117
CONCLUSIONS
During the last past decade, cytosolic 5’-nucleotidase II has drawn the attention of different research groups interested in its physiological role and in its involvement in prodrug metabolism. Despite there has been dramatic progress in the understanding of the structure of cN-II, many questions are still waiting for unambiguous answers. Our work attempted to clear up how and where the known regulators exert their effect and if there is a correlation between the oligomerization state and the presence of the effectors. But this was only the first stage of a wider project whose goal was to shed light on the metabolic role of cN-II and to give some guidance for the tailoring of chemotherapy. In succession, I will give a general outline of the findings obtained during this research doctorate project and of their possible implications.
1. Structural and regulatory features
Cytosolic 5’-nucleotidase II is submitted to a complex regulatory system, being activated by a large variety of phosphorylated compounds such as Ap4A, ATP, ADP and 2,3-BPG. Orthophosphate,
instead, exerts an inhibitory effect. It has been demonstrated that ATP stabilizes an enzyme form with high kcat, while Pi causes the stabilization of a cN-II form with high Km. If ATP and Pi are
present together, a form with high kcat and high Km is observed, indicating that these two molecules
probably have different binding sites (Pesi et al., 1994). Furthermore, kinetic studies suggested that cN-II has at least three different effector sites, one for ATP, one for ADP and one for 2,3-BPG (Pesi et al., 1994). Kinetic characterization showed that ATP and inorganic phosphate reciprocally affect subunit association of human recombinant cN-II, the first promoting the oligomerisation of the protein and the second decreasing the size of protein aggregates (Spychala et al., 1999).
The first chapter of the experimental excursus we followed concerns the identification of the regulatory sites. On the basis of the crystal structure of human cytosolic 5’-nucleotidase II, we generated several point mutants on residues that constitute the putative effector site 1 and 2 (Walldén et al., 2007b). Molecular modeling suggested that the effector site 1 might be the binding site for Ap4A: Ap4A may bind between two subunits with one adenosine moiety in each subunit;
our results confirmed this and, besides, suggested that this regulatory site is probably specific for the binding of ADP. Moreover, our data showed that ATP and 2,3-BPG probably interact with some other site: it is unlikely that this is the effector site 2; several doubts remain on its identity as a
118 site of cN-II activity regulation. In fact, point mutants at this level showed either a generalized impairment in the enzyme regulation or characteristics very similar to WT. These data were partially confirmed by further crystallographic studies published last year (Pesi et al., 2010; Walldén et al., 2011).
There is an unmet need for the development of new antimetabolites and for technologies allowing a more suitable and effective administration of nucleoside analogues for the treatment of cancer patients. The identification of the effector site 1 as one of the activation sites of cN-II provides useful information for the design of compounds that selectively modulate cN-II as a way to enhance the therapeutic power of anticancer prodrugs (leaving the other 5’-nucleotidases unaffected!). The targeting of the allosteric site of cN-II might, therefore, be an avenue to reduce drug resistance against nucleoside analogs.
The second main research line we carried out was the clarification of the relationship between cN-II oligomerization state and activity. From all the sources cytosolic 5’-nucleotidase II has been purified, it has always been described as a homotetramer (Pinto et al., 1987; Spychala et al., 1988; Itoh et Yamada, 1990). Our preliminary gel filtration experiments performed on recombinant WT and mutant cN-II showed that this enzyme exists in different aggregation states ranging from a dodecameric to a dimeric form; anyway, the most represented state is the tetramer. It could be possible that oligomerization states higher than the tetrameric one depend on the high concentration of the recombinant purified protein; furthermore, it is very probable that aggregation states different from tetramer are not naturally expressed. In fact, all the mutants and the WT showed a prevalence of the tetrameric form; only the mutations of the residues located in interface B have the dimer as the prevalent form, as expected, but without consequences on the catalytic activity, contrasting the hypothesis which correlates specific activity to the aggregation extent (Spychala et al., 1999). In order to verify if there was any relationship between the enzyme aggregation state and the presence of the effectors, and so the specific activity, we carried out further studies: both FPLC and light scattering experiments performed in the absence/presence of the activators/inhibitor underlined that cN-II subunit association is irrespective of the effectors. Moreover, we were able to assess, for the first time, that the smallest active quaternary structure is the dimer and not the tetramer as it has always been supposed. To demonstrate this point, not only we performed FPLC analysis, but we also set up a very useful and cheap method, which combines discontinuos native gradient gel electrophoresis, western blotting and detection of 5’-nucleotidase activity. This new protocol overcomes the disadvantages of common gel-filtration and light-scattering methods, such as time of
119 execution, specific protein concentrations and high sensitivity to sample impurities, as well those of the radiochemical assays; further advantages are the easiness and rapidity of performance and the possibility to test several samples at the same time. Moreover, this method requires only common laboratory equipment to be performed. This technique might be turned into a diagnostic tool: in fact, performing the assay in the presence of specific activators/inhibitors, it would be possible to identify which nucleotidase is altered in cells of patients suffering from neurological and developmental disorders, helping diagnose and treatment (and without the need of immunoblotting analysis!).
2. Physiological aspects
cN-II has been described as a part of the purine nucleoside cycle (PNC), whose central position between PRPP-mediated purine salvage and Rib1-P-mediated pyrimidine salvage pathways underlines its importance for the balance of dNTP intracellular pools (Ipata and Tozzi, 2006). Thus an impairment of cytosolic 5’-nucleotidase II activity may cause an outstanding alteration of dNTP pools, potentially leading to serious consequences. In this line it is the observation that an overexpression of cN-II in human embryonal kidney cells of more than 10-fold was impossible to obtain probably because of an adverse effect on cell viability and that the knockdown of cN-II led astrocytoma cell line (ADF) to apoptosis (Rampazzo et al., 1999; Careddu et al., 2008).
