Differential control of cell cycle,proliferation, and survival of primary T lymphocytes by purine and pyrimidine nucleotides

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G(1) to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.     

The PRPP synthetase spectrum: what does it demonstrate about nucleotide syndromes?

Defects in X-linked phosphoribosylpyrophosphate synthetase 1 (PRPS1) manifest as follows: (1) PRS-I enzyme "superactivity" (gain-of-function mutations affecting allosteric regions); (2) PRS-I overexpression (which may be linked to miRNA mutation); (3) severe PRS-I deficiency/Arts syndrome (missense mutations producing loss-of-function); (4) moderate PRS-I deficiency/Charcot-Marie-Tooth disease-5 (less severe loss-of-function mutations); and (5) mild PRS-I deficiency/Deafness-2 (mutations producing slight destabilization). Similar to Lesch-Nyhan disease, PRPS1-related disorders arise from phosphoribosyl-pyrophosphate (PRPP)-dependent nucleotide "depletion" of purine nucleotides (e.g., ATP, GTP). S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion. But pyrimidine salvage from uridine does not require PRPP, and this nucleoside is transported freely to pyrimidine-depleted tissues. Regulation of nicotinamide nucleotides is less clear; synthesis from pyridine nucleobases is PRPP dependent. Nucleotide "depletion" contrasts with nucleotide "toxicity," exemplified by the purine disorders adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies or by pyrimidine nucleotidase deficiency. These are characterized by the accumulation of one or more abnormal nucleotides such as succinyl- or deoxy-nucleotides or their metabolites, which interrupt other nucleotide or related pathways or are toxic to specific cell types. Theoretically, purine toxicity disorders would not be ameliorated by SAMe therapy, and this was confirmed for one adenylosuccinate lyase-deficient child. Nucleotide defects may also be seen as an aspect of mitochondrial disease, with SAMe-based mitochondrial therapy perhaps meriting further investigation.     

Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus

Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2'-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2'-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.     

Stereospecific synthesis of sugar-1-phosphates and their conversion to sugar nucleotides

As Leloir glycosyltransferases are increasingly being used to prepare oligosaccharides, glycoconjugates, and glycosylated natural products, efficient access to stereopure sugar nucleotide donor substrates is required. Herein, the rapid synthesis and purification of eight sugar nucleotides is described by a facile 30 min activation of nucleoside 5'-monophosphates bearing purine and pyrimidine bases with trifluoroacetic anhydride and N-methylimidazole, followed by a 2 h coupling with stereospecifically prepared sugar-1-phosphates. Tributylammonium bicarbonate and tributylammonium acetate were the ion-pair reagents of choice for the C18 reversed-phase purification of 6-deoxysugar nucleotides, and hexose or pentose-derived sugar nucleotides, respectively.     

Regulation of Escherichia coli pyrC by the purine regulon repressor protein.

The purine regulon repressor, PurR, was identified as a component of the Escherichia coli regulatory system for pyrC, the gene that encodes dihydroorotase, an enzyme in de novo pyrimidine nucleotide synthesis. PurR binds to a pyrC control site that resembles a pur regulon operator and represses expression by twofold. Mutations that increase binding of PurR to the control site in vitro concomitantly increase in vivo regulation. There are completely independent mechanisms for regulation of pyrC by purine and pyrimidine nucleotides. Cross pathway regulation of pyrC by PurR may provide one mechanism to coordinate synthesis of purine and pyrimidine nucleotides.     

Optimal alphabets for an RNA world

Experiments have shown that the canonical AUCG genetic alphabet is not the only possible nucleotide alphabet. In this work we address the question ''is the canonical alphabet optimal?'' We make the assumption that the genetic alphabet was determined in the RNA world. Computational tools are used to infer the RNA secondary structure (shape) from a given RNA sequence, and statistics from RNA shapes are gathered with respect to alphabet size. Then, simulations based upon the replication and selection of fixed-sized RNA populations are used to investigate the effect of alternative alphabets upon RNA''s ability to step through a fitness landscape. These results show that for a low copy fidelity the canonical alphabet is fitter than two-, six- and eight-letter alphabets. In higher copy-fidelity experiments, six-letter alphabets outperform the four-letter alphabets, suggesting that the canonical alphabet is indeed a relic of the RNA world.     

