To explore the effects of heroin on purine nucleotides metabolism in rat brain.

Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.

As second messengers, the cyclic purine nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) play an essential role in intracellular signaling. Recent data suggest that the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) also act as second messengers. Hydrolysis by phosphodiesterases (PDEs) is the most important degradation mechanism for cAMP and cGMP. Elimination of cUMP and cCMP is not completely understood, though. We have shown that human PDEs hydrolyze not only cAMP and cGMP but also cyclic pyrimidine nucleotides, indicating that these enzymes may be important for termination of cCMP- and cUMP effects as well. However, these findings were acquired using a rather expensive HPLC/mass spectrometry assay, the technical requirements of which are available only to few laboratories. N'-Methylanthraniloyl-(MANT-)labeled nucleotides are endogenously fluorescent and suitable tools to study diverse protein/nucleotide interactions. In the present study, we report the synthesis of new MANT-substituted cyclic purine- and pyrimidine nucleotides that are appropriate to analyze substrate specificity and kinetics of PDEs with more moderate technical requirements. MANT-labeled nucleoside 3',5'-cyclic monophosphates (MANT-cNMPs) are shown to be substrates of various human PDEs and to undergo a significant change in fluorescence upon cleavage, thus allowing direct, quantitative and continuous determination of hydrolysis via fluorescence detection. As substrates of several PDEs, MANT-cNMPs show similar kinetics to native nucleotides, with some exceptions. Finally, they are shown to be also appropriate tools for PDE inhibitor studies.

Translation fidelity relies essentially on the ability of ribosomes to accurately recognize triplet interactions between codons on mRNAs and anticodons of tRNAs. To determine the codon-anticodon pairs that are efficiently accepted by the eukaryotic ribosome, we took advantage of the IRES from the intergenic region (IGR) of the Cricket Paralysis Virus. It contains an essential pseudoknot PKI that structurally and functionally mimics a codon-anticodon helix. We screened the entire set of 4096 possible combinations using ultrahigh-throughput screenings combining coupled transcription/translation and droplet-based microfluidics. Only 97 combinations are efficiently accepted and accommodated for translocation and further elongation: 38 combinations involve cognate recognition with Watson-Crick pairs and 59 involve near-cognate recognition pairs with at least one mismatch. More than half of the near-cognate combinations (36/59) contain a G at the first position of the anticodon (numbered 34 of tRNA). G34-containing tRNAs decoding 4-codon boxes are almost absent from eukaryotic genomes in contrast to bacterial genomes. We reconstructed these missing tRNAs and could demonstrate that these tRNAs are toxic to cells due to their miscoding capacity in eukaryotic translation systems. We also show that the nature of the purine at position 34 is correlated with the nucleotides present at 32 and 38.

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.

Surface-enhanced Raman scattering (SERS) is a highly sensitive technique used in diverse biomedical applications including rapid antibiotic susceptibility testing (AST). However, signal fluctuation in SERS, particularly the widespread of signals measured from different batches of SERS substrates, compromises its reliability and introduces potential errors in SERS-AST. In this study, we investigate the use of purine as an internal standard (IS) to recalibrate SERS signals and quantify the concentrations of two important purine derivatives, adenine and hypoxanthine, which are the most important biomarkers used in SERS-AST. Our findings demonstrate that purine IS effectively mitigates SERS signal fluctuations and enables accurate prediction of adenine and hypoxanthine concentrations across a wide range (5 orders of magnitude). Calibrations with purine as an IS outperform those without, exhibiting a 10-fold increase in predictive accuracy. Additionally, the calibration curve obtained from the first batch of SERS substrates remains effective for 64 additional substrates fabricated over a half-year period. Measurements of adenine and hypoxanthine concentrations in bacterial supernatants using SERS with purine IS closely align with the liquid chromatography-mass spectrometry results. The use of purine as an IS offers a simple and robust platform to enhance the speed and accuracy of SERS-AST, while also paving the way for in situ SERS quantification of purine derivatives released by bacteria under various stress conditions.

Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is different from that of more common carbohydrate biomass polymers, materials based on nucleic acids may exhibit new, attractive characteristics. In this study, fluorescent nanoparticles (biodots) were prepared by a hydrothermal (HT) method from various nucleic acids (DNA, RNA, nucleotides, and nucleosides) to establish the relationship between the structure of precursors and fluorescent properties of biodots and to optimize conditions for preparation of the most fluorescent product. HT treatment of nucleic acids results in decomposition of sugar moieties and depurination/depyrimidation of nucleobases, while their consequent condensation and polymerization gives fluorescent nanoparticles. Fluorescent properties of DNA and RNA biodots are drastically different from biodots synthesized from individual nucleotides. In particular, biodots synthesized from purine-containing nucleotides or nucleosides show up to 50-fold higher fluorescence compared to analogous pyrimidine-derived biodots. The polymeric nature of a precursor disfavors formation of a bright fluorescent product. The reported effect of the structure of the nucleic acid precursor on the fluorescence properties of biodots should help designing and synthesizing brighter fluorescent nanomaterials with broader specification for bioimaging, sensing, and other applications.

Polymerization of nucleotides under prebiotically plausible conditions has been a focus of several origins of life studies. Non-activated nucleotides have been shown to undergo polymerization under geothermal conditions when subjected to dry-wet cycles. They do so by a mechanism similar to acid-catalyzed ester-bond formation. However, one study showed that the low pH of these reactions resulted in predominantly depurination, thereby resulting in the formation of abasic sites in the oligomers. In this study, we aimed to systematically characterize the nature of the oligomers that resulted in reactions that involved one or more of the canonical ribonucleotides. All the reactions analyzed showed the presence of abasic oligomers, with purine nucleotides being affected the most due to deglycosylation. Even in the reactions that contained nucleotide mixtures, the presence of abasic oligomers was detected, which suggested that information transfer would be severely hampered due to losing the capacity to base pair via H-bonds. Importantly, the stability of the N-glycosidic linkage, under conditions used for dry-wet cycling, was also determined. Results from this study further strengthen the hypothesis that chemical evolution in a pre-RNA World would have been vital for the evolution of informational molecules of an RNA World. This is evident in the high degree of instability displayed by N-glycosidic bonds of canonical purine ribonucleotides under the same geothermal conditions that otherwise readily favors polymerization. Significantly, the resultant product characterization in the reactions concerned underscores the difficulty associated with analyzing complex prebiotically relevant reactions due to inherent limitation of current analytical methods.

Microsporidia are strict obligate intracellular parasites that infect a wide range of eukaryotes including humans and economically important fish and insects. Surviving and flourishing inside another eukaryotic cell is a very specialised lifestyle that requires evolutionary innovation. Genome sequence analyses show that microsporidia have lost most of the genes needed for making primary metabolites, such as amino acids and nucleotides, and also that they have only a limited capacity for making adenosine triphosphate (ATP). Since microsporidia cannot grow and replicate without the enormous amounts of energy and nucleotide building blocks needed for protein, DNA, and RNA biosynthesis, they must have evolved ways of stealing these substrates from the infected host cell. Providing they can do this, genome analyses suggest that microsporidia have the enzyme repertoire needed to use and regenerate the imported nucleotides efficiently. Recent functional studies suggest that a critical innovation for adapting to intracellular life was the acquisition by lateral gene transfer of nucleotide transport (NTT) proteins that are now present in multiple copies in all microsporidian genomes. These proteins are expressed on the parasite surface and allow microsporidia to steal ATP and other purine nucleotides for energy and biosynthesis from their host. However, it remains unclear how other essential metabolites, such as pyrimidine nucleotides, are acquired. Transcriptomic and experimental studies suggest that microsporidia might manipulate host cell metabolism and cell biological processes to promote nucleotide synthesis and to maximise the potential for ATP and nucleotide import. In this review, we summarise recent genomic and functional data relating to how microsporidia exploit their hosts for energy and building blocks needed for growth and nucleic acid metabolism and we identify some remaining outstanding questions.

In all kingdoms of life, cellular replication relies on the presence of nucleosides and nucleotides, the building blocks of nucleic acids and the main source of energy. In bacteria, the availability of metabolites sometimes directly regulates the expression of enzymes and proteins involved in purine salvage, biosynthesis, and uptake through riboswitches. Riboswitches are located in bacterial mRNAs and can control gene expression by conformational changes in response to ligand binding. We have established an inverse reporter gene system in Bacillus subtilis that allows us to monitor riboswitch-controlled gene expression. We used it to investigate the activity of five potential purine riboswitches from Bacillus anthracis in response to different purines and pyrimidines. Furthermore, in vitro studies on the aptamer domains of the riboswitches reveal their variation in guanine binding affinity ranging from namomolar to micromolar. These data do not only provide insight into metabolite sensing but can also aid in engineering artificial cell regulatory systems.

