Adenosine Nucleotides Research Articles

Synthesis of Terminal Ribose Analogues of Adenosine 5'-Diphosphate Ribose as Probes for the Transient Receptor Potential Cation Channel TRPM2.

TRPM2 (transient receptor potential cation channel, subfamily M, member 2) is a nonselective cation channel involved in the response to oxidative stress and in inflammation. Its role in autoimmune and neurodegenerative diseases makes it an attractive pharmacological target. Binding of the nucleotide adenosine 5′-diphosphate ribose (ADPR) to the cytosolic NUDT9 homology (NUDT9H) domain activates the channel. A detailed understanding of how ADPR interacts with the TRPM2 ligand binding domain is lacking, hampering the rational design of modulators, but the terminal ribose of ADPR is known to be essential for activation. To study its role in more detail, we designed synthetic routes to novel analogues of ADPR and 2′-deoxy-ADPR that were modified only by removal of a single hydroxyl group from the terminal ribose. The ADPR analogues were obtained by coupling nucleoside phosphorimidazolides to deoxysugar phosphates. The corresponding C2″-based analogues proved to be unstable. The C1″- and C3″-ADPR analogues were evaluated electrophysiologically by patch-clamp in TRPM2-expressing HEK293 cells. In addition, a compound with all hydroxyl groups of the terminal ribose blocked as its 1″-β-O-methyl-2″,3″-O-isopropylidene derivative was evaluated. Removal of either C1″ or C3″ hydroxyl groups from ADPR resulted in loss of agonist activity. Both these modifications and blocking all three hydroxyl groups resulted in TRPM2 antagonists. Our results demonstrate the critical role of these hydroxyl groups in channel activation.

Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir.

Remdesivir (GS-5734) is a 1′-cyano-substituted adenosine nucleotide analogue prodrug that shows broad-spectrum antiviral activity against several RNA viruses. This compound is currently under clinical development for the treatment of Ebola virus disease (EVD). While antiviral effects have been demonstrated in cell culture and in non-human primates, the mechanism of action of Ebola virus (EBOV) inhibition for remdesivir remains to be fully elucidated. The EBOV RNA-dependent RNA polymerase (RdRp) complex was recently expressed and purified, enabling biochemical studies with the relevant triphosphate (TP) form of remdesivir and its presumptive target. In this study, we confirmed that remdesivir-TP is able to compete for incorporation with adenosine triphosphate (ATP). Enzyme kinetics revealed that EBOV RdRp and respiratory syncytial virus (RSV) RdRp incorporate ATP and remdesivir-TP with similar efficiencies. The selectivity of ATP against remdesivir-TP is ~4 for EBOV RdRp and ~3 for RSV RdRp. In contrast, purified human mitochondrial RNA polymerase (h-mtRNAP) effectively discriminates against remdesivir-TP with a selectivity value of ~500-fold. For EBOV RdRp, the incorporated inhibitor at position i does not affect the ensuing nucleotide incorporation event at position i+1. For RSV RdRp, we measured a ~6-fold inhibition at position i+1 although RNA synthesis was not terminated. Chain termination was in both cases delayed and was seen predominantly at position i+5. This pattern is specific to remdesivir-TP and its 1′-cyano modification. Compounds with modifications at the 2′-position show different patterns of inhibition. While 2′-C-methyl-ATP is not incorporated, ara-ATP acts as a non-obligate chain terminator and prevents nucleotide incorporation at position i+1. Taken together, our biochemical data indicate that the major contribution to EBOV RNA synthesis inhibition by remdesivir can be ascribed to delayed chain termination. The long distance of five residues between the incorporated nucleotide analogue and its inhibitory effect warrant further investigation.

