GTP Pool Size Research Articles

Cytoophidium assembly reflects upregulation of IMPDH activity.

ABSTRACTCytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH) (both of which have two isoforms) can form fiber-like subcellular structures termed ‘cytoophidia’ under certain circumstances in mammalian cells. Although it has been shown that filamentation of CTPS downregulates its activity by disturbing conformational changes, the activity of IMPDH within cytoophidia is still unclear. Most previous IMPDH cytoophidium studies were performed under conditions involving inhibitors that impair GTP synthesis. Here, we show that IMPDH forms cytoophidia without inhibition of GTP synthesis. First, we find that an elevated intracellular CTP concentration or treatment with 3′-deazauridine, a CTPS inhibitor, promotes IMPDH cytoophidium formation and increases the intracellular GTP pool size. Moreover, restriction of cell growth triggers the disassembly of IMPDH cytoophidia, implying that their presence is correlated with active cell metabolism. Finally, we show that the presence of IMPDH cytoophidia in mouse pancreatic islet cells might correlate with nutrient uptake in the animal. Collectively, our findings reveal that formation of IMPDH cytoophidia reflects upregulation of purine nucleotide synthesis, suggesting that the IMPDH cytoophidium plays a role distinct from that of the CTPS cytoophidium in controlling intracellular nucleotide homeostasis.

Guanosine supplementation reduces the antiproliferative and apoptotic effects of the IMPDH inhibitor gnidilatimonoein in K562 cells

IMPDH (inosine 5'-monophosphate dehydrogenase) is the rate-limiting enzyme in the de novo biosynthetic pathway of guanine nucleotides, which is usually up-regulated in human leukaemia cell lines. Our previous studies have classified gnidilatimonoein, isolated from Daphne mucronata, as an IMPDH inhibitor and a strong antiproliferative agent among several types of leukaemia cells. In the present study, we investigated the effects of gnidilatimonoein on intracellular GTP pool size and its link to differentiation and apoptosis of K562 cells. It was found that gnidilatimonoein inhibited cell proliferation and induced G0/G1 cell cycle arrest in K562 cells after 24 h exposure to a single dose of gnidilatimonoein (1.5 μM), while no significant effects were observed on unstimulated and phytohaemagglutinin-stimulated peripheral blood lymphocyte cells at the gnidilatimonoein dose (1.5 μM) used. Based on the morphological changes, Wright-Giemsa staining, benzidine assay and the expression of cell surface markers [GPIIb (glycoprotein IIb) and glycophorin A], as analysed by flow cytometry, we found that K562 cells had differentiated towards megakaryocytic lineage. In addition, gnidilatimonoein induced apoptosis among K562 cells based on Acridine Orange/ethidium bromide and annexin V/propidium iodide double-staining observations. These changes, which were abrogated by the addition of guanosine, became evident when the intracellular GTP level decreased to approx. 20-35% of the untreated control level. Based on these findings, it can be concluded that gnidilatimonoein induces differentiation and apoptosis in K562 cells through perturbation of GTP metabolism, as one of its routes of action.

Induction of apoptosis by novel inosine monophosphate dehydrogenase, 3-hydrogenkwadaphnin