Regulation of Salmonella typhimurium pyr gene expression: effect of changing both purine and pyrimidine nucleotide pools

The synthesis of the pyrimidine biosynthetic enzymes is repressed by the pyrimidine nucleotide end-products of the pathway. However, purine nucleotides also play a role. In this study, I have measured expression of the pyr genes (pyrA-E) in Salmonella typhimurium strains harbouring mutations that permit manipulation of the intracellular pools of both pyrimidine and purine nucleotides. The results identify the effectory purine compound as being a guanine nucleotide; it is probably GTP, but it may be GDP or GMP. The synthesis of carbamoylphosphate synthase, encoded by pyrA, and particularly dihydroorotase, encoded by pyrC, and dihydroorotate dehydrogenase, encoded by pyrD, is stimulated by the guanine nucleotide, while the synthesis of aspartate transcarbamoylase, encoded by pyrBI, and orotate phosphoribosyltransferase, encoded by pyrE, is inhibited by guanine nucleotides. The regulatory pattern of each pyr gene is discussed in relation to present knowledge on gene structure and regulatory mechanism.     

Effect of nucleotides 37 and 38 on cleavage of tRNA~(Phe) precursors

Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNAPhe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N37 and N38 were found to be the best substrates for the enzyme. When N37 and N38 were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.     

Nucleoside deoxyribosyltransferase-II from Lactobacillus helveticus Substrate specificity studied. Pyrimidine bases as acceptors.

The nucleoside deoxyribosyltransferase (nucleoside:purine (pyrimidine) deoxyribosyltransferase, EC 2.4.2.6) fraction catalyzing specifically the transfer of the deoxyribosyl moiety from a purine (or a pyrimidine) to a pyrimidine (or a purine) exhibits a broad specificity for the acceptor base. With a pyrimidine base as the acceptor a -OH or -SH group adjacent to the N-1 atom is essential. A substituent on position 6 hinders the reaction. On positions 4 and 5 various substituent were found to influence the reaction rate and some of them give non-competent substrates. A few anomalous cases are also discussed in relation with the role of N-3. Deoxyribonucleosides can also be obtained with non-pyrimidine rings.     

Molecular mechanics calculations on a triple stranded DNA involving C+.G-T and T.A+-C mismatched base triples

We have carried out molecular modeling of a triple stranded pyrimidine(Y). purine(R): pyrimidine(Y) (where ':' refers to Watson-Crick and '.' to Hoogsteen bonding) DNA, formed by a homopurine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC). Molecular mechanics calculations using NMR constraints have provided a detailed three dimensional structure of the triplex. The entire stretches of purine and the pyrimidine nucleotides have a conformation close to B-DNA. The three strands are held by the canonical C+.G:C and T.A:T hydrogen bonds. The structure also contains two mismatch C+.G-T and T.A+-C base triples which have been characterized for the first time. In the A+-C base-pair of the T.A+-C triple, both hydrogen donors are situated on the purine (A+(1N) and A+(6N)). We observe a unique hydrogen bonding interaction scheme in case of C+.G-T where one acceptor, G(60), is bonded to three donors (C+(3NH), C+(4NH2) and T(3NH)). Though the C+.G-T base triple is less stable than C+.G:C, it is significantly more stable than T.A:T. On the other hand, T.A+-C is as stable as the T.A:T base triad.     

