Variation of Ap4A and other dinucleoside polyphosphates in stressed Drosophila cells

The unusual bis(5'-nucleosidyl)oligophosphates: Ap4A, Ap4G, Ap3A, and Ap3G, have been measured in cultures of Drosophila cells. Exponentially growing cells contain concentrations of 0.25, 0.31, 0.87, and 2.25 microM, respectively. These nucleotides have been followed after stressing the cells either by CdCl2 addition or by heat-shock treatment. Their concentrations are not affected by exposure to 500 microM CdCl2 during 6 h. Beyond this threshold of cadmium concentration, the nucleotides increase. With 5 mM CdCl2, an enhancement by 2 orders of magnitude of all the dinucleoside tri- and tetra-phosphates is observed. Upon heat-shock from 19 to 37 degrees C, Ap4A, Ap3A, and Ap3G increase up to 2.2, 3, and 3.3 times their initial levels, respectively. The increase is achieved within 1 h.     

Lack of correlation between extensive accumulation of bisnucleoside polyphosphates and the heat-shock response in eukaryotic cells

The accumulation in large amounts of bisnucleoside polyphosphates (Ap4X) after heat shock in Xenopus laevis oocytes or cultured hepatoma cells (HTC cells) is observed after exposure to temperatures of 45 degrees C or higher. The accumulation is a transient phenomenon, with the collapse in cellular ATP concentration severely affecting the rate of synthesis of Ap4X, allowing degrading activities to empty the pool of these compounds under prolonged heat shock. This accumulation of Ap4X to high levels, compared to the basic content, is only observed under conditions leading to irreversible damage, ultimately resulting in the death of the cell. It is shown that the increase in Ap4X after hyperthermia is due to the partial or almost complete inhibition of their degradation pathways, rather than to a stimulation of their rate of synthesis. Finally, the synthesis of heat-shock proteins could be observed under conditions which do not lead to important accumulation of Ap4X, therefore ruling out the possibility that these adenylylated nucleotides would behave as chemical signals ("alarmones") triggering the synthesis of heat-shock proteins. Nevertheless, on the basis of our earlier results (Guédon, G., Sovia, D., Ebel, J. P., Befort, D., and Remy, P. (1985) Embo J. 4, 3743-3749), it cannot be excluded that Ap4X might play a role in the regulation of the heat-shock response; this would, however, rely on variations in Ap4X concentrations which do not exceed a factor of 2.     

Regulation of hepatic parenchymal and non-parenchymal cell function by the diadenine nucleotides Ap3A and Ap4A

The diadenine nucleotides diadenosine 5',5"-P1,P3-triphosphate (Ap3A) and diadenosine 5',5"-P1,P4-tetraphosphate (Ap4A) can be released from platelets and were shown to act as long-lived signal molecules. Accordingly, we studied their potential effect on hepatic metabolism. In isolated perfused rat liver, Ap3A and Ap4A increase the portal pressure, lead to a transient net release of Ca2+, complex net K+ movement across the liver plasma membrane and stimulate hepatic glucose output and 14CO2 production from [1-14C]glutamate. These responses resemble that obtained with extracellular ATP. This and studies on the additivity of ATP and Ap4A effects suggest similar mechanisms mediating the ATP and diadenine nucleotide effects in the liver. Ap3A and Ap4A increased the activity of glycogen phosphorylase a in isolated hepatocyte suspensions by about 100%, pointing to a direct effect of these nucleotides on hepatic parenchymal cells. A response of hepatic non-parenchymal cells to diadenine nucleotide infusion is suggested by a marked stimulation of thromboxane and prostaglandin D2 release from perfused liver. Studies with the thromboxane A2 receptor antagonist BM 13.177 (20 microM) show that the pressure and glucose response to the diadenine nucleotides is partially mediated by this thromboxane formation. Studies with retrograde and sequential liver perfusions suggest a less efficient degradation of the diadenine nucleotides during a single liver passage compared to extracellular ATP. The data suggest that Ap3A and Ap4A are potential regulators of hepatic hemodynamics and metabolism, involving complex interactions between hepatic parenchymal cells and hepatic non-parenchymal cells, including eicosanoids as signal molecules.     