Hypoxia Inducible Factor Activation Prevents Renal Mitochondria Dysfunction and Improves Cortical Oxygenation in Type 1 Diabetic Mice

Hypoxia‐inducible factors (HIFs) are transcription factors that are activated during hypoxia in order to regulate a hypoxic gene response. During normoxia, prolyl hydroxylase domain‐containing protein 2 (PHD2) is one of three isoforms that acts on HIF as a first step towards its degradation and its knockdown consequently results in HIF activation. Previous studies have demonstrated that diabetes induces mitochondria dysfunction resulting in renal cortical hypoxia, yet studies indicate that hyperglycemia impairs HIF stabilization resulting in suboptimal activation and an inability to correct renal hypoxia. In this study, we hypothesize that increased HIF‐activation resulting from PHD2 knockdown, prevents mitochondria dysfunction and improves renal oxygenation in type 1 diabetic mice.Balb/c mice from four groups (Wild type normoglycemic (n=16), wild type diabetic (n=10), PHD2+/− normoglycemic (n=16) and PHD2+/− diabetic (n=11)) were placed in metabolic cages for 24 h urine collection followed by conscious determination glomerular filtration rate (GFR), using a single bolus injection of FITC‐inulin. Diabetes was induced by a low dose injection of strepzotocin (i.p.) for 5 consecutive days and diabetes duration was 5–6 weeks. Renal cortical oxygenation was measured with Clark electrodes and kidney mitochondria function, including mitochondria proton leak, was assessed by high resolution respirometry. Urinary excretion of albumin and thiobarbituric acid reactive species were measured and corrected for 24 h urine volume.Diabetes resulted in increased GFR in both groups compared to normoglycemic mice. Wild type diabetic mice presented with increased total mitochondrial leak respiration compared to normoglycemic mice, which was prevented in the PHD2+/− mice. Interestingly, regulated leak, defined as mitochondrial proton leak regulated by uncoupling proteins and adenosine nucleotide translocase, was increased in diabetic PHD2+/− only, while unregulated leak, defined as total leak minus regulated leak, was increased in wild type diabetic mice. Both diabetic groups displayed increased albuminuria and thiobarbituric acid reactive species compared to normoglycemic control mice.In conclusion, activation of HIF by reducing PHD2 expression prevents mitochondria dysfunction and renal cortical hypoxia in type 1 diabetic mice. This could imply a novel target for the PHD inhibitors currently in clinical trials.Support or Funding InformationThis study was supported financially by the Swedish Research Council, the Swedish Diabetes Foundation and the Family Ernfors Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hydrogen exchange reveals Hsp104 architecture, structural dynamics, and energetics in physiological solution

Hsp104 is a large AAA+ molecular machine that can rescue proteins trapped in amorphous aggregates and stable amyloids by drawing substrate protein into its central pore. Recent cryo-EM studies image Hsp104 at high resolution. We used hydrogen exchange mass spectrometry analysis (HX MS) to resolve and characterize all of the functionally active and inactive elements of Hsp104, many not accessible to cryo-EM. At a global level, HX MS confirms the one noncanonical interprotomer interface in the Hsp104 hexamer as a marker for the spiraled conformation revealed by cryo-EM and measures its fast conformational cycling under ATP hydrolysis. Other findings enable reinterpretation of the apparent variability of the regulatory middle domain. With respect to detailed mechanism, HX MS determines the response of each Hsp104 structural element to the different bound adenosine nucleotides (ADP, ATP, AMPPNP, and ATPγS). They are distinguished most sensitively by the two Walker A nucleotide-binding segments. Binding of the ATP analog, ATPγS, tightly restructures the Walker A segments and drives the global open-to-closed/extended transition. The global transition carries part of the ATP/ATPγS-binding energy to the somewhat distant central pore. The pore constricts and the tyrosine and other pore-related loops become more tightly structured, which seems to reflect the energy-requiring directional pull that translocates the substrate protein. ATP hydrolysis to ADP allows Hsp104 to relax back to its lowest energy open state ready to restart the cycle.

Structural basis for RNA translocation by DEAH-box ATPases.