Blood uridine concentration may be an indicator of the degradation of pyrimidine nucleotides during physical exercise with increasing intensity

During prolonged maximal exercise, oxygen deficits occur in working muscles. Progressive hypoxia results in the impairment of the oxidative resynthesis of ATP and increased degradation of purine nucleotides. Moreover, ATP consumption decreases the conversion of UDP to UTP, to use ATP as a phosphate donor, resulting in an increased concentration of UDP, which enhances pyrimidine degradation. Because the metabolism of pyrimidine nucleotides is related to the metabolism of purines, in particular with the cellular concentration of ATP, we decided to investigate the impact of a standardized exercise with increasing intensity on the concentration of uridine, inosine, hypoxanthine, and uric acid. Twenty-two healthy male subjects volunteered to participate in this study. Blood concentrations of metabolites were determined at rest, immediately after exercise, and after 30 min of recovery using high-performance liquid chromatography. We also studied the relationship between the levels of uridine and indicators of myogenic purine degradation. The results showed that exercise with increasing intensity leads to increased concentrations of inosine, hypoxanthine, uric acid, and uridine. We found positive correlations between blood uridine levels and indicators of myogenic purine degradation (hypoxanthine), suggesting that the blood uridine level is related to purine metabolism in skeletal muscles.     

In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases

In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.     

Structures of purine 2'-deoxyribosyltransferase, substrate complexes, and the ribosylated enzyme intermediate at 2.0 A resolution

The structure of class I N-deoxyribosyltransferase from Lactobacillus helveticus was determined by X-ray crystallography. Unlike class II N-deoxyribosyltransferases, which accept either purine or pyrimidine deoxynucleosides, class I enzymes are specific for purines as both the donor and acceptor base. Both class I and class II enzymes are highly specific for deoxynucleosides. The class I structure reveals similarities with the previously determined class II enzyme from Lactobacillus leichmanni [Armstrong, S. A., Cook, W. J., Short, S. A., and Ealick, S. E. (1996) Structure 4, 97-107]. The specificity of the class I enzyme for purine deoxynucleosides can be traced to a loop (residues 48-62), which shields the active site in the class II enzyme. In the class I enzyme, the purine base itself shields the active site from the solvent, while the smaller pyrimidine base cannot. The structure of the enzyme with a bound ribonucleoside shows that the nucleophilic oxygen atom of Glu101 hydrogen bonds to the O2' atom, rendering it unreactive and thus explaining the specificity for 2'-deoxynucleosides. The structure of a ribosylated enzyme intermediate reveals movements that occur during cleavage of the N-glycosidic bond. The structures of complexes with substrates and substrate analogues show that the purine base can bind in several different orientations, thus explaining the ability of the enzyme to catalyze alternate deoxyribosylation at the N3 or N7 position.     

Nucleosome Positioning by Sequence,State of the Art and Apparent Finale

Abstract

All major suggestions about the nucleosome positioning sequence pattern(s) are overviewed. Two basic binary periodical patterns are well established: in purine/pyrimidine alphabet—YRRRRRYYYYYR and in strong/weak alphabet-SWWWWWSSSSSW. Their merger in four-letter alphabet sequence coincides with first ever complete matrix of nucleosome DNA bendability derived from very large database of nucleosome DNA sequences. Its simplified linear form is CGGAAATTTCCG. Several independent ways of derivation of the same pattern are described. It appears that the pattern represents an ultimate solution of long-standing problem of nucleosome positioning, and provides simple means for nucleosome mapping on sequences with single-base resolution.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Purine metabolism in trypanosomatids.

