Novel fluorescent labelled affinity probes for diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A)-binding studies

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.     

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k(cat) = 0.29+/-0.05 s(-1)) the transfer of AMP from the E-AMP-ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra- or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.     

Characterization of the interaction of P1,P4-diadenosine 5'-tetraphosphate with luciferase

Adenylated dinucleotides (Ap(n)A) are regulatory molecules that control various cellular processes. A very likely intracellular target for Ap(4)A are enzymes that require ATP as either substrate or modulator. We report the results of new biochemical studies aimed at characterizing the Ap(4)A interaction with firefly luciferase, by using the luminometric and thin layer chromatography techniques. The data presented herein demonstrate that Ap(4)A is a noncompetitive inhibitor for the ATP-induced luminescence. These results together with our previous findings that Ap(4)A is a luciferase substrate [Nucleosides Nucleotides Nucleic Acids 23 (2004) in press.] support the notion that, similar to its interaction with P(2) receptors, Ap(4)A also has a dual interaction with luciferase. Other Ap(n)As (n = 2, 5, and 6) also inhibited the ATP-luciferase interaction. Since Ap(n)As may have similar interactions with other intracellular ATP-requiring enzymes, the study presented herein validates ulterior investigations of the Ap(n)A interaction with such enzymes, and opens the way to a better understanding of their intracellular roles.     

Mapping eIF5A binding sites for Dys1 and Lia1: in vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal alpha-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required.     

The binding activities of proteins that bind Ap4A, an alarmone, are stimulated in the presence of ethanol or phosphatidylethanolamine

Three proteins binding Ap4A which is known to increase in the heat-shocked cells or to trigger DNA synthesis in G1-arrested eukaryotic cells were purified from E. coli cell extract. For the binding activities of the proteins, glutathione or dithiothreitol and manganese or iron ion were absolutely required. Glutathione, which exists in relatively high concentration in the cells and had been reported to be related to oxidant shock, was far more effective than an artificial antioxidant, dithiothreitol. Ethanol, which has an effect similar to heat or oxidant shock on microbial or eukaryotic cells, enhanced several fold the Ap4A-binding activity. Phosphatidylethanolamine, a major component of phospholipids of cytoplasma and membrane of E. coli cell also stimulated the Ap4A-binding activity.     

Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1)

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA-amido conjugates, the most important of which appears to be JA-Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono- and dinucleoside polyphosphates, which are side-reaction products of many enzymes forming acyl approximately adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile-conjugates was observed for (+/-)-JA and 9,10-dihydro-JA, while the rate of conjugation with 12-hydroxy-JA and OPC-4 (3-oxo-2-(2Z-pentenyl)cyclopentane-1-butyric acid) was only about 1-2% that for (+/-)-JA. Of the two stereoisomers of JA, (-)-JA and (+)-JA, rate of synthesis of the former was about 100-fold faster than for (+)-JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg(2+), (-)-JA and tripolyphosphate the ligase produces adenosine 5'-tetraphosphate (p(4)A); (2) addition of isoleucine to that mixture halts the p(4)A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap(3)A) nor diadenosine tetraphosphate (Ap(4)A) and (4) Ap(4)A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA-Ile.     

Inhibition of ADP-ribosylation of histone by diadenosine 5', 5"' -p (1), p(4)-tetraphosphate

Diadenosine 5′, 5?-p¹, p⁴-tetraphosphate (Ap4A) strongly inhibited ADP-ribosylation reaction of histone by purified bovine thymus poly(ADP-ribose) polymerase. This compound showed a relatively weak inhibitory effect on Mg²⁺-dependent, enzyme-bound poly(ADP-ribose) synthesis. Among various adenine nucleotides tested, including several diadenosine nucleotides with varying phosphate chain length, Ap4A was the most effective inhibitor of the histone-modification reaction. Ap5A and Ap6A showed slightly lower inhibitory effect than Ap4A. Kinetic analysis of the inhibitor (Ap4A) with varying concentration of substrate (NAD⁺) revealed that this compound is a “mixed type inhibitor”, with a Ki value of 5.1 μM.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