DEAH-box adenosine triphosphatases (ATPases) play a crucial role in the spliceosome-mediated excision of pre-mRNA introns. Recent spliceosomal cryo-EM structures suggest that these proteins utilize translocation to apply forces on ssRNAs rather than direct RNA duplex unwinding to ensure global rearrangements. By solving the crystal structure of Prp22 in different adenosine nucleotide-free states, we identified two missing conformational snapshots of genuine DEAH-box ATPases that help to unravel the molecular mechanism of translocation for this protein family. The intrinsic mobility of the RecA2 domain in the absence of adenosine di- or triphosphate (ADP/ATP) and RNA enables DEAH-box ATPases to adopt different open conformations of the helicase core. The presence of RNA suppresses this mobility and stabilizes one defined open conformation when no adenosine nucleotide is bound. A comparison of this novel conformation with the ATP-bound state of Prp43 reveals that these ATPases cycle between closed and open conformations of the helicase core, which accommodate either a four- or five-nucleotide stack in the RNA-binding tunnel, respectively. The continuous repetition of these states enables these proteins to translocate in 3′-5′ direction along an ssRNA with a step-size of one RNA nucleotide per hydrolyzed ATP. This ATP-driven motor function is maintained by a serine in the conserved motif V that senses the catalytic state and accordingly positions the RecA2 domain.

Several Polyphosphate Kinase 2 Enzymes Catalyse the Production of Adenosine 5'-Polyphosphates.

Polyphosphate kinases (PPKs) are involved in many metabolic processes; enzymes of the second family (PPK2) are responsible for nucleotide synthesis fuelled by the consumption of inorganic polyphosphate. They catalyse the phosphorylation of nucleotides with various numbers of phosphate residues, such as monophosphates or diphosphates. Hence, these enzymes are promising candidates for cofactor regeneration systems. Besides adenosine 5'-triphosphate, PPK2s also catalyse the synthesis of highly phosphorylated nucleotides in vitro, as shown here for adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate. These unusually phosphorylated adenosine 5'-polyphosphates add up to 50 % of the whole adenosine nucleotides in the assay. The two new products were chemically synthesised to serve as standards and compared with the two enzymatically produced compounds by high-performance ion chromatography and 31 P NMR analysis. This study shows that PPK2s are highly suitable for biocatalytic synthesis of different phosphorylated nucleotides.

The Cfd1 Subunit of the Nbp35-Cfd1 Iron Sulfur Cluster Scaffolding Complex Controls Nucleotide Binding.

The cytosolic iron sulfur cluster assembly (CIA) scaffold biosynthesizes iron sulfur cluster cofactors for enzymes residing in the cytosol and the nucleus. In fungi and animals, it comprises two homologous ATPases, called Nbp35 and Cfd1 in yeast, which can form homodimeric and heterodimeric complexes. Both proteins are required for CIA function, but their individual roles are not well understood. Here we investigate the nucleotide affinity of each form of the scaffold for ATP and ADP to reveal any differences that could shed light on the functions of the different oligomeric forms of the protein or any distinct roles of the individual subunits. All forms of the CIA scaffold are specific for adenosine nucleotides and not guanosine nucleotides. Although the Cfd1 homodimer has no detectable ATPase activity, it binds ATP with an affinity comparable to that of the hydrolysis competent forms, Nbp352 and Nbp35-Cfd1. Titrations to determine the number of nucleotide binding sites combined with site-directed mutagenesis demonstrate that the nucleotide must bind to the Cfd1 subunit of the heterodimer before it can bind to Nbp35 and that the Cfd1 subunit is hydrolysis competent when bound to Nbp35 in the heterodimer. Altogether, our work reveals the distinct roles of the Nbp35 and Cfd1 subunits in their heterodimeric complex. Cfd1 controls nucleotide binding, and the Nbp35 subunit is required to activate nucleotide hydrolysis.

Multiple covalent fluorescence labeling of eukaryotic mRNA at the poly(A) tail enhances translation and can be performed in living cells.

Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length. These mechanisms play crucial roles in the dynamics of cell polarization and embryonic development. Furthermore, mRNAs are emerging therapeutics and chemical alterations to increase their translational efficiency are highly sought after. We show that yeast poly(A) polymerase can be used to install multiple azido-modified adenosine nucleotides to luciferase and eGFP-mRNAs. These mRNAs can be efficiently reacted in a bioorthogonal click reaction with fluorescent reporters without degradation and without sequence alterations in their coding or untranslated regions. Importantly, the modifications in the poly(A) tail impact positively on the translational efficiency of reporter-mRNAs in vitro and in cells. Therefore, covalent fluorescent labeling at the poly(A) tail presents a new way to increase the amount of reporter protein from exogenous mRNA and to label genetically unaltered and translationally active mRNAs.

The Effects of Oxygenation on Ex Vivo Kidneys Undergoing Hypothermic Machine Perfusion.

Supplemental oxygenation of the standard hypothermic machine perfusion (HMP) circuit has the potential to invoke favorable changes in metabolism, optimizing cadaveric organs before transplantation. Eight pairs of porcine kidneys underwent 18 hours of either oxygenated (HMP/O2) or aerated (HMP/Air) HMP in a paired donation after circulatory death model of transplantation. Circulating perfusion fluid was supplemented with the metabolic tracer universally labeled glucose.Perfusate, end-point renal cortex, and medulla samples underwent metabolomic analysis using 1-dimension and 2-dimension nuclear magnetic resonance experiments in addition to gas chromatography-mass spectrometry. Analysis of C-labeled metabolic products was combined with adenosine nucleotide levels and differences in tissue architecture. Metabolomic analysis revealed significantly higher concentrations of universally labeled lactate in the cortex of HMP/Air versus HMP/O2 kidneys (0.056 mM vs 0.026 mM, P < 0.05). Conversely, newly synthesized [4,5-C] glutamate concentrations were higher in the cortex of HMP/O2 kidneys inferring relative increases in tricarboxylic acid cycle activity versus HMP/Air kidneys (0.013 mmol/L vs 0.003 mmol/L, P < 0.05). This was associated with greater amounts of adenoside triphosphate in the cortex HMP/O2 versus HMP/Air kidneys (19.8 mmol/mg protein vs 2.8 mmol/mg protein, P < 0.05). Improved flow dynamics and favorable ultrastructural features were also observed in HMP/O2 kidneys. There were no differences in thiobarbituric acid reactive substances and reduced glutathione levels, tissue markers of oxidative stress, between groups. The supplementation of perfusion fluid with high-concentration oxygen (95%) results in a greater degree of aerobic metabolism versus aeration (21%) in the nonphysiological environment of HMP, with reciprocal changes in adenoside triphosphate levels.

Effect of the sulfur atom on S2 and S4 positions of the uracil ring in different DNA:RNA hybrid microhelixes with three nucleotide base pairs

The effect of the sulphur atom on the uracil ring was analyzed in different DNA:RNA microhelixes with three nucleotide base-pairs, including uridine, 2-thiouridine, 4-thiouridine, 2,4-dithiouridine, cytidine, adenosine and guanosine. Distinct backbone and helical parameters were optimized at different density functional (DFT) levels. The Watson-Crick pair with 2-thiouridine appears weaker than with uridine, but its interaction with water molecules appears easier. Two types of microhelixes were found, depending on the H-bond of H2' hydroxyl atom: A-type appears with the ribose ring in 3 E-envelope C3' -endo, and B-type in 2 E-envelope C2' -endo. B-type is less common but it is more stable and with higher dipole-moment. The sulphur atoms significantly increase the dipole-moment of the microhelix, as well as the rise and propeller twist parameters. Simulations with four Na atoms H-bonded to the phosphate groups, and further hydration with explicit water molecules were carried out. A re-definition of the numerical value calculation of several base-pair and base-stacking parameters is suggested.

Protective Effects of Apocynum venetum Against Pirarubicin-Induced Cardiotoxicity.