Today’s medical word is highly dependent on severel natural products (e.g. taxol) in fighting different kinds of cancer and in constantly working to found new compounds. In this respect, 3-hydrogenkwadaphnin is a new diterpene ester isolated from Dendrostellera lessertii (Thymelaceae). It has been previously shown that this new compound has high anti-tumor activity and the capability of arresting the growing leukemic cells at G1/S phase of their progression cycle, which finally leads to cell death. Further studies revealed 3-hydrogenkwadaphnin decreased both DNA synthesis and inosine monophosphate dehydrogenase (IMPDH) activity in K562 and HL-60 cells. Here we decided to interrogate the mechanism (s) and mode of cytotoxic effects of this compound. Inosine monophosphate dehydrogenase is the key regulatory and the rate-limiting enzyme of the de novo biosynthetic pathway of purines. Apparently, the activity of this enzyme significantly increases in tumor cells in proportion to the proliferation. We hypothesized that inhibition of IMPDH and consequently depletion of guanine nucleotide pool is responsible of cytotoxic effect with characteristic of apoptosis. HL-60 and K562 leukemia cells were treated with increasing doses of 3-hydrogenkwadaphnin. Thereafter, the mode of its cytotoxic action was determined by Acridine orange/Ethedium bromide double staining, DNA fragmentation, sub-G1 flow cytometry and electron microscopic. Involvement of guanine nucleotide pool was tested by co-provision of guanosine and deoxyguanosine that replead GTP and dGTP pool size, respectively. 3-hydrogenkwadaphnin at low doses (10 nM) enhanced fragment of the apoptotic HL-60 cells within 72 hours of treatment. Higher doses (25 nM) led to rapid induction of apoptosis, which could be detected as early as 24 h. The majority (85%) of the treated HL-60 cells died by apoptosis, 72 hours after exposure to 3-hydrogenkwadaphnin. Althought K562 cells are generally more resistant to a variety of apoptotic stimuli, to our surprising 3-hydrogenkwadaphnin was equally effective in this cell line as compared to HL-60. 3-hydrogenkwadaphnin-induced apoptosis was almost totally prevented by supplementation with guanosine, but not with adenosine or deoxyguanosine, indicating the specific effect of 3-hydrogenkwadaphnin in reducing the pool size of GTP. The results indicate that 3-hydrogenkwadaphnin decreases GTP pool size and depletion of this pool, and not dGTP, is probably the cause of cell death through apoptosis. Induction of apoptosis is a major goal of chemotherapy. GTP depletion induced apoptosis is selective for tumor cells; thereby 3-hydrogenkwadaphnin could be presented as a powerful drug for treatment of cancer, especially leukemia.

Induction of Megakaryocytic Differentiation in Chronic Myelogenous Leukemia Cell K562 by 3-Hydrogenkwadaphnin

3-Hydrogenkwadaphnin (3-HK) is a daphnane-type diterpene ester isolated from Dendrostellera lessertii (Thymelaeaceae) with high differentiation and apoptotic potency in leukemic cells without any measurable adverse effects on normal cells (Moosavi et al., 2005b). In this study, we report that 3-HK (12 nM) has the ability to cease proliferation, induce differentiation and apoptosis in chronic myelogenous leukemia (CML) K562 cell line. The treated cells lost erythroid properties and differentiated along the megakaryocytic lineage based on the morphological features apparent after Wright-Giemsa staining, DNA content analysis and the expression of cell surface marker glycoprotein IIb as analyzed by flow cytometry. Moreover, using Hoechst 33258 and Annexin V double staining indicated the occurrence of apoptosis among the treated cells. On the other hand, restoration of the depleted GTP pool size by exogenous addition of guanosine (50 microM) reduced the effect of the drug regarding the extent of differentiation while no further enhancement of 3-HK effect was obtained by addition of exogenous hypoxanthine (100 microM). These interesting results necessitate further investigation regarding the mechanism of action of this unique anti-leukemic agent.

Physiological Analysis of the Stringent Response Elicited in an Extreme Thermophilic Bacterium,Thermus thermophilus

