Nuclear Location of a Diadenosine 5′,5′”-P1,P4Tetraphosphate (Ap4A) Hydrolase in Tomato Cells Grown in Suspension Cultures1

Diadenosine 5′,5′”-P¹,P⁴-tetraphosphate (Ap₄A) cleaving enzymes are assumed to regulate intracellular levels of Ap₄A, a compound known to affect cell proliferation and stress responses. From plants an Ap₄A hydrolase was recently purified using tomato cells grown in suspension. It was partially sequenced and a peptide antibody was prepared (Feussner et al., 1996). Using this polyclonal monospecific antibody, an abundant nuclear location of Ap₄A hydrolase in 4-day-old cells of atomato cell suspension culture is demonstrated here by means of immunocytochemical techniques using FITC (fluorescein-5-isothiocyanate) labeled secondary antibodies. The microscopic analysis of the occurrence of Ap₄A hydrolase performed for different stages of the cell cycle visualized by parallel DAPI (4,6-diamidino-2-phenylindole) staining revealed that the protein accumulates within nuclei of cells in the interphase, but is absent in the nucleus as well as cytoplasm during all stages of mitosis. This first intracellular localization of an Ap₄A degrading enzyme within the nucleus and its pattern of appearance during the cell cycle is discussed in relation to the suggested role of Ap₄A in triggering DNA synthesis and cell proliferation.     

Enzymatic characteristics of an ApaH-like phosphatase,PrpA, and a diadenosine tetraphosphate hydrolase,ApaH, from Myxococcus xanthus

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap₄A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards Ap_nA and ATP. In the presence of Mn²⁺, PrpA hydrolyzed Ap₄A into AMP and ATP, whereas in the presence of Co²⁺ PrpA hydrolyzed Ap₄A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards Ap_nA and ATP. Mn²⁺ was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co²⁺ was required for Ap_nA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an Ap_nA hydrolase, respectively.     

Synthesis and Use of a Chromogenic Substrate Analog for Ap4A Catabolic Enzymes

ATP was coupled with 5-bromo-4-chloro-3-indolyl phosphate using a water-soluble carbodiimide to yield 5-bromo-4-chloro-3-indolyl tetraphospho-5′-adenosine (BClp₄A) which is an analog of diadenosine 5′,5′″-P¹,P⁴-tetraphosphate (Ap₄A). BClp₄A is a chromogenic substrate for three different types of Ap₄A catabolic enzyme in alkaline phosphatase-coupled reactions. Ap₄A phosphorylase I from Saccharomyces cerevisiae was used as a model enzyme to demonstrate that BClp₄A stains for enzymic activity in polyacrylamide gels under nondenaturing conditions. A yeast colony assay was developed to detect Ap₄A phosphorylase I activity in situ using BClp₄A as a chromogenic substrate. Ap₄A phosphorylase I was assayed in situ in yeast transformed with a multicopy plasmid containing APA1, the gene encoding Ap₄A phosphorylase I. BClp₄A should facilitate screening of genomic or cDNA libraries for genes encoding Ap₄A catabolic enzymes.     

Synthesis of hybrid bisnucleoside 5′, 5‴ — P1, P4 — tetraphosphates by aminoacyl — tRNA synthetases

Aminoacyl tRNA synthetases, by means of a back reaction, are able to synthesize certain 5, 5 - P¹, P⁴ — bisnucleoside tetraphosphates of biological importance, such as ANA. Here it is shown that HisRS and TrpRS (Bacillus stearothermophilus) and AlaRS (E. coli) also synthesize the hybrid compounds Ap₄G, Ap₄C, and Ap₄U. GInRS (E. coli) is unable to synthesize any of the above compounds.AlaRS synthesizes Ap₄U very poorly, and Ap₄C and Ap₄G almost as effectively as Ap₄A. HisRS and TrpRS synthesize Ap₄G, Ap₄U and Ap₃U quite effectively, and Ap₄C very poorly. The fact that hybrid bisnucleoside tetraphosphates can be made by the same enzymes, and at rates comparable to Ap₄A, suggests that these compounds may also occur in vivo.Abbreviations HPLC high pressure liquid chromatography - PEI polyethyleneimine - RS aminoacyl tRNA synthetase     

Purification of tubulin from rat pancreas

Diadenosine triphosphate (Ap₃A) has been identified and quantified in human platelets using a coupled enzymatic assay specific for Ap₃A, after fractionation of acidic extracts with high-performence liquid chromatography. Upon thrombin-induced aggregation, Ap₃A is released together with the homologue diadenosine tetraphosphate (Ap₄A).Extracts of human platelets do also contain enzymatic activities that degrade diadenosine tetraphosphate as well as diadenosine triphosphate. These enzymes, however, are not released during thrombin-induced aggregation of the platelets.     