Purine nucleotide biosynthesis was studied in culture forms of Trypanosoma cruzi strain Y, Crithidia deanei (a reduviid trypanosomatid with an endosymbiote) and an aposymbiotic strain of C. deanei (obtained by curing C. deanei with chloramphenicol). Trypanosoma cruzi was found to synthesize purine nucleotides only fring incorporated into both adenine and guanine nucleotides. Similar results were obtained with guanine, indicating that this flagellate has a system for the interconversion of purine nucleotides. Crithidia deanei was able to synthesize purine and pyrimidine nucleotides from glycine ("de novo" pathway) and purine nucleotides from adenine and guanine ("salvage" pathway). Adenine was incorporated into both adenine and guanine nucleotides, while guanine was incorporated into guanine nucleotides only, indicating the presence of a metabolic block at the level of GMP reductase. The aposymbiotic C. deanei strain was unable to utilize glycine for the synthesis of purine nucleotides, although glycine was utilized for synthesizing pyrimidine nucleotides. These results suggest that the endosymbiote is implicated in the de novo purine nucleotide pathway of the C. deanei-endosymbiote complex. The incorporation of adenine and guanine by aposymbiotic C. deanei strain followed a pattern similar to that observed for C. deanei.     

Effect of nucleotides 37 and 38 on cleavage of tRNAPhe precursors

Splicing is required for tRNA maturation when the precursors contain the introns. In order to determine whether nucleotides 37 and 38 affect splicing, yeast tRNA^Phe precursors with different nucleotides 37 and 38 were prepared by in vitro mutagenesis and cleaved by the purified yeast tRNA-splicing endonuclease. The precursors with purine nudeolides at N₃₇ and N₃₈ were found to be the best substrates for the enzyme. When N₃₇ and N₃₈ were replaced by pyrimidine nucleotides, few precursors could be cleaved by the endonuclease. If one is pyrimidine nucleotide, the other one is purine nudeotide at these positions, the cleavage efficiencies are between the two groups of precursors stated above. The pyrimidine nucleotides at these positions might affect the fine structures of the precursors or the distance between the splicing sites, so that the precursors can not be fixed or anchored on the enzyme well, leading to the poor cutting.     

Partial purification and properties of diglyceride kinase from Escherichia coli.

Diglyceride kinase (diacylglycerol kinase, E.C. 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.     

Energy of interaction in actinomycin-nucleotide complexes

Complexes of the natural heterocyclic antibiotic actinomycin D (AMD) with its putative carriers: purine and pyrimidine nucleotides, as well as with fragmented DNA and phospholipid liposomes have been studied by high-sensitivity spectrophotometry. The antibiotic is not only adsorbed onto the surface of purine clusters but also is incorporated into them. It is especially readily incorporated into unwound DNA regions. The incorporation is accompanied by a long-wavelength shift of the absorption spectrum. From the magnitude of the shift, the energy of interaction was calculated. In the case of AMD in the complex with caffeine and adenosine, it is 2.4 and 2.7 kcal/mol, and in the complex with guanosine and fragmented DNA it is considerably higher, 3.3 and 3.7 kcal/mol. It is assumed that guanosine, adenosine, caffeine and fragmented DNA may serve as carriers of the antibiotic.     

Analysis of adenosine-mediated pyrimidine starvation using cultured wild-type and mutant mouse T-lymphoma cells.

Using the S49 T-cell lymphoma system for the study of immunodeficiency diseases, we characterized several variants in purine salvage and transport pathways and studied their responses to the cytotoxic action of adenosine (5-20 micron) in the presence of adenosine deaminase (ADA) inhibitors. Both an adenosine transport deficient mutant and a mutant lacking adenosine (ado) kinase activity are resistant to the cytotoxic effects of adenosine up to 15 micron. Variants lacking hypoxanthine-guanine phosphoribosyl transferase or adenine phosphoribosyltransferase are sensitive to the killing action of adenosine. We monitored the intracellular concentrations of purine and pyrimidine nucleotides, orotate, and PPriboseP in mutant and wild-type cells following the addition of adenosine and an ADA inhibitor. We conclude that at low concentrations, adenosine must be phosphorylated to deplete the cell of pyrimidine nucleotides and PPriboseP and to promote the accumulation of orotate. These alterations account for one mechanism of adenosine toxicity.