Synthesis of bisphosphonate derivatives of ATP by T4 RNA ligase

T4 RNA ligase catalyzes the synthesis of ATP beta,gamma-bisphosphonate analogues, using the following substrates with the relative velocity rates indicated between brackets: methylenebisphosphonate (pCH(2)p) (100), clodronate (pCCl(2)p) (52), and etidronate (pC(OH)(CH(3))p) (4). The presence of pyrophosphatase about doubled the rate of these syntheses. Pamidronate (pC(OH)(CH(2)-CH(2)-NH(2))p), and alendronate (pC(OH)(CH(2)-CH(2)-CH(2)-NH(2))p) were not substrates of the reaction. Clodronate displaced the AMP moiety of the complex E-AMP in a concentration dependent manner. The K(m) values and the rate of synthesis (k(cat)) determined for the bisphosphonates as substrates of the reaction were, respectively: methylenebisphosphonate, 0.26+/-0.05 mM (0.28+/-0.05 s(-1)); clodronate, 0.54+/-0.14 mM (0.29+/-0.05 s(-1)); and etidronate, 4.3+/-0.5 mM (0.028+/-0.013 s(-1)). In the presence of GTP, and ATP or AppCCl(2)p the relative rate of synthesis of adenosine 5',5'-P(1),P(4)-tetraphosphoguanosine (Ap(4)G) was around 100% and 33%, respectively; the methylenebisphosphonate derivative of ATP (AppCH(2)p) was a very poor substrate for the synthesis of Ap(4)G. To our knowledge this report describes, for the first time, the synthesis of ATP beta,gamma-bisphosphonate analogues by an enzyme different to the classically considered aminoacyl-tRNA synthetases.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Mammalian mitochondrial and microsomal cytochromes b(5) exhibit divergent structural and biophysical characteristics

The only outer mitochondrial membrane cytochrome b(5) examined to date, from rat (rOM b(5)), exhibits greater stability than known mammalian microsomal (Mc) isoforms, as well as a much higher kinetic barrier for hemin dissociation and a more negative reduction potential. A BlastP search of available databases using the protein sequence of rOM b(5) as template revealed entries for analogous proteins from human (hOM b(5)) and mouse (mOM b(5)). We prepared a synthetic gene coding for the heme-binding domain of hOM b(5), and expressed the protein to high levels. The hOM protein exhibits stability, hemin-binding, and redox properties similar to those of rOM b(5), suggesting that they are characteristic of the OM b(5) subfamily. The divergence in properties between the OM and Mc b(5) isoforms in mammals can be attributed, at least in part, to the presence of two extended hydrophobic patches in the former. The biophysical properties characteristic of the OM proteins may be important in facilitating the two functions proposed for them so far, reduction of ascorbate radical and stimulation of androgen synthesis.     

The comparative study on the solution structures of the oxidized bovine microsomal cytochrome b5 and mutant V45H

A comparative study on the solution structures of bovine microsomal cytochrome b5 (Tb5) and the mutant V45H has been achieved by 1D and 2D 1H-NMR spectroscopy to clarify the differences in the solution conformations between these two proteins. The results reveal that the global folding of the V45H mutant in solution is unchanged, but the subtle changes exist in the orientation of the axial ligand His39, and heme vinyl groups. The side chain of His45 in V45H mutant extends to the outer edge of the heme pocket leaving a cavity at the site originally occupied by the inner methyl group of Val45 residue. In addition, the imidazole ring of axial ligand His39 rotates counterclockwise by approximately 3 degrees around the His-Fe-His axis, and the 4-heme vinyl group turns to the space vacated by the removed side chain due to the mutation. Furthermore, the helix III of the heme pocket undergoes outward displacement, while the linkage between helix II and III is shifted leftward. These observations are not only consistent with the pattern of the pseudocontact shifts of the heme protons, but also well account for the lower stability of V45H mutant against heat and urea.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Ap4A is not an efficient Zn(II) binding agent. A concerted potentiometric, calorimetric and NMR study

