IMP preferring cytosolic 5'-nucleotidase (cN-II) is an ubiquitous nucleotide hydrolysing enzyme. The enzyme is widely distributed and its amino acid sequence is highly conserved among vertebrates. Fluctuations of cN-II activity have been associated with the pathogenesis of neurological disorders. The enzyme appears to be involved in the regulation of the intracellular availability of the purine precursor IMP and also of GMP and AMP, but the contribution of this activity and of its regulation to cell metabolism and to CNS cell functions remains uncertain. To address this issue, we used a vector based short hairpin RNA (shRNA) strategy to knockdown cN-II activity in human astrocytoma cells. Our results demonstrated that 53 h after transduction, cN-II mRNA was reduced to 17.9+/-0.03% of control cells. 19 h later enzyme activity was decreased from 0.7+/-0.026 mU/mg in control ADF cells to 0.45+/-0.046 mU/mg, while cell viability (evaluated by the MTT reduction assay) decreased up to 0.59+/-0.01 (fold vs control) and caspase 3 activity increased from 136+/-5.8 pmol min(-1) mg(-1) in control cells to 639+/-37.5 pmol min(-1) mg(-1) in silenced cells, thus demonstrating that cN-II is essential for cell survival. The decrease of enzyme activity causes apoptosis of the cultured cells without altering intracellular nucleotide and nucleoside concentration or energy charge. Since cN-II is highly expressed in tumour cells, our finding offers a new possible therapeutical approach especially against primary brain tumours such as glioblastoma, and to ameliorate chemotherapy against leukemia.

Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-haloacid dehalogenase superfamily identified three highly conserved motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the measured kinetic parameters, caused by conservative and nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of the covalent enzyme-phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho-enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for catalysis. These findings were consistent with data already determined for P-type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5¢-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal region of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded as regulatory phenomena.Keywords: catalytic residues; HAD; nucleotidase; regulation; site-directed mutagenesis.Mammalian 5¢-nucleotidases (eN, cN-Ia, cN-Ib, cN-II, cN-III, cdN and mdN) make up a family of proteins with different subcellular locations and remarkably low sequence similarities [1]. Besides ectosolic 5¢-nucleotidase, one mitochondrial and five cytosolic enzymes have been described to date. According to its substrate specificity and tissue distribution, each protein seems to play a specific role within the cell. In fact, cN-Is, which is highly expressed in skeletal muscle, heart and testis, is specific for AMP and seems to be involved in adenosine production during hypoxia or ischemia, because it mediates the cell response to low energy charges [2]. On the other hand, cN-II is more specific for inosine monophosphate (IMP) and GMP, and is a ubiquitous enzyme involved in the regulation of intracellular IMP and GMP concentrations [3]. Furthermore, cN-III, which is expressed in red blood cells and is specific for pyrimidines, seems to participate in RNA degradation during erythrocyte maturation [4]. Likewise, cytosolic and mitochondrial deoxynucleotidases (cdN and mdN) regulate nucleotide pools in their respective compartments [1].cN-II was the first member of the cytosolic 5¢-nucl...

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. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011572: cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) and cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by dynamic light scattering (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0038)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011493, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8011481: cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) and cN‐II (uniprotkb:http://www.uniprot.org/uniprot/O46411) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)