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

Abstract

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.

Metabolic Aspects of Adenosine Functions in the Brain

“…NT5C1, which has been mainly studied in skeletal muscle, and NT5C2, which is ubiquitously expressed, are the major cytosolic NT5Cs acting on intracellular nucleotides (Camici et al, 2020). Among purine nucleotides, AMP, with a K M in the millimolar range (Hunsucker et al, 2001;Tkacz-Stachowska et al, 2005) is the preferred substrate for NT5C1, while IMP and GMP are better substrates for NT5C2 (K M in the micromolar range) (Tozzi et al, 2013;Pesi et al, 2021), but this enzyme catalyzes also the hydrolysis of the phosphoester bond of AMP (with a K M in the millimolar range) (Tozzi et al, 2013). The intracellular concentrations of IMP, GMP and also AMP depend on the rate of the AMP-IMP-GMP cycles (Figure 2) which in turn depends on NT5C2 activity (Barsotti et al, 2003).…”

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

“…Structural analysis confirmed the existence of the two distinct conformations of cN-II obtained in the presence of allosteric effectors [7]. Oxidative stress causes the irreversible inactivation of the enzyme [8,9]. Therefore, K M and k cat of cN-II depend on the cell energy charge and on reactive oxygen species [8].…”

“…We have previously reported that, in several cell models, cN-II silencing is followed by an increase in oxidative metabolism and of antioxidant defenses [8,9,16]. Therefore, we measured reduced thiols in NCI-H292 cells and found that also in this cell model, they were slightly more abundant in pScNII cells and the incubation with pifithrin-α was able to reduce their level both in pScont and pScNII cells (Figure 4A).…”

“…In this respect, in a breast cancer cell line (MCF-7), an antagonist of A1 adenosine receptor has been found to cause a significant increase of p53 expression [35]. Besides, cN-II may interact with a plethora of other proteins (for details see the following databases BioGRID; IntAct-EMBL-EBI) including enzymes, intracellular inflammatory receptors [14], transcription factors and even tyrosine kinase receptors, presumably with relevant regulatory functions [9]. Therefore, we suggest that the level of cN-II expression might be relevant in determining protein-protein interactions with consequences on both cN-II and interacting protein activities possibly interfering with mechanisms involved in p53 expression and function.…”

“…In this condition, adenosine can be generated by AMP hydrolysis mediated by cN-I, an isoenzyme specific for AMP which is activated by ADP [10,13]. We have previously demonstrated that cN-II interacts with Ice protease-activating factor (IPAF), a protein involved in the innate immunity and inflammation, only in the conformation determined by high energy charge [9,14]. In addition, using different techniques, more than 40 different proteins are expected to interact with cN-II, such as proteins involved in the regulation of protein degradation and synthesis, transcription factors, tyrosine kinase receptors and their regulators (for a better insight see the following databases BioGRID; IntAct-EMBL-EBI).…”