Transcription of the phosphoglycerate kinase gene of Saccharomyces cerevisiae increases when fermentative cultures are stressed by heat-shock

The single gene for phosphoglycerate kinase (PGK) in the haploid genome of Saccharomyces cerevisiae is expressed to a very high level in cultures fermenting glucose. Despite this it responds to heat-shock. When S. cerevisiae growing exponentially on glucose media was shifted from 25 degrees C to 38 degrees C transient increases of 6-7-fold in cellular PGK mRNA were observed. This elevation in PGK mRNA still occurred in the presence of the protein-synthesis inhibitor cycloheximide, but was not observed in cells bearing the rna1.1 mutation. From the kinetics of continuous labelling of PGK mRNA, relative to the labelling of other RNAs in the same cultures whose levels do not alter with heat-shock, it was shown that the elevation in PGK mRNA in response to temperature upshift reflects primarily an increased synthesis of this mRNA and not an alteration of its half-life. PGK mRNA synthesis is therefore one target of a response mechanism to thermal stress. Synthesis of PGK enzyme in glucose-grown cultures is efficient after mild (25 degrees C to 38 degrees C) or severe (25 degrees C to 42 degrees C) heat-shocks. Following the severe shock, the synthesis of most proteins is abruptly terminated, but synthesis of PGK and a few other glycolytic enzymes continues at levels comparable to the levels of synthesis of most of those proteins dramatically induced by heat (heat-shock proteins). Cells that overproduce PGK due to the presence of multiple copies of the PGK gene on a high-copy-number plasmid continue their overproduction of this enzyme during severe thermal stress. Therefore PGK mRNA is both elevated in level in response to heat-shock and translated efficiently at supra-optimal temperatures.     

Unproportionally high concentrations of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) in heavy platelets. Consequences for in vitro studies with human platelets

Platelets from whole blood were separated into five density subpopulations using a discontinuous Percoll gradient. The content of diadenosine triphosphate (Ap3A), diadenosine tetraphosphate (Ap4A), ADP and ATP were determined in the subfractions. The dinucleotides were directly measured in neutralized, acid-soluble extracts of human platelets with a bioluminescence method not requiring any chromatographic step. When comparing the nucleotide contents of the density subpopulations it became evident that all nucleotides steadily increased with increasing density. Ap3A, Ap4A, ADP and ATP were present in 10-, 7-, 4- and 2-fold higher amounts in the heaviest platelets, respectively, as compared to the subfraction with the lowest density. This finding is practically relevant since the most dense platelet subpopulations may be lost during conventional centrifugation to obtain platelet-rich plasma. Therefore we compared a platelet population obtained from PRP with the platelet population, which had been prepared from whole blood by means of a continuous Percoll gradient. All the four nucleotides investigated were represented in 1.5- to 2-fold higher amounts in the whole blood platelet population. This indicates that PRP does not contain a representative population but lacks part of the large heavy platelets containing the highest amounts of nucleotides.     

Variation in intracellular P1P4-bis(5''-adenosyl) tetraphosphate (Ap4A) in virus-infected cells.

The effect of virus infection on the intracellular concentration of the proposed stress alarmone P1P4-bis(5'-adenosyl) tetraphosphate (Ap4A) has been examined in Vero cells. Compared with exposure to 0.8 mM-Cd2+, which causes a 30-fold increase in Ap4A, infection with simian virus 40 and poliovirus causes only a 2-fold increase, whereas herpes simplex virus type 1 results in a decrease in Ap4A during the course of the infection.     

Synthesis, modification and structural binding of heat-shock proteins in tomato cell cultures

