Rossana Pesi

Cytosolic 5′-nucleotidase hyperactivity in erythrocytes of Lesch–nyhan syndrome patients

Lesch–Nyhan syndrome is a metabolic–neurological syndrome caused by the X-linked deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Metabolic consequences of HGPRT deficiency have been clarified, but the connection with the neurological manifestations is still unknown. Much effort has been directed to finding other alterations in purine nucleotides in different cells of Lesch–Nyhan patients. A peculiar finding was the measure of appreciable amount of Z-nucleotides in red cells. We found significantly higher IMP-GMP-specific 5′-nucleotidase activity in the erythrocytes of seven patients with Lesch–Nyhan syndrome than in healthy controls. The same alteration was found in one individual with partial HGPRT deficiency displaying a severe neurological syndrome, and in two slightly hyperuricemic patients with a psychomotor delay. Since ZMP was a good substrate of 5′-nucleotidase producing Z-riboside, we incubated murine and human cultured neuronal cells with this nucleoside and found that it is toxic for our models, promoting apoptosis. This finding suggests an involvement of the toxicity of the Z-riboside in the pathogenesis of neurological disorders in Lesch–Nyhan syndrome and possibly in other pediatric neurological syndromes of uncertain origin.

Serum deprivation increases ceramide levels and induces apoptosis in undifferentiated HN9.10e cells

Sphingolipid metabolites have been involved in the regulation of proliferation, differentiation and apoptosis. While cellular mechanisms of these processes have been extensively analysed in the post-mitotic neurons, little is known about proliferating neuronal precursors. We have taken as a model of neuroblasts the embryonic hippocampal cell line HN9.10e. Apoptosis was induced by serum deprivation and by treatment with N-acetylsphingosine (C2-Cer), a membrane-permeant analogue of the second messenger ceramide. Following C2-Cer addition, cytochrome c was released from mitochondria, [Ca(2+)](i) and caspase-3-like activity increased. Both cytochrome c release and rise of [Ca(2+)](i) occurred before caspase-3 activation and nuclear condensation. The intracellular levels of ceramide peaked at 1h following the serum deprivation. These results indicate that the serum deprivation induces a rise in the intracellular ceramide level, and that increased ceramide concentration leads to calcium dysregulation and release of cytochrome c followed by caspase-3 activation. We show that cytochrome c is released without a loss of mitochondrial transmembrane potential.

5 ′-Amino-4-Imidazolecarboxamide Riboside Induces Apoptosis in Human Neuroblastoma Cells Via the Mitochondrial Pathway

5′-Amino-4-imidazolecarboxamide (AICA) riboside induces apoptosis in neuronal cell models. In order to exert its effect, AICA riboside must enter the cell and be phosphorylated to the ribotide. In the present work, we have further studied the mechanism of apoptosis induced by AICA riboside. The results demonstrate that AICA riboside activates AMP-dependent protein kinase (AMPK), induces release of cytochrome c from mitochondria and activation of caspase 9. The role of AMPK in determining cell fate is controversial. In fact, AICA riboside has been reported to be neuroprotective or to induce apoptosis depending on its concentration, cell type or apoptotic stimuli used. In order to clarify whether the activation of AMPK is related to apoptosis in our model, we have used another AMPK stimulator, metformin, and we have analysed its effects on cell viability, nuclear morphology and AMPK activity. Five mM metformin increased AMPK activity, inhibited viability, and increased the number of apoptotic nuclei. AICA riboside, which can be generated from the ribotide (an intermediate of the purine de novo synthesis) by the action of the ubiquitous cytosolic 5′-nucleotidase (cN-II), may accumulate in those individuals in which an inborn error of purine metabolism causes both a building up of intermediates and/or an increase of the rate of de novo synthesis, and/or an overexpression of cN-II. Therefore, our results suggest that the toxic effect of AICA riboside on some types of neurons may participate in the neurological manifestations of syndromes related to purine dismetabolisms.

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.

