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.

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.

Initial studies to define the physiologic role of cN-II.

IMP preferring cytosolic 5'-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy.

Active and regulatory sites of cytosolic 5'-nucleotidase.

Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap4A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap4A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.