Synthesis of about 30 acidic and 18 basic heat-shock proteins (hsps) is induced in suspension cultures of tomato (Lycopersicon peruvianum) if subjected to supraoptimal temperature conditions (35-40 degrees C). A characteristic aspect of the plant heat-shock response is the formation of cytoplasmic granular aggregates, heat-shock granules, containing distinct heat-shock proteins as major structural components and, in addition, several hitherto undetected minor acidic and basic heat-shock proteins. Structural binding of heat-shock proteins, i.e. assembly of heat-shock granules, is dependent on the persistance of supraoptimal temperature conditions. Despite the ongoing synthesis also at 25 degrees C, e.g. in pulse heat-shocked cultures, these proteins are accumulated exclusively in soluble form. Individual heat-shock proteins are characterized by their kinetics of synthesis and are classified by their compartmentation behaviour into class A proteins (exclusively found in soluble form, e.g. hsps 95 and 80), class B proteins (5-10% bound to heat-shock granules, e.g. hsps 70, 68), class C proteins (30-80% bound to heat-shock granules, e.g. hsps 21, 17, 15) and class D proteins, which are minor heat-shock proteins only detected in structure-bound form. Major representatives are modified proteins, i.e. hsps 95, 80, 70 and 68 are phosphorylated and hsps 80, 74, 70 and 17 are methylated proteins (numbers 70, 80 etc. refer to 10(-3) Mr). Under heat-shock conditions synthesis of the proteins detected in control cells (25 degrees C proteins) exhibits two patterns. There are proteins with continued and proteins with discontinued synthesis. Synthesis of most of the latter proteins is resumed very rapidly after shift-down to 25 degrees C, even in the presence of actinomycin D. We conclude that reversible segregation of distinct mRNA species from the translation apparatus contributes to the heat-shock-specific pattern of protein synthesis in plants also.     

Interleukin-2 regulation of diadenosine 5',5'-p1 p4-tetraphosphate (Ap4A) levels and DNA synthesis in cloned murine T lymphocytes

The levels or diadenosine 5', 5'-p1, p4, tetraphosphate (Ap4A), a putative signal molecule associated with DNA synthesis, has been measured in murine T lymphocytes. The level or Ap4A detected correlated with the stimulation of DNA synthesis in murine T lymphocytes. In interleukin-2 (IL-2) dependent cells previously deprived of IL-2, new DNA synthesis can be induced by adding IL-2; the synthesis of DNA is preceded by an increase in Ap4A levels. A significant increase in DNA synthesis was observed after the Ap4A concentration exceeded the Kd of DNA polymerase alpha for Ap4A. Similarly, in cells blocked from synthesizing DNA by hydroxyurea, the levels or Ap4A are maintained only in the presence of IL-2. Once IL-2 is removed, the potential to synthesize DNA decreases and is preceded by decreases in the level or Ap4A. The DNA synthesis potential decreases rapidly after the Ap4A concentration fell below the Kd of DNA polymerase alpha for Ap4A. It is possible that Ap4A is a second messenger molecule required for the proliferation of lymphocytes and that the production of Ap4A in IL-2 dependent murine T lymphocytes is regulated by the homologous growth factor.     

Design and characterization of Escherichia coli mutants devoid of Ap4N-hydrolase activity

Escherichia coli strains with abnormally high concentrations of bis(5'-nucleosidyl)-tetraphosphates (Ap4N) were constructed by disrupting the apaH gene that encodes Ap4N-hydrolase. Variation deletions and insertions were also introduced in apaG and ksgA, two other cistrons of the ksgA apaGH operon. In all strains studied, a correlation was found between the residual Ap4N-hydrolase activity and the intracellular Ap4N concentration. In cells that do not express apaH at all, the Ap4N concentration was about 100-fold higher than in the parental strain. Such a high Ap4N level did not modify the bacterial growth rate in rich or minimal medium. However, while, as expected, the ksgA- and apaG- ksgA- strains stopped growing in the presence of this antibiotic at 600 micrograms/ml. The were not sensitive to kasugamycin, the apaH- apaG- ksgA- strain filamented and stopped growing in the presence of this antibiotic at 600 micrograms/ml. The growth inhibition was abolished upon complementation with a plasmid carrying an intact apaH gene. Trans addition of extra copies of the heat-shock gene dnaK also prevented the kasugamycin-induced filamentation of apaH- apaG- ksgA- strains. This result is discussed in relation to the possible involvement of Ap4N in cellular adaptation following a stress.     

Phosphorylation of threonyl- and seryl-tRNA synthetase by cAMP-dependent protein kinase. A possible role in the regulation of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) synthesis