Pirarubicin (THP) is an anthracycline antibiotic, frequently used for the treatment of various human cancers. Unfortunately, the clinical effectiveness of THP is limited by its dose-related cardiotoxicity. Apocynum leaf extract is an extract of the dried leaves of Apocynum venetum L. (a member of the Apocynaceae family, AVLE) that has many positive effects on the cardiovascular system and is widely consumed as tea in China. In this study we established a cardiactoxicity rat model, which showed that pretreatment with AVLE attenuated THP-induced myocardial histopathological injury, electrocardiogram abnormalities, and cardiac dysfunction. AVLE also significantly reduced serum levels of malondialdehyde (MDA), brain natriuretic peptide (BNP), creatine kinase (CK-MB), cardiac troponin (CTnT), and lactate dehydrogenase (LDH); and increased serum superoxide dismutase (SOD) levels. Treatment with AVLE or dexrazoxane (DZR) resulted in an increase Cytochrome C (cytc) in the mitochondria and reduced Cytc and cleaved-caspase-3 levels () in cytoplasm. We also found that AVLE significantly reduced voltage-dependent anion channel 1 (VDAC1), adenosine nucleotide transporter 1 (ANT1), and cyclophilin D (CYPD) mRNA expression (). Furthermore, AVLE appeared to exert therapeutic effects in a dose-dependent manner. Our study suggests the anti-oxidant and anti-apoptotic properties of AVLE may be responsible for the observed cardioprotective effects.

Single-Molecule Kinetic Fingerprinting for the Ultrasensitive Detection of Small Molecules with Aptasensors.

Aptamers have emerged as promising molecular tools for small-molecule analyte sensing. However, the performance of such aptasensors is generally limited by leakage since it has been difficult to completely suppress signal in the absence of analyte, resulting in a compromise between sensitivity and specificity. Here, we describe a methodology for the ultrasensitive detection of analytes combining aptasensors with single-molecule kinetic fingerprinting. A short, fluorescently labeled DNA probe is utilized to detect the structural changes upon ligand binding to the designed hairpin-shaped aptasensor probe. The Poisson statistics of binding and dissociation events of the DNA probe to single surface-immobilized aptasensor molecules is monitored by total internal reflection fluorescence microscopy, permitting the high-accuracy discrimination of the ligand bound and ligand-free states, resulting in zero background. The programmable dynamics of the hairpin enables fine-tuning of the hybridization kinetics of the fluorescent probe, rendering the acquisition time sufficiently flexible to optimize discrimination. Remarkable detection limits are achieved for a diverse set of analytes when spiked into chicken meat extract: the nucleotide adenosine (0.3 pM), the insecticide acetamiprid (0.35 pM), and the dioxin-like toxin PCB-77 (0.72 pM), which is superior to recently reported aptasensors. Our generalizable method significantly improves the performance of aptasensors, with the potential to extend to other molecular biomarkers.

Gene2vec: gene subsequence embedding for prediction of mammalian N6-methyladenosine sites from mRNA.

N6-Methyladenosine (m6A) refers to methylation modification of the adenosine nucleotide acid at the nitrogen-6 position. Many conventional computational methods for identifying N6-methyladenosine sites are limited by the small amount of data available. Taking advantage of the thousands of m6A sites detected by high-throughput sequencing, it is now possible to discover the characteristics of m6A sequences using deep learning techniques. To the best of our knowledge, our work is the first attempt to use word embedding and deep neural networks for m6A prediction from mRNA sequences. Using four deep neural networks, we developed a model inferred from a larger sequence shifting window that can predict m6A accurately and robustly. Four prediction schemes were built with various RNA sequence representations and optimized convolutional neural networks. The soft voting results from the four deep networks were shown to outperform all of the state-of-the-art methods. We evaluated these predictors mentioned above on a rigorous independent test data set and proved that our proposed method outperforms the state-of-the-art predictors. The training, independent, and cross-species testing data sets are much larger than in previous studies, which could help to avoid the problem of overfitting. Furthermore, an online prediction web server implementing the four proposed predictors has been built and is available at http://server.malab.cn/Gene2vec/.