Damaged tissues and cells release intracellular purine nucleotides, which serve as intercellular signaling factors. We previously showed that exogenously added adenine nucleotide (250 μM ATP) suppressed the activation of murine splenic T lymphocytes. Here, we examined the effects of other purine nucleotides/nucleosides on mouse T cell activation. First, we found that pretreatment of mouse spleen T cells with 250 μM GTP, GDP, GMP, guanosine, ITP, IDP, IMP or inosine significantly reduced the release of stimulus-inducible cytokine IL-2. This suppression of IL-2 release was not caused by induction of cell death. Further studies with GTP, ITP, guanosine and inosine showed that pretreatment with these nucleotides/nucleosides also suppressed release of IL-6. However, these nucleotides/nucleosides did not suppress stimulus-induced phosphorylation of ERK1/2, suggesting that the suppression of the release of inflammatory cytokines does not involve inhibition of ERK1/2 signaling. In contrast to ATP pretreatment at the same concentration, guanine or inosine nucleotides/nucleosides did not attenuate the expression of CD25. Our findings indicate that exogenous guanine or inosine nucleotides/nucleosides can suppress inflammatory cytokine release from T cells, and may be promising candidates for use as supplementary agents in the treatment of T cell-mediated immune diseases.

Ribonucleases play an important role in the RNA metabolism which is critical for the localization, stability and function of mature RNA transcripts. More and more ribonucleases were discovered in recent years with the progress of technology. In the present study, we found that the uncharacterized C19orf43, a novel interacting protein of human telomerase RNA (hTR), digested T7 transcribed RNA, total cellular RNA and RNA oligos but not DNA. Thus we named this new RNase as hTRIR (human telomerase RNA interacting RNase). Genetic analysis showed that hTRIR is conserved among eukaryotic species and widely expressed in different cell lines. The RNase activity of hTRIR works in a broad temperature and pH range while divalent cations are not required. The conserved C-terminus of C19orf43 is necessary for its activity. Finally, we found that hTRIR cleaves all four unpaired RNA nucleotides from 5' end or 3' end with higher efficiency for purine bases, which suggested that hTRIR is an exoribonuclease. Taken together, our study showed the first evidence of the novel function of hTRIR in vitro, which provides clue to study the regulatory mechanism of hTR homeostasis in vivo.

Choline is an essential nutrient for cellular metabolism, including the biosynthesis of phospholipids, neurotransmitters, and one-carbon metabolism. A critical step of choline catabolism is the mitochondrial import and synthesis of chorine-derived methyl donors, such as betaine. However, the underlying mechanisms and the biological significance of mitochondrial choline catabolism remain insufficiently understood. Here, we report that a mitochondrial inner-membrane protein SLC25A48 controls mitochondrial choline transport and catabolism in vivo. We demonstrate that SLC25A48 is highly expressed in brown adipose tissue and required for whole-body cold tolerance, thermogenesis, and mitochondrial respiration. Mechanistically, choline uptake into the mitochondrial matrix via SLC25A48 facilitates betaine synthesis and one-carbon metabolism. Importantly, cells lacking SLC25A48 exhibited reduced synthesis of purine nucleotides and failed to initiate the G1-to-S phase transition, thereby leading to cell death. Taken together, the present study identified SLC25A48 as a mitochondrial carrier that mediates choline import and plays a critical role in mitochondrial respiratory capacity, purine nucleotide synthesis, and cell survival.

The recent development of a sequential, high-yielding route to activated pyrimidine nucleotides, under conditions thought to be prebiotic, is an encouraging step toward the greater goal of a plausible prebiotic pathway to RNA and the potential for an RNA world. However, this synthesis has led to a disparity in the methodology available for stepwise construction of the canonical pyrimidine and purine nucleotides. To address this problem, and further explore prebiotically accessible chemical systems, we have developed a high-yielding, aqueous, one-pot, multicomponent reaction that tethers masked-sugar moieties to prebiotically plausible purine precursors. A pH-dependent three-component reaction system has been discovered that utilizes key nucleotide synthons 2-aminooxazole and 5-aminoimidazoles, which allows the first divergent purine/pyrimidine synthesis to be proposed. Due to regiospecific aminoimidazole tethering, the pathway allows N9 purination only, thus suggesting the first prebiotically plausible mechanism for regiospecific N9 purination.