Evidence for the Involvement of Cytosolic 5′-Nucleotidase (cN-II) in the Synthesis of Guanine Nucleotides from Xanthosine

In this paper, we show that in vitro xanthosine does not enter any of the pathways known to salvage the other three main natural purine nucleosides: guanosine; inosine; and adenosine. In rat brain extracts and in intact LoVo cells, xanthosine is salvaged to XMP via the phosphotransferase activity of cytosolic 5′-nucleotidase. IMP is the preferred phosphate donor (IMP + xanthosine → XMP + inosine). XMP is not further phosphorylated. However, in the presence of glutamine, it is readily converted to guanyl compounds. Thus, phosphorylation of xanthosine by cytosolic 5′-nucleotidase circumvents the activity of IMP dehydrogenase, a rate-limiting enzyme, catalyzing the NAD+-dependent conversion of IMP to XMP at the branch point of de novo nucleotide synthesis, thus leading to the generation of guanine nucleotides. Mycophenolic acid, an inhibitor of IMP dehydrogenase, inhibits the guanyl compound synthesis via the IMP dehydrogenase pathway but has no effect on the cytosolic 5′-nucleotidase pathway of guanine nucleotides synthesis. We propose that the latter pathway might contribute to the reversal of the in vitro antiproliferative effect exerted by IMP dehydrogenase inhibitors routinely seen with repletion of the guanine nucleotide pools.

Deoxyadenosine metabolism in a human colon-carcinoma cell line (LoVo) in relation to its cytotoxic effect in combination with deoxycoformycin

We have assessed the intracellular metabolism of 2′‐deoxyadenosine in a human colon‐carcinoma cell line (LoVo), both in the absence and in the presence of deoxycoformycin, the powerful inhibitor of adenosine deaminase. The combination of 2′‐deoxyadenosine and deoxycoformycin has been reported to inhibit the growth of LoVo cells in culture. In this paper we demonstrate that the observed toxic effect is strictly dependent on cell density. In the absence of deoxycoformycin, 2′‐deoxyadenosine is primarily deaminated to 2′‐deoxyinosine and then converted into hypoxanthine. In the presence of the inhibitor, the deoxynucleoside, in addition to a phosphorylation process, undergoes phosphorolytic cleavage giving rise to adenine. The conversion of 2′‐deoxyadenosine to adenine might represent a protective device, emerging when the activity of adenosine deaminase is reduced or inhibited. There is much evidence to indicate that the enzyme catalyzing this process may be distinct from methylthioadenosine phosphorylase and S‐adenosyl homocysteine hydrolase, which are the enzymes reported to be responsible for the formation of adenine from 2′‐deoxyadenosine in mammals. Int. J. Cancer 75:713–720, 1998.© 1998 Wiley‐Liss, Inc.

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.

Identification of the Nucleotidase Responsible for the AMP Hydrolysing Hyperactivity Associated with Neurological and Developmental Disorders

Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5′-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5′-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5′-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5′-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.

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) NN 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 H+ released by the hydrolysis of the 16 ATP molecules needed to fix the atmospheric N2

6-Thioguanine resistance in a human colon carcinoma cell line with unaltered levels of hypoxanthine guanine phosphoribosyltransferase activity

Cell populations resistant to high doses (30 μM) of 6‐thioguanine (6‐TG, 6‐TGr cells) were selected from a human colon carcinoma cell line, LoVo. This cell line, which lacks hMSH2, a component of the human mismatch binding heterodimer hMutSα, is resistant to low doses of 6‐TG. The level of activity of hypoxanthine‐guanine phosphoribosyltransferase, the enzyme responsible for the phosphoribosylation of the thiopurine, was comparable to that expressed in the parental cells. No significant difference was found in the levels of enzyme activities involved in the conversion of 6‐TG or its derivatives into non‐toxic compounds. In contrast, a significant difference was found in the uptake kinetics of 6‐TG in the 2 cell types. Net uptake of 6‐TG ceased after 100‐sec incubation in the 6‐TGr cells, while it appeared to continue throughout the 10‐min incubation in the wild‐type cells. As a consequence, after 10‐min incubation, the total amount of 6‐TG taken up by the parental LoVo cells was approximately 3 times higher than that present in the 6‐TGr cells. Int. J. Cancer 82:556–561, 1999.