• cN-II interfaces
• cN-II physiological role
• cN-II regulatory sites
• cN-II silencing
• cN-II structure
• cytosolic 5'-nucleotidase II

ABSTRACT

5’-Nucleotidases, which dephosphorylate non-cyclic (deoxy)ribonucleoside monophosphates to nucleosides and orthophosphate, constitute a heterogeneous family of widespread enzymes that vary in subcellular location, tissue-specific expression and substrate specificity.
Seven human 5’-nucleotidases have been isolated, five of which are located in the cytosol (cN-IA, cN-IB, cN-II, cN-II, cdN), one in the mitochondrial matrix (mdN) and one is anchorated to the outer plasma membrane by a glycosyl phosphatidylinositol anchor (eN). By opposing the phosphorylation of nucleosides by kinases, intracellular 5’-nucleotidases take part in substrate cycles thereby regulating the intracellular levels of (deoxy)ribonucleoside pools.
The focus of this research project is the cytosolic 5’-nucleotidase II (cN-II), a widespread enzyme with a remarkable sequence conservation through evolution. The Mg2+-dependent cN-II is a bifunctional enzyme, since it can transfer the orthophosphate from a nucleoside monophosphate (preferentially (d)IMP and (d)GMP) not only to water, but also to the 5’ position of a nucleoside acceptor, usually (d)inosine, by a ping-pong reaction mechanism; this is due to the formation of a pentacovalent phosphoester enzyme intermediate, a feature of all members of the HAD superfamily cN-II belongs to. CN-II is submitted to an intricate allosteric regulation, being activated by several phosphorylated compounds (Ap4A, dATP, ATP, ADP, 2,3-BPG) and inhibited by inorganic phosphate; moreover adenylate energy charge is able to affect its activity. In particular, at physiological values of adenylate energy charge, pH and Pi, if a suitable nucleoside acceptor is available, cN-II behaves mainly as a phosphotransferase. Nevertheless, it is unknown whether the transfer of orthophosphate normally occurs in vivo, as Km values for nucleoside substrates are quite high. In vivo, in fact, hydrolysis seems to be cN-II major activity.
CN-II has always been described as a homotetramer of 60 kDa subunits: the crystallized structure of cN-II highlighted the residues responsible for the interaction of two single subunits at interface A and those which hold the two dimers together at interface B. Besides, also the presence of two putative effector sites has been hypothesized. Moreover, it has been proposed by other authors that the effectors influence cN-II oligomerization state: the activators would promote subunit aggregation, while Pi woud act in the opposite way.
Clinical interest in this enzyme increases enormously in these last years, as it seems that cN-II is involved in anticancer and antiviral prodrug metabolism: in fact, not only it could be responsible for resistance to nucleoside analogues with its catabolic function, but it could also be able to activate them by its phosphotransferase activity. Besides mRNA level of cN-II probably has a prognostic value in adult acute myeloid leukemia.
In the light of this, any findings concerning how cN-II activity is regulated and its physiological role will offer new possible therapeutical approaches. Therefore, we designed this research project combining two experimental branches: one focused on the regulatory features of cN-II, seeking to ascertain if the effectors are able to affect cN-II subunit association and enzymatic activity; the other, instead, directed towards the clarification of the role of cN-II in purine metabolism.
In this way, the results obtained would help to improve existing therapeutic treatments leading to the design of personalized chemotherapy; moreover, the cellular systems we built could be very important for the determination not only of the role of cN-II in purine metabolism but also of the relationship between its altered activity and neurological and/or neoplastic pathologies.