Regulation of Ryanodine Receptor Calcium Release Channels by Diadenosine Polyphosphates

Abstract: [³H]Ryanodine binding to, as well as functions of, ryanodine receptor intracellular Ca²⁺ release channel complexes are modulated by several adenosine-based compounds. In this study, we determined the effects of endogenous compounds termed diadenosine polyphosphates (Ap_nAs; n = 2–6 phosphate groups) on [³H]ryanodine binding to membranes prepared from rat brain and skeletal and cardiac muscle. Under low ionic strength buffer conditions, [³H]ryanodine binding to brain membranes was significantly increased by 171% with 333 µMP¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) and by 209% with the same concentration of the metabolism-resistant ATP analogue βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) compared with control values for [³H]ryanodine binding of 9.6 ± 1.8 fmol/mg of protein. Dose-related increases in [³H]ryanodine binding were observed for all five Ap_nAs tested [P¹,P²-di(adenosine-5′) pyrophosphate (Ap₂A), P¹,P³-di(adenosine-5′) triphosphate (Ap₃A), P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A), Ap₅A, and P¹,P⁶-di(adenosine-5′) hexaphosphate (Ap₆A)] as well as AMP-PCP; oxidized salts of Ap_nAs stimulated [³H]ryanodine binding to a greater degree than did nonoxidized Ap_nAs. The apparent rank order for the capacity of these agents to increase [³H]-ryanodine binding was oxidized Ap₄A = oxidized Ap₅A > oxidized Ap₃A > Ap₆A > AMP-PCP > Ap₅A > Ap₂A. Addition of the approximate EC₅₀ dose of oxidized Ap₄A (37 µM) increased the affinity (K_D) of ryanodine receptors from 34 ± 7 to 12 ± 2 nM; the apparent binding site density (B_max) was not significantly different from control values of 107 ± 33 fmol/mg of protein. Increases in [³H]-ryanodine binding by either oxidized Ap₄A or nonoxidized Ap₅A were not further enhanced by coincubation with AMP-PCP, which suggests a similar site of action for the Ap_nAs and AMP-PCP. [³H]Ryanodine binding to skeletal and cardiac muscle membranes was enhanced by addition of oxidized Ap₄A, Ap₅A, and AMP-PCP. Oxidized Ap₄A increased the specific binding by ninefold in skeletal muscle and by threefold in cardiac muscle. These results suggest that Ap_nAs, at physiologically relevant concentrations, may serve as endogenous modulators of ryanodine receptor-gated Ca²⁺ release channels.     

Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Chemical proteomics reveals interactors of the alarmone diadenosine triphosphate in the cancer cell line H1299†

Intracellular dinucleoside polyphosphates (Np_nNs) have been known for decades but the functional role remains enigmatic. Diadenosine triphosphate (Ap₃A) is one of the most prominent examples, and its intercellular concentration was shown to increase upon cellular stress. By employment of previously reported Ap₃A-based photoaffinity-labeling probes (PALPs) in chemical proteomics, we investigated the Ap₃A interactome in the human lung carcinoma cell line H1299. The cell line is deficient of the fragile histidine triade (Fhit) protein, a hydrolase of Ap₃A and tumor suppressor. Overall, the number of identified potential interaction partners was significantly lower than in the previously investigated HEK293T cell line. Gene ontology term analysis revealed that the identified proteins participate in similar pathways as for HEK293T, but the percentage of proteins involved in RNA-related processes is higher for H1299. The obtained results highlight similarities and differences of the Ap₃A interaction network in different cell lines and give further indications regarding the importance of the presence of Fhit.     

Diadenosine 5′,5‴-P1,P4-tetraphosphate- (Ap4A-) mediated stimulation of DNA synthesis in extracts from Xenopus laevis oocytes

Diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A) stimulates DNA synthesis in Xenopus laevis oocytes in the presence of activated DNA as template. Besides Ap₄A, other analogues such as Ap₃A, ATP and other derivatives are able to stimulate DNA polymerase activity. The effect of Ap₄A on DNA synthesis is observed with poly(dT) and poly(dT)-poly(dA) as templates, while no effect is found with poly(dA)(dT)_12–18 and poly(dC)(dG)_12–18. In the presence of a poly(dT) template, the oocyte extract is able to utilize Ap₄A as primer and to form a covalent bond between this dinucleotide and the nascent poly(dA) chain. An Ap₄A-binding protein present in the system has been purified and separated from DNA polymerase α-primase after phosphocellulose chromatography. After this separation, Ap₄A is no longer able to stimulate the polymerase activity, or to be utilized as primer by DNA polymerase α-primase.     