PO-053 Plasma Metabolic Profiles of Elite Rowers Response to the Early Phase of Altitude Training Based on LC-MS

Objective When arriving at altitude, because of the dry air, strong ultraviolet and especially the low oxygen et.al., athletes who living on the plain always exhibited kinds of stress reactions such as hyperventilation, tachycardia, dizziness, sleep-disorder, gastrointestinal disturbance and cognitive impairment in the early phase. Except these stress reactions, some studies reported the carbohydrate and lipid metabolism show significant change. Acute exposure to hypoxia could strengthen the glycolysis and suppress hepatic gluconeogenesis. The expression of some genes involved in lipid oxidation was down-regulated. However, it’s also found the increased activity of some enzymes took part in aerobic metabolism in muscle after long term acclimatization to altitude. These inconsistent conclusions make us confusion. Train load is an important factor influence the stress reactions which isn’t like as altitude travel for normal people. What would happen in the body of athletes when altitude training? We still know very little. Metabonomic give us a new tool to understand the whole map of body’s reaction to altitude training. In this pilot study, we aimed to explore the plasma profiles changes of elite rowers in the early two-weeks training at 2300m plateau using metabolic tool based on LC-MS.&#x0D; Methods Sixteen male elite rowers(age 25.56±3.44y, height 189.06±5.37cm, weight 82.81±12.25kg, training years 10.31±2.52y) from China National team took part in this research. This altitude training camp was organized in the preparation period of a new season. It lasted six weeks. The acute phase (AP) which was the first three days after arriving at 2300m altitude base was composed by regenerative training and low-intensity aerobic training sessions. During the next ten days (chronic phase, CP), the intensity of most sessions including three low-weight resistance training sets were low to middle aerobic. After resting half day, 5 ml venous blood was collected into heparin anticoagulant tubes and then centrifuged at 8000rpm in 4℃ lasting 15 minutes to separate plasma in the 4th day and 14th morning. Plasma was stored in -80℃ to measure metabolic profile by LC-MS. The data was performed feature extraction and preprocessed with Compound Discoverer 2.0 software (Thermo), and then normalized and edited into two-dimensional data matrix by excel 2010 software, including Retention time(RT), Compound Molecular Weight (compMW), Observations(samples) and peak intensity. The feature data after editing were performed Multivariate Analysis (MVA) using SIMCA-P software (Umetrics AB, Umea, Sweden).&#x0D; Results Compared with pre-altitude, 26 and 30 features at (ESI+) ion mode, 57 and 49 features at (ESI-) ion mode were found in the AP and CP respectively. Compared AP with CP, there were 46 features at (ESI+) ion mode and 67 features at (ESI-) ion mode. In AP, plasma benzamide and indole-3-acetaldehyde increased 35.16 fold and 16.54 fold respectively. Plasma phenethylamine, phenol, indole, piperidine, leucine, 4-chlorbenzoic acid and benzoic acid increased 4.55 to 8.22 fold compared with pre-altitude. Top three decreased features were dibutyl sebacate, arbinosylhypoxanthine and cholesterol hydrogen sulfate which decreased 1.76 to 3.85 fold in AP. After a longer adaption, in the 14th day of altitude training, plasma benzamide, indole-3-acetaldehyde, phenethylamine, indole, 4-chlorbenzoic acid and benzoic acid still increased but the amplitude reduced compared with the 4th day. Ingenuity Pathways Analysis (IPA) suggested that the top 5 canonical pathways were tyrosine biosynthesis Ⅳ, phenylalanine degradationⅠ, protein kinase A signaling, tRNA charging and phenylalanine degradation Ⅳ in AP. In CP, the top 5 canonical pathways were tRNA charging, urate biosynthesis, tryptophan degradation Ⅹ, guanosine nucleotides degradation Ⅲ and adenosine nucleotides degradationⅡ.&#x0D; Conclusions In this pilot study, we found 83 and 79 plasma feature in acute and chronic phase respectively. Considering the sharp elevation, plasma benzamide and indole-3-acetaldehyde which involved in the regulation of energy metabolism of brain may be the sensitive makers in acute adaption to altitude for athletes. As the extension of time, the increased amplitude came down in the 14th day. It suggested that the energy metabolism of brain may take significant change. Central nerve system should be paid more attention during altitude training especially in the early phase.