Threonyl-tRNA synthetase has been shown to be phosphorylated in reticulocytes (Dang, C. V., Tan, E. M., and Traugh, J. A., (1988) FASEB J. 2, 2376-2379). Upon incubation of reticulocytes with 8-bromo-cAMP, phosphorylation of threonyl-tRNA synthetase is stimulated approximately 2-fold, an increase similar to that observed with ribosomal protein S6. To analyze the effects of phosphorylation on activity, threonyl-tRNA synthetase has been purified to apparent homogeneity from rabbit reticulocytes utilizing a four-step purification procedure with the simultaneous purification of seryl-tRNA synthetase. Both synthetases are phosphorylated in vitro by the cAMP-dependent protein kinase. Prior to phosphorylation, the two synthetases produce significant amounts of P1, P4-bis(5'-adenosyl)-tetraphosphate (Ap4A) in the presence of the cognate amino acid and ATP, with activities comparable to that of lysyl-tRNA synthetase. Phosphorylation has no effect on aminoacylation, but an increase in Ap4A synthesis of up to 6-fold is observed with threonyl-tRNA synthetase and 2-fold with seryl-tRNA synthetase. Thus, cAMP-mediated phosphorylation of specific aminoacyl-tRNA synthetases appears to be a potential mode of regulation of Ap4A synthesis in mammals.     

High diadenosine tetraphosphate (Ap4A) level in germ cells and embryos of sea urchin and Xenopus and its effect on DNA synthesis

Ap4A levels in sperms, eggs and different developmental stages of sea urchin (Psammechinus miliaris) and (Xenopus laevis) were determined by a method based on ATP measurement with luciferin/luciferase after splitting diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) into ATP and AMP. Appreciable storage pools of Ap4A were found in unfertilized eggs of Psammechinus and Xenopus as well as in sea urchin sperms. The actual Ap4A concentration of 28 microM in sperm represents the highest Ap4A level so far observed in eukaryotic cells. Upon fertilization an instant onset of de novo synthesis of Ap4A was demonstrated. Ap4A levels during early embryogenesis of P. miliaris and X. laevis (2.5-4 microM) are higher than those in exponentially growing mammalian culture cells and mammalian fetuses. Microinjection of Ap4A into unfertilized eggs of Psammechinus miliaris caused a 3-7 fold increase of DNA synthesis in comparison with mock-injected eggs.     

A paradoxical increase of a metabolite upon increased expression of its catabolic enzyme: the case of diadenosine tetraphosphate (Ap4A) and Ap4A phosphorylase I in Saccharomyces cerevisiae.

The APA1 gene in Saccharomyces cerevisiae encodes Ap4A phosphorylase I, the catabolic enzyme for diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A). APA1 has been inserted into a multicopy plasmid and into a centromeric plasmid with a GAL1 promoter. Enhanced expression of APA1 via the plasmids resulted in 10- and 90-fold increases in Ap4A phosphorylase activity, respectively, as assayed in vitro. However, the intracellular concentration of Ap4A exhibited increases of 2- and 15-fold, respectively, from the two different plasmids. Intracellular Ap4A increased 3- to 20-fold during growth on galactose of a transformant with APA1 under the control of the GAL1 promoter. Intracellular adenosine 5'-P1-tetraphospho-P4-5"'-guanosine (Ap4G) and diguanosine 5',5"'-P1,P4-tetraphosphate (Gp4G) also increased in the transformant under these conditions. The chromosomal locus of APA1 has been disrupted in a haploid strain. The Ap4A phosphorylase activity decreased by 80% and the intracellular Ap4A concentration increased by a factor of five in the null mutant. These results with the null mutant agree with previous results reported by Plateau et al. (P. Plateau, M. Fromant, J.-M. Schmitter, J.-M. Buhler, and S. Blancquet, J. Bacteriol. 171:6437-6445, 1989). The paradoxical increase in Ap4A upon enhanced expression of APA1 indicates that the metabolic consequences of altered gene expression may be more complex than indicated solely by assay of enzymatic activity of the gene product.     

Effect of diadenosine oligophosphates (Ap4A and Ap3A) and their phosphonate analogs on catalytic properties of phenylalanyl-tRNA synthetase from E. coli

The influence of P1,P3-bis(5'-adenosyl)triphosphate (Ap3A), P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and its analogues, containing a residue of methylenediphosphonic acid in various positions of the oligophosphate chain, on the reactions catalysed by phenylalanyl-tRNA synthetase from E. coli MRE-600 has been studied. The compounds do not affect significantly the rate of ATP-[32P]PPi-exchange nor maintain this reaction in the absence of ATP. The diadenosineoligophosphates are shown to be noncompetitive inhibitors of ATP in the tRNA aminoacylation by phenylalanine (for Ap4A Ki = 1,45.10(-3) M). The phosphonate analogues of Ap4A inhibit the synthesis of Ap3A depending on their structure. The conclusion is thus drawn that the E. coli MRE-600 phenylalanyl-tRNA synthetase does not interact property with Ap4A and its phosphonate analogues.     

