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Tesi etd-02042008-112738


Thesis type
Tesi di dottorato di ricerca
Author
BALESTRI, FRANCESCO
URN
etd-02042008-112738
Title
role of pentose phosphates in nucleoside catabolism and interconversion
Settore scientifico disciplinare
BIO/10
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Commissione
Relatore Prof.ssa Camici, Marcella
Parole chiave
  • neurological disorders
  • central nervous system
Data inizio appello
13/03/2008;
Consultabilità
completa
Riassunto analitico
Purine and pyrimidine are the basic constituents of the polynucleotides DNA and RNA; they are considered of predominant importance also as information molecules, energy transducers, antioxidants and they have also important roles in cellular signalling processes. Reports suggest protective roles for purines and pyrimidines in various pathological conditions ranging from cancer, to ischemia-associated injury, traumatic tissue damage, bone resorption, stress and haemorrhagic shock. Some papers have shown that adenosine and inosine protect neural cells during hypoxia/ischemia in vivo and in vitro. While in some cases the action of adenosine is receptor-mediated, to explain the effect of its deamination product, inosine, the contribution of hitherto unknown specific receptors has been invoked. On the other hand, several papers report a receptor-independent mechanism of nucleoside action, which indicates that nucleosides donate their ribose moiety to the pentose phosphate pathway, where it is converted to intermediates for entry into the glycolytic pathway for anaerobic production of ATP.<br>To shed light on the mechanism underlying the protective role of purine and pyrimidine during ischemia or brain insults, we have used a human astrocytoma cell line which has been subjected to metabolic stress conditions by exclusion of glucose and pre-incubation with oligomycin. This treatment brings a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. <br>Additionally, the phosphorylated ribose moiety of nucleosides may be used itself for the salvage of pyrimidine nucleosides or can be converted by 5-phosphoribosyl-1-pyrophoshate (PRPP) synthetase, into PRPP which is an essential compound for the salvage pathway of purines. In fact, adenine and hypoxanthine, in the presence of PRPP, are substrates of adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, which catalyze the formation of adenosine and inosine monophosphate, respectively. On the other hand, uridine can be both phosphorolytically cleaved by uridine phosphorylase (UPase) to ribose-1-phosphate (Rib-1-P) and the uracil base, or can be converted by uridine kinase (UK) into uridine nucleotides. It has been shown that disruption of uridine homeostasis by deletion of rat UPase gene leads to disorders of both pyrimidine and purine nucleotide synthesis, thus suggesting a linkage between the two metabolic processes. We have hypothesized that the UPase–UK enzyme system, which maintains uridine homeostasis, regulates the processes of both purine and pyrimidine salvage. Exogenous uridine and, to a lesser extent inosine, activate the salvage of exogenous adenine in human astrocytoma cells in a concentration-dependent manner. Moreover, uridine is also able to activate the salvage of exogenous hypoxanthine. When uridine and inosine are present, more Rib-1-P becomes available, through the action of UPase and purine nucleoside phosphorylase, for the PRPP-mediated adenine and hypoxanthine salvage. Moreover, exogenous inosine favours not only uracil salvage but also 5-FU activation through a Rib-1-P-mediated process. The pre-treatment of the cells with cytidine brings about an inhibition of the pyrimidine salvage and an activation of the purine salvage in the presence of uridine as ribose phosphate donor. In fact, cytidine enters the cells and is converted into CTP which inhibits UK and this inhibition causes a shift of the equilibrium of the reversible UPase reaction towards uridine phosphorolysis. The Rib-1-P formed is then converted into PRPP, which is used for the purine salvage synthesis. Conversely, when the concentration of CTP is relatively low, the fully active UK, which catalyzes a virtual irreversible reaction, drives uridine towards uridine nucleotide formation, thus lowering the rate of purine synthesis. Therefore, the ribose phosphate stemming from the phosphorolysis of purine and pyrimidine nucleosides not only can be converted into energetic intermediates in order to restore the ATP pool during cellular stress but it can be considered a link between the purine and pyrimidine salvage and these two processes are regulated at the level of UPase–UK enzyme system by the relative pyrimidine nucleoside triphosphate concentration. <br><br><br>
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