Diadenosine 5',5'-P(1)P(4) tetraphosphate (Ap(4)A) has been considered as an intracellular partner for Zn(II). We applied potentiometry, ITC and NMR to study protonation equilibria of Ap(4)A and Zn(II) complexation by this dinucleotide. The values of binding constants obtained by these three techniques under various experimental conditions coherently demonstrated that Ap(4)A binds Zn(II) weakly, with an apparent binding constant of ca. 10(4) at neutral pH. Such a low stability of Zn(II) complexes with Ap(4)A excludes a possibility for interactions between these two agents in vivo.     

Diadenosine 5', 5"'-P1,P4-tetraphosphate stimulates processing of adp-ribosylated poly(ADP-ribose) polymerase

The effect of diadenosine 5', 5"'-P1,P4-tetraphosphate (Ap4A) on the time course and acceptors of poly(ADP-ribose) synthesis was studied in undamaged and N-methyl-N'-nitro-N-nitrosoguanidine-treated human lymphocytes. Analysis of protein acceptors of poly(ADP-ribose) revealed that treatment with Ap4A stimulated ADP-ribosylation of bands at molecular weights of 96,000, 79,000, and 62,000. Pulse-chase studies showed that these bands were produced as a result of an effect of Ap4A on the processing of ADP-ribosylated proteins rather than on the synthesis of newly ADP-ribosylated proteins. By incubating permeabilized cells in the absence or presence of Ap4A and purified poly(ADP-ribose) polymerase auto-ADP-ribosylated with [32P]NAD+, we showed that the Mr = 96,000, 79,000, and 62,000 bands were derivatives of the prelabeled enzyme. Our results indicate that normal human lymphocytes process auto-ADP-ribosylated poly(ADP-ribose) polymerase to specific lower molecular weight products and that this processing is stimulated by Ap4A.     

Fhit proteins can also recognize substrates other than dinucleoside polyphosphates

We show here that Fhit proteins, in addition to their function as dinucleoside triphosphate hydrolases, act similarly to adenylylsulfatases and nucleoside phosphoramidases, liberating nucleoside 5'-monophosphates from such natural metabolites as adenosine 5'-phosphosulfate and adenosine 5'-phosphoramidate. Moreover, Fhits recognize synthetic nucleotides, such as adenosine 5'-O-phosphorofluoridate and adenosine 5'-O-(gamma-fluorotriphosphate), and release AMP from them. With respect to the former, Fhits behave like a phosphodiesterase I concomitant with cleavage of the P-F bond. Some kinetic parameters and implications of the novel reactions catalyzed by the human and plant (Arabidopsis thaliana) Fhit proteins are presented.     

Effects of charged amino-acid mutation on the solution structure of cytochrome b5 and binding between cytochrome b5 and cytochrome c

The solution structure of oxidized bovine microsomal cytochrome b(5) mutant (E48, E56/A, D60/A) has been determined through 1524 meaningful nuclear Overhauser effect constraints together with 190 pseudocontact shift constraints. The final family of 35 conformers has rmsd values with respect to the mean structure of 0.045+/-0.009 nm and 0.088+/-0.011 nm for backbone and heavy atoms, respectively. A characteristic of this mutant is that of having no significant changes in the whole folding and secondary structure compared with the X-ray and solution structures of wild-type cytochrome b(5). The binding of different surface mutants of cytochrome b(5) with cytochrome c shows that electrostatic interactions play an important role in maintaining the stability and specificity of the protein complex formed. The differences in association constants demonstrate the electrostatic contributions of cytochrome b(5) surface negatively charged residues, which were suggested to be involved in complex formation in the Northrup and Salemme models, have cumulative effect on the stability of cyt c-cyt b(5) complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry proposed by Northrup, which is involved in the participation of Glu48, Glu56, Asp60, and heme propionate of cytochrome b(5), do occur in the association between cytochrome b(5) and cytochrome c.