This is the scientific context of the other main experimental branch of this doctorate research project, which was the building of eukaryotic systems characterized by different cN-II expression levels: these would have made possible the temporal and quantitative control of cN-II and so the clarification of the role of cN-II in purine metabolism, for the first time, in ex vivo models. The recent development of tetracycline-regulated trans-activation system for inducible gene expression was a very helpful tool for the set up of such experiments in human cell lines, as well as galactose-inducible expression system in yeasts.
These two experimental approaches brought several clues of the pivotal role played by cN-II in cell metabolism and indicated how fundamental cN-II is for cell viability. First of all, we indirectly demonstrated that cN-II is involved in the regulation of the intracellular availability of nucleotides: in fact, after the exposure to increasing concentrations of the alkylating agent MMS, the diploid strain RS112 of S. cerevisiae expressing cN-II had a lower capability to repair DNA damage in comparison with the control, probably due to an alteration of nucleotide pools. Second,
tetracycline-120 controllable cN-II knockdown demonstrated that cN-II is essential for cell survival, as downregulating cN-II activity to 64% of the control led cells to apoptosis.
Moreover, preliminary HPCE analysis of the intracellular nucleoside and nucleotide pools both from yeasts and from ADF cells seems to highlight the involvement of cN-II in purine metabolism. The identification by mass spectrometry of the peaks whose area varies in dependence on the expression grade of cN-II will help to complete the puzzle.
Actually, another clue arose from this research doctorate project is that cN-II could possess an extra function in addition to the enzymatic one: is cN-II a nucleic acid binding protein?
Taken together, our findings and systems could be very important for the determination of the physiological role of cN-II in regulating the intracellular availability of nucleosides and nucleotides and of the relationship between its altered activity and neurological and/or neoplastic pathologies; furthermore these data might be very important for the design of a personalized chemotherapy. Besides, the eukaryotic models we designed are a useful tool to study the involvement of cN-II in prodrug metabolism. In fact, even if the role of cN-II in nucleoside analogue resistance has been extensively studied, further investigation is required to throw light on several aspects which still remain unclear; in addition, there is little evidence from in vivo studies that 5′-nucleotidases actually phosphorylate (deoxy)nucleoside analogues.
The data obtained during the present doctorate project let me and the team I worked with to published some scientific articles and to partecipate to (inter)national congresses as shown below:
ARTICLES
R. Pesi, S. Allegrini, M.G.Careddu, D.N. Filoni, M. Camici and M.G, Tozzi (2010) Active and regulatory sites of cytosolic 5’-nucleotidase, FEBS J., 277(23):4863-72;
D.N. Filoni, R. Pesi, M.G. Careddu, S. Allegrini, A. Collavoli, I. Scarfone, F. Zucchi, A. Galli and M.G. Tozzi (2011) Initial studies to define the physiologic role of cN-II, Nucleosides, Nucleotides and Nucleic Acids, NNNA, 30, 12, 1155-60.
121 In preparation, D.N. Filoni, A. Galli, A. Collavoli, S. Allegrini, R. Pesi, M.G. Tozzi, Expression of bovine cytosolic 5’-nucleotidase (cN-II) in yeast: effect on DNA repair (to be sent to “Purinergic Signal”).
ABSTRACTS
D.N. Filoni, R. Pesi, M. Camici, S. Allegrini, M.G. Tozzi, Structural and functional studies of cytosolic 5’-Nucleotidase II, 34th FEBS Congress, Prague, Czech Republic, 4th-9th July 2009
S. Allegrini, D.N. Filoni, R. Pesi, M. Camici, M.G. Tozzi, cN-II: structural and functional studies, 13th International Symposium on Purine and Pyrimidine Metabolism in Man, Skogshem-Wijk, Lidingo, Stockholm, Sweden, 21st-24th June 2009
D.N. Filoni, R. Pesi, M. Camici, S. Allegrini, A. Collavoli, A. Galli and M.G. Tozzi ,Cytosolic 5’-Nucleotidase’s adventures in Metaboland, 10th
YSF (FEBS GRANT for YSF) and 35th FEBS Congress, Gotheburg, Sweden, 23rd-26th June and 26th June-1st July 2010
D.N. Filoni, R. Pesi, M. Camici, S. Allegrini, A.Collavoli, A. Galli, M.G. Tozzi, A Janus’ scientific approach for cN-II, 55th National Meeting of the Italian Society of Biochemistry and Molecular Biology ( SIB ), Milano, Italy, 14th-17th September 2010
M.G. Tozzi, D.N. Filoni, R. Pesi, M.G. Careddu, M. Camici, S. Allegrini, A. Collavoli, A. Galli, Face to face with cN-II, 14th International Symposium on Purine and Pyrimidine Metabolism in Man, Tokio, Japan, 18th-21st February 2011
D.N. Filoni, R. Pesi, S. Allegrini, M. Camici, P.L Ipata, M.G. Tozzi, Puzzling cN-II, 36th FEBS
Congress, Torino, Italy, 25th-30th June 2011
D.N. Filoni, R. Pesi, M. Camici, S. Allegrini, M.G. Tozzi, Cytosolic 5’-Nucleotidase II, an intriguing enzyme, 3rd EMBO Meeting, Vienna, Austria, 10th-13th September 2011