A Convenient Method for the Synthesis of ATP and Ap4A

Abstract

A bifunctional phosphorylating reagent, O-8-(5-chloroquinolyl) S-phenyl phosphorothioate (1) was employed for the synthesis of adenosine 5′-triphosphate (ATP) and diadenosine 5′-tetraphosphate (Ap₄A) from adenosine 5′-phosphate (AMP) on a large scale.     

Structure determination of the adduct formed between the signal nucleotide diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) and cis-[Pt(NH3)2Cl2

Diadenosine 5′,5?-P₁,P₄-tetraphosphate (Ap₄A), an intracellular regulatory nucleotide, has been found to react with the antitumor drug cis-diamminedichloroplatinum(II) and its aqua derivative to form a single complex. This complex has been purified by high-performance liquid chromatography and characterized by ¹H-nmr and CD spectroscopy. In this complex, Ap₄A takes a very particular conformation. It is an N7-N7 chelate of the metal with the two adenines in a head-to-head arrangement and an anti–anti conformation of the adenosines. Platinum chelation leads to a large decrease of the Ap₄A conformational flexibility.     

Promiscuous esterase activities of the C–C hydrolases from <Emphasis Type="Italic">Dyella ginsengisoli</Emphasis>

A C–C hydrolase gene (bphD _LA-4 ) from strain Dyella ginsengisoli LA-4 was cloned and expressed in Escherichia coli BL21 (DE3). BphD_LA-4 together with another hydrolase MfphA_LA-4, which derived from the same strain, possessed esterase activities. p-Nitrophenyl butyrate was the best substrate for both enzymes. BphD_LA-4 had high catalytic efficiency to p-nitrophenyl benzoate, whereas MfphA_LA-4 had no activity. Homology modeling and docking studies demonstrated that the proper hydrogen bond interaction was important for the reactivity of specific substrate.     

Dinucleoside 5′,5-P,P-Triphosphate Hydrolase from Yellow Lupin (Lupinus luteus) Seeds: Purification to Homogeneity and Hydrolysis of mRNA 5′-Cap Analogs

Three separable forms of diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A)-degrading activity were revealed when proteins obtained from the meal of yellow lupin seeds by ammonium sulfate precipitation were chromatographed on a DEAE-Sephacel column. The major form, which eluted first at the lowest salt concentration (0.15MKCl), was free of any activity converting the reaction products, ADP and AMP. Its further purification by gel filtration on Sephadex G-200 and by affinity elution from an AMP-agarose column yielded homogeneous protein as demonstrated on SDS–polyacrylamide gel electrophorograms. The enzyme is a single polypeptide chain ofM_r41 kDa. Eleven guanosine-containing dinucleoside triphosphates, including analogs of the mRNA 5′-cap structure, have been tested as potential substrates of the lupin “Ap₃A hydrolase.” All have been hydrolyzed yielding mixtures of corresponding nucleoside mono- and diphosphates. Asymmetrical compounds gave four products; m⁷Gp₃G, et⁷Gp₃G, and bz⁷Gp₃G were hydrolyzed randomly, whereas m⁷Gp₃A, m⁷Gp₃C, and m⁷Gp₃U yielded at least 80% m⁷GMP plus corresponding NDP and 20% or less NMP plus m⁷GDP. Hydrolysis of the guanosine-containing hybrids, Ap₃G, Cp₃G, and Up₃G, yielded at least 85% GMP plus corresponding NDP. All dinucleotides containing the m⁷G- moiety were hydrolyzed 2- to 4.5-fold faster than Ap₃A. Thus a general name, “dinucleoside triphosphate hydrolase,” is more appropriate for the enzymatic activity described.     

Diadenosine polyphosphate-activated inward and outward currents in follicular oocytes of Xenopus laevis

Ionic currents evoked by α,ω-adenine dinucleotides (Ap_XA; X = 2–6) in follicular oocytes of Xenopus laevis were studied under voltage-clamp conditions. Dinucleotides evoked inward and outward currents in Xenopus oocytes by activating native P₁ and P₂ purinoceptors known to be present on the follicle cell monolayer enveloping oocytes. Inward currents were mediated by a suramin-sen skive P₂ purinoceptor which showed an agonist potency order (at 10μM): Ap₄A>ATP>Ap₃A>Ap₅A, while Ap₂A and Ap₆A were inactive. Outward currents were mediated by a novel theophylline-sensitive P₁ purinoceptor which showed an agonist potency order (at 10μM): Ap₂A>ATP>Ap₄A=Ap₅A=Ap₆A>Ap₃A. Chromatographic analysis confirmed ectonucleotidase activity at the follicle cell layer of oocytes but at a very low rate of dinucleotide cleavage, indicating that currents evoked by dinucleotides resulted from a direct activation of oocyte P₁ and P₂ purinoceptors and not through their breakdown to ATP, ADP and AMP. There was no evidence for specific receptors (i.e., P₄ purinoceptors) for diadenosine polyphosphates in Xenopus oocytes.     