In vivo levels of diadenosine tetraphosphate and adenosine tetraphospho-guanosine in Physarum polycephalum during the cell cycle and oxidative stress.

Cellular levels of diadenosine tetraphosphate (Ap4A) and adenosine tetraphospho-guanosine (Ap4G) were specifically measured during the cell cycle of Physarum polycephalum by a high-pressure liquid chromatographic method. Ap4A was also measured indirectly by a coupled phosphodiesterase-luciferase assay. No cell cycle-specific changes in either Ap4A or Ap4G were detected in experiments involving different methods of assay, different strains of P. polycephalum, or different methods of fixation of macroplasmodia. Our results on Ap4A are in contrast with those reported previously (C. Weinmann-Dorsch, G. Pierron, R. Wick, H. Sauer, and F. Grummt, Exp. Cell Res. 155:171-177, 1984). Weinmann-Dorsch et al. reported an 8- to 30-fold increase in Ap4A in early S phase in P. polycephalum, as measured by the phosphodiesterase-luciferase assay. We also measured levels of Ap4A, Ap4G, and ATP in macroplasmodia treated with 0.1 mM dinitrophenol. Ap4A and Ap4G transiently increased three- to sevenfold after 1 h and then decreased concomitantly with an 80% decrease in the level of ATP after 2 h in the presence of dinitrophenol. These results do not support the hypothesis that Ap4A is a positive pleiotypic activator that modulates DNA replication, but they are consistent with the hypothesis proposed for procaryotes that Ap4A and Ap4G are signal nucleotides or alarmones of oxidative stress (B.R. Bochner, P.C. Lee, S.W. Wilson, C.W. Cutler, and B.N. Ames, Cell 37:225-232, 1984).     

Defect in expression of heat-shock proteins at high temperature in xthA mutants.

Escherichia coli mutants lacking exonuclease III (xthA) are defective in the induction of heat-shock proteins upon severe heat-shock treatment (upshift from 30 to 50 degrees C) but not mild heat-shock treatment (upshift from 30 to 42 degrees C). We show that this defect is due to the xthA mutation by complementation. Furthermore, increasing the gene dosage of xthA+ prolongs the synthesis of heat shock proteins seen after a shift to 42 degrees C. Increasing the gene dosage of htpR+ partially suppresses the defect of xthA mutants in the synthesis of heat-shock proteins at 50 degrees C. When an xthA strain was incubated at 42 degrees C before a shift to 50 degrees C, it was then able to carry out the synthesis of heat-shock proteins at 50 degrees C.     

Gelsolin and plasminogen activator inhibitor-1 are Ap3A-binding proteins

Previous data on the accumulation of diadenosine 5',5"'-P1,P3-triphosphate (Ap3A) in cells in response to various physiological factors raised the issue of identification of Ap3A binding proteins as potential targets for Ap3A. Ap3A binding proteins were isolated from a human leukocyte lysate by affinity chromatography through Ap3A-aga-rose. Two proteins, gelsolin and plasminogen activator inhibitor-1 (PAI-1), were tentatively identified by in-gel tryptic digestion and mass fingerprint analysis by MALDI-TOF mass spectrometry. The ability of the pure proteins to bind Ap3A was confirmed. Scatchard analysis of [3H]Ap3A binding data yielded dissociation constants of 0.3 microM for gelsolin and 4.1 microM for PAI-1. Binding was saturable at 0.78 mol Ap3A/mol of gelsolin and 0.68 mol Ap3A/mol PAI-1. The binding was non-covalent and insensitive to the presence of divalent metal ions. In neither case was binding affected by a 100-fold molar excess of ATP, ADP and AMP or Ap4A, suggesting a high degree of specificity for Ap3A. Ap3A produced significant effects on cell morphology when added at 10 microM to reversibly permeabilized CEM-SS cells, suggesting that it might influence cytoskeletal disruption by activating gelsolin. Ap3A added externally to HL60 promyelocytic cells reduced the inhibitory effect of PAI-1 on VP16-induced apoptosis. These findings provide new information about intra- and extracellular targets of Ap3A.     

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.