Pharmacologic agents that interfere with nucleotide metabolism constitute an important class of anticancer agents. Recent studies have demonstrated that mTOR complex 1 (mTORC1) inhibitors suppress de novo biosynthesis of pyrimidine and purine nucleotides. Here, we demonstrate that mTORC1 itself is suppressed by drugs that reduce intracellular purine nucleotide pools. Cellular treatment with AG2037, an inhibitor of the purine biosynthetic enzyme GARFT, profoundly inhibits mTORC1 activity via a reduction in the level of GTP-bound Rheb, an obligate upstream activator of mTORC1, because of a reduction in intracellular guanine nucleotides. AG2037 treatment provokes both mTORC1 inhibition and robust tumor growth suppression in mice bearing non-small-cell lung cancer (NSCLC) xenografts. These results indicate that alterations in purine nucleotide availability affect mTORC1 activity and suggest that inhibition of mTORC1 contributes to the therapeutic effects of purine biosynthesis inhibitors.

In vivo imaging of biological processes is an important asset of modern cell biology. Selectively reacting fluorophores herein are an important tool and click chemistry reactions take a large share in these events. 5-Ethynyl-2'-deoxyuridine (EdU) is well known for visualizing DNA replication, but does not show any selectivity for incorporation into DNA. Striving for specific visualization of virus replication, in particular HIV replication, a series of propargylated purine deoxynucleosides were prepared aiming for selective incorporation by HIV reverse transcriptase (RT). We here report on the synthesis and preliminary biological effects (cellular toxicity, HIV inhibitory effects, and feasibility of the click reaction) of these nucleoside analogues.

Nucleic acids that exhibit a high affinity toward noble and transition metal ions have attracted growing attention in the fields of metal ion sensing, toxic metal ion removal, and the construction of functional metal nanostructures. In this study, fluorescent nanoparticles (biodots) were synthesized from DNA, RNA, and RNA nucleotides (AMP, GMP, UMP, and CMP) using a hydrothermal (HT) method, in order to study their metal ion sensing characteristics. The fluorescent properties of biodots differ markedly between those prepared from purine and pyrimidine nucleobases. All biodots demonstrate a high sensitivity to the presence of mercury cations (Hg2+), while biodots prepared from DNA, RNA, and guanosine monophosphate (GMP) are also sensitive to Ag+ and Cu2+ ions, but to a lesser extent. The obtained results show that biodots inherit the metal ion recognition properties of nucleobases, while the nucleobase composition of biodot precursors affects metal ion sensitivity and selectivity. A linear response of biodot fluorescence to Hg2+ concentration in solution was observed for AMP and GMP biodots in the range 0-250 μM, which can be used for the analytic detection of mercury ion concentration. A facile paper strip test was also developed that allows visual detection of mercury ions in solutions.

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.

Cytostasis is the most salient manifestation of the potent antimicrobial activity of nitric oxide (NO), yet the mechanism by which NO disrupts bacterial cell division is unknown. Here, we show that in respiring Escherichia coli, Salmonella, and Bacillus subtilis, NO arrests the first step in division, namely, the GTP-dependent assembly of the bacterial tubulin homolog FtsZ into a cytokinetic ring. FtsZ assembly fails in respiring cells because NO inactivates inosine 5'-monophosphate dehydrogenase in de novo purine nucleotide biosynthesis and quinol oxidases in the electron transport chain, leading to drastic depletion of nucleoside triphosphates, including the GTP needed for the polymerization of FtsZ. Despite inhibiting respiration and dissipating proton motive force, NO does not destroy Z ring formation and only modestly decreases nucleoside triphosphates in glycolytic cells, which obtain much of their ATP by substrate-level phosphorylation and overexpress inosine 5'-monophosphate dehydrogenase. Purine metabolism dictates the susceptibility of early morphogenic steps in cytokinesis to NO toxicity.