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A,Mg2+ Ap5A,and Mn2+ Ap5A reveal an intermediate lid position and six coordinate octahedral geometry for bound Mg2+ and Mn2+

Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap₅A, Mn²⁺ Ap₅A, and Mg²⁺ Ap₅A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap₅A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn²⁺ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg²⁺ Ap₅A and Mn²⁺ Ap₅A structures, Mg²⁺ and Mn²⁺ demonstrate six coordinate octahedral geometry. The interactions of the Mg²⁺-coordinated water molecules with the protein and Ap₅A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg²⁺) rather than α-γ (preferred by Mn²⁺) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276–288, 1998. © 1998 Wiley-Liss, Inc.     

(S)-1-(Pent-4′-enoyl)-4-(hydroxymethyl)-azetidin-2-one derivatives as inhibitors of human fatty acid amide hydrolase (hFAAH): synthesis,biological evaluation and molecular modelling

Abstract

A series of lipophilic ester derivatives (2a–g) of (S)-1-(pent-4′-enoyl)-4-(hydroxymethyl)-azetidin-2-one has been synthesised in three steps from (S)-4-(benzyloxycarbonyl)-azetidin-2-one and evaluated as novel, reversible, β-lactamic inhibitors of endocannabinoid-degrading enzymes (human fatty acid amide hydrolase (hFAAH) and monoacylglycerol lipase (hMAGL)). The compounds showed IC₅₀ values in the micromolar range and selectivity for hFAAH versus hMAGL. The unexpected 1000-fold decrease in activity of 2a comparatively to the known regioisomeric structure 1a (i.e. lipophilic chains placed on N₁ and C₃ positions of the β-lactam core) could be explained on the basis of docking studies into a revisited model of hFAAH active site, considering one or two water molecules in interaction with the catalytic triad.     

Presence of diadenosine polyphosphates in microdialysis samples from rat cerebellum in vivo: effect of mild hyperammonemia on their receptors

Diadenosine triphosphate (Ap₃A), diadenosine tetraphosphate (Ap₄A), and diadenosine pentaphosphate (Ap₅A) have been identified in microdialysis samples from the cerebellum of conscious freely moving rats, under basal conditions, by means of a high-performance liquid chromatography method. The occurrence of Ap₃A in the cerebellar microdyalisates is noteworthy, as the presence of this compound in the interstitial medium in neural tissues has not been previously described. The concentrations measured for the diadenosine polyphosphates in the cerebellar dialysate were (in nanomolar) 10.5 ± 2.9, 5.4 ± 1.2, and 5.8 ± 1.3 for Ap₃A, Ap₄A, and Ap₅A, respectively. These concentrations are in the range that allows the activation of the presynaptic dinucleotide receptor in nerve terminals. However, a possible interaction of these dinucleotides with other purinergic receptors cannot be ruled out, as rat cerebellum expresses a variety of P2X or P2Y receptors susceptible to be activated by diadenosine polyphosphates, such as the P2X1-4, P2Y₁, P2Y₂, P2Y₄, and P2Y₁₂ receptors, as demonstrated by quantitative real-time PCR. Also, the ecto-nucleotide pyrophosphatases/phosphodiesterases NPP1 and NPP3, able to hydrolyze the diadenosine polyphosphates and terminate their extracellular actions, are expressed in the rat cerebellum. All these evidences contribute to reinforce the role of diadenosine polyphosphates as signaling molecules in the central nervous system. Finally, we have analyzed the possible differences in the concentration of diadenosine polyphosphates in the cerebellar extracellular medium and changes in the expression levels of their receptors and hydrolyzing enzymes in an animal model of moderate hyperammonemia.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9382-3) contains supplementary material, which is available to authorized users.     

Hydrolysis of some mRNA 5′-Cap Analogs Catalyzed by the Human Fhit Protein - and Lupin ApppA Hydrolases

Abstract

Hydrolysis of the following four cap analogs: m⁷G(5′)ppp(5′)A, m⁷G(5′)ppp(5′)m⁶A, m⁷G(5′)ppp(5′)m^2′OG and m⁷G(5′)ppp(5′)2′dG catalyzed by homogeneous human Fhit protein and yellow lupin Ap₃A hydrolase has been investigated. The hydrolysis products were identified by HPLC analysis and the K_m and V_max values calculated based on the data obtained by the fluorimetric method.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.