research outputs found
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
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
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 enzym
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
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
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
Nucleoside recycling in the brain and the nucleosidome: a complex metabolic and molecular cross-talk between the extracellular nucleotide cascade system and the intracellular nucleoside salvage
The transports of nucleosides from blood into
neurons and astrocytes are essential prerequisites to enter
their metabolic utilization in brain. Adult brain does not
possess the de novo nucleotide synthesis, and maintains its
nucleotide pools by salvaging preformed nucleosides
imported from liver. Once nucleosides enter the brain
through the blood brain barrier and the nucleoside transporters,
they become obligatory precursors for the synthesis
of RNA and DNA and a plethora of other important
functions. However, an aliquot of nucleotides are transferred
into vesicular nucleotide transporters, and then in the
extracellular space by exocytosis of the vesicles, where
ATP and UTP interact with a vast heterogeneity of purine
and pyrimidine receptors. Their signal actions are terminated
by the ectonucleotidase cascade system, which
degrades ATP and UTP into adenosine and uridine,
respectively. The low specificity of the vesicular nucleotide
transporters may explain the presence in the extracellular
space of GTP and CTP, which are equally degraded to their
respective nucleosides by the ectonucleotidases. The main
four nucleosides are re-imported either into the same cell,
or in adjacent cells, e.g. between two astrocytes, or
between a neuron and an astrocyte, to regenerate nucleoside
triphosphates. The molecular network of this metabolic
cross-talk, involving the ectonucleotidases, the
nucleoside transporters, the nucleotide salvage system, the
nucleotide transport into the vesicular nucleotide transporters,
and the exocytotic release of nucleotides, called by
us the ‘‘nucleosidome’’, serves the nucleoside recycling in
the brain, with a considerable spatial–temporal advantage
Metabolic regulation of uridine in the brain
The importance of nucleoside metabolism in brain followed the recognition that i) adult
nervous system maintains its nucleotide pools in the proper qualitative and quantitative balance by salvaging
preformed purine and pyrimidine rings, rather than by synthesizing nucleosides de novo from
simple precursors, ii) adenosine, a purine nucleoside, acts as an extracellular signal, and exerts its protective
effects by interacting with plasmamembrane bound purinergic G-protein coupled P2X receptors.
More recently uridine, a pyrimidine nucleoside, has received considerable attention. Most of the
uridine content of brain is supplied by its uptake from the plasma. An increasing body of evidence
suggests that uridine exerts its function intracellularly in three distinct ways. It is phosphorylated to
UTP, a pyrimidine nucleotide acting as a precursors for RNA and DNA synthesis, and as an extracellular neurotrophic
signal. In combination with the -3 fatty acid decosahexaenoic acid and choline, uridine accelerates formation of synaptic
membrane, being an obligatory precursor for CDP-choline synthesis. Finally, uridine can preserve the ATP pool via the
conversion of its ribose-1-phosphate moiety into energetic intermediates of glycolysis. This article summarizes our present
knowledge on uridine metabolism in the brain, with special emphasis on the mechanisms maintaining its intracellular
homeostasis and on the cross talk between intracellular and extracellular uridine metabolism
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) NBN 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 H1 released by the
hydrolysis of the 16 ATP molecules needed to fix the
atmospheric N2 VC 2015 by the International Union of Biochemistry
and Molecular Biology, 43(3):142–144, 2015
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