Calcium-regulated mitochondrial ATP-Mg/Pi carriers evolved from a fusion of an EF-hand regulatory domain with a mitochondrial ADP/ATP carrier-like domain.

The mitochondrial ATP‐Mg/Pi carrier is responsible for the calcium‐dependent regulation of adenosine nucleotide concentrations in the mitochondrial matrix, which allows mitochondria to respond to changing energy requirements of the cell. The carrier is expressed in mitochondria of fungi, plants and animals and belongs to the family of mitochondrial carriers. The carrier is unusual as it consists of three separate domains: (i) an N‐terminal regulatory domain with four calcium‐binding EF‐hands similar to calmodulin, (ii) a loop domain containing an amphipathic α‐helix and (iii) a mitochondrial carrier domain related to the mitochondrial ADP/ATP carrier. This striking example of three domains coming together from different origins to provide new functions represents an interesting quirk of evolution. In this review, we outline how the carrier was identified and how its physiological role was established with a focus on human isoforms. We exploit the sequence and structural information of the domains to explore the similarities and differences to their closest counterparts; mitochondrial ADP/ATP carriers and proteins with four EF‐hands. We discuss how their combined function has led to a mechanism for calcium‐regulated transport of adenosine nucleotides. Finally, we compare the ATP‐Mg/Pi carrier with the mitochondrial aspartate/glutamate carrier, the only other mitochondrial carrier regulated by calcium, and we will argue that they have arisen by convergent rather than divergent evolution. © 2018 The Authors. IUBMB Life published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 70(12):1222–1232, 2018

Ultrafast electronic deactivation of UV-excited adenine and its ribo- and deoxyribonucleosides and -nucleotides: A comparative study

The high stability of the DNA bases after UV photoexcitation is generally ascribed to their intrinsic ability to dissipate the supplied electronic energy on ultrafast time scales. The sugar-phosphate backbone of the related nucleosides and nucleotides may modulate the efficiency of these relaxation processes by hydrogen bonding and/or intramolecular energy transfer. To elucidate these effects, we performed a comparative study of the excited-state lifetimes of 9H-adenine, its nucleosides adenosine and deoxyadenosine, and its nucleotides adenosine monophosphate and deoxyadenosine monophosphate by transient electronic absorption spectroscopy under otherwise constant experimental conditions. All five adenine derivatives were found to exhibit virtually identical spectro-temporal behavior with uniform excited-state lifetimes of τ1=0.19±0.03 ps and τ2=0.45±0.05 ps. The results support a common deactivation mechanism within the adenine unit that leaves the DNA sugar-phosphate backbone groups idle.

A tale of not-so-pure tails

RNA Metabolism The poly(A) tail of mRNA has been thought to be a pure stretch of adenosine nucleotides with little informational content except for length. Lim et al. identified enzymes that can decorate poly(A) tails with non-A nucleotides. The noncanonical poly(A) polymerases, TENT4A and TENT4B, incorporate intermittent non-A residues (G, U, or C) with a preference for guanosine, which results in a heterogenous poly(A) tail. Deadenylases trim poly(A) tails to initiate mRNA degradation but stall at the non-A residues. In effect, the not-so-pure tail stabilizes mRNAs by slowing down deadenylation. Science , this issue p. [701][1] [1]: /lookup/doi/10.1126/science.aam5794

Modulatory effect of eugenol on arginase, nucleotidase, and adenosine deaminase activities of platelets in a carrageenan-induced arthritis rat model: A possible anti-arthritic mechanism of eugenol

This study investigated the effect of eugenol on arginase, nucleotidase and adenosine deaminase activities in platelets of carrageenan-induced arthritic rat model to explain a possible anti-arthritic mechanism of eugenol. Fifty adult female rats (140–250 g) were divided into ten (10) groups (n = 5). Group I received oral administration of corn oil, group II received 2.50 mg/kg of eugenol, group III and IV rats received oral administration of 5.0 and 10.0 mg/kg of eugenol respectively, group V received 0.20 mg/kg of dexamethasone orally, group VI rats was injected with 1% carrageenan (arthritic rats) and received saline solution orally (arthritic control rat group), group VII, VIII and IX: arthritic rats received 2.50, 5.0 or 10 mg/kg of eugenol orally respectively, group X: arthritic rats was administered with 0.20 mg/kg of dexamethasone orally. The animals were treated for 21 days, thereafter, tibiofemoral histological examination, thiobabituric acid reactive substances level, arginase, nucleoside triphosphate diphosphohydrolase, 5´-nucleotidase and adenosine deaminase activities were assessed. Tibiofemoral histological examination result showed that infiltration of inflammatory cells was significantly decreased with an increase in eugenol dose. Activities of arginase, adenosine triphosphate and adenosine monophosphate hydrolyses were significantly decreased while adenosine diphosphate hydrolysis and adenosine deaminase activities were significantly increased in arthritic rat groups administered with different doses of eugenol. Therefore, eugenol might be a natural complement and alternative promising anti-arthritic agent. These possible anti-arthritic mechanisms may be partly through the modulation of arginase and adenosine nucleotides hydrolyzing enzyme activities as well as the antioxidative action of eugenol.

Mixed tailing by TENT4A and TENT4B shields mRNA from rapid deadenylation.

RNA tails play integral roles in the regulation of messenger RNA (mRNA) translation and decay. Guanylation of the poly(A) tail was discovered recently, yet the enzymology and function remain obscure. Here we identify TENT4A (PAPD7) and TENT4B (PAPD5) as the enzymes responsible for mRNA guanylation. Purified TENT4 proteins generate a mixed poly(A) tail with intermittent non-adenosine residues, the most common of which is guanosine. A single guanosine residue is sufficient to impede the deadenylase CCR4-NOT complex, which trims the tail and exposes guanosine at the 3' end. Consistently, depletion of TENT4A and TENT4B leads to a decrease in mRNA half-life and abundance in cells. Thus, TENT4A and TENT4B produce a mixed tail that shields mRNA from rapid deadenylation. Our study unveils the role of mixed tailing and expands the complexity of posttranscriptional gene regulation.

Structural basis for ADP-dependent glucokinase inhibition by 8-bromo–substituted adenosine nucleotide

In higher eukaryotes, several ATP-utilizing enzymes known as hexokinases activate glucose in the glycolysis pathway by phosphorylation to glucose 6-phosphate. In contrast to canonical hexokinases, which use ATP, ADP-dependent glucokinase (ADPGK) catalyzes noncanonical phosphorylation of glucose to glucose 6-phosphate using ADP as a phosphate donor. Initially discovered in Archaea, the human homolog of ADPGK was described only recently. ADPGK's involvement in modified bioenergetics of activated T cells has been postulated, and elevated ADPGK expression has been reported in various cancer tissues. However, the physiological role of ADPGK is still poorly understood, and effective ADPGK inhibitors still await discovery. Here, we show that 8-bromo-substituted adenosine nucleotide inhibits human ADPGK. By solving the crystal structure of archaeal ADPGK in complex with 8-bromoadenosine phosphate (8-Br-AMP) at 1.81 Å resolution, we identified the mechanism of inhibition. We observed that 8-Br-AMP is a competitive inhibitor of ADPGK and that the bromine substitution induces marked structural changes within the protein's active site by engaging crucial catalytic residues. The results obtained using the Jurkat model of activated human T cells suggest its moderate activity in a cellular setting. We propose that our structural insights provide a critical basis for rational development of novel ADPGK inhibitors.