Guanosine tetraphosphate (ppGpp) is a key mediator of stringent control, an adaptive response of bacteria to amino acid starvation, and has thus been termed a bacterial alarmone. Previous X-ray crystallographic analysis has provided a structural basis for the transcriptional regulation of RNA polymerase activity by ppGpp in the thermophilic bacterium Thermus thermophilus. Here we investigated the physiological basis of the stringent response by comparing the changes in intracellular ppGpp levels and the rate of RNA synthesis in stringent (rel(+); wild type) and relaxed (relA and relC; mutant) strains of T. thermophilus. We found that in wild-type T. thermophilus, as in other bacteria, serine hydroxamate, an amino acid analogue that inhibits tRNA(Ser) aminoacylation, elicited a stringent response characterized in part by intracellular accumulation of ppGpp and that this response was completely blocked in a relA-null mutant and partially blocked in a relC mutant harboring a mutation in the ribosomal protein L11. Subsequent in vitro assays using ribosomes isolated from wild-type and relA and relC mutant strains confirmed that (p)ppGpp is synthesized by ribosomes and that mutation of RelA or L11 blocks that activity. This conclusion was further confirmed in vitro by demonstrating that thiostrepton or tetracycline inhibits (p)ppGpp synthesis. In an in vitro system, (p)ppGpp acted by inhibiting RNA polymerase-catalyzed 23S/5S rRNA gene transcription but at a concentration much higher than that of the observed intracellular ppGpp pool size. On the other hand, changes in the rRNA gene promoter activity tightly correlated with changes in the GTP but not ATP concentration. Also, (p)ppGpp exerted a potent inhibitory effect on IMP dehydrogenase activity. The present data thus complement the earlier structural analysis by providing physiological evidence that T. thermophilus does produce ppGpp in response to amino acid starvation in a ribosome-dependent (i.e., RelA-dependent) manner. However, it appears that in T. thermophilus, rRNA promoter activity is controlled directly by the GTP pool size, which is modulated by ppGpp via inhibition of IMP dehydrogenase activity. Thus, unlike the case of Escherichia coli, ppGpp may not inhibit T. thermophilus RNA polymerase activity directly in vivo, as recently proposed for Bacillus subtilis rRNA transcription (L. Krasny and R. L. Gourse, EMBO J. 23:4473-4483, 2004).

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.

ADP-ribosylation of proteins in Bacillus subtilis and its possible importance in sporulation.

Endogenous ADP-ribosylation was detected in Bacillus subtilis, as determined in vitro with crude cellular extracts. The ADP-ribosylated protein profile changed during growth in sporulation medium, displaying a temporary appearance of two ADP-ribosylated proteins (36 and 58 kDa) shortly after the end of exponential growth. Mutants resistant to 3-methoxybenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained, and a significant proportion (15%) were found to be defective in both sporulation and antibiotic production. These mutants failed to ADP-ribosylate the 36- and 58-kDa proteins. The parent strain also lost the ability to ADP-ribosylate these proteins when grown in the presence of 3-methoxybenzamide at a concentration at which sporulation but not cell growth was severely inhibited. Results from genetic transformations showed that the mutation conferring resistance to 3-methoxybenzamide, named brgA, was cotransformed with the altered phenotypes, i.e., defects in ADP-ribosylation and sporulation. spoOA and spoOF mutants displayed an ADP-ribosylation profile similar to that of the parent strain, but a spoOH mutant failed to ADP-ribosylate any proteins, including the 36- and 58-kDa proteins. The significance of protein ADP-ribosylation in sporulation was further indicated by the observation that ADP-ribosylation of the 36-kDa protein could be induced by treatment with decoyinine, an inhibitor of GMP-synthetase, and by amino acid limitation, both of which resulted in an immediate decrease in GTP pool size eventually leading to massive sporulation. We propose that a new sporulation gene, which presumably controls sporulation via ADP-ribosylation of certain functional proteins, exists.

Physiological analysis of bicozamycin high-producing Streptomyces griseoflavus used at industrial level.

Streptomyces griseoflavus, a bicozamycin-producing wild type strain and its high-producing one derived from it by multiple (greater than 15) mutagenic treatments, were analyzed physiologically and biochemically. The high-producing strain was characterized by: (1) An increased pool size of amino acids including leucine and isoleucine, precursors for bicozamycin synthesis, (2) an earlier and greater accumulation of intracellular ppGpp, (3) a more accentuated decrease in GTP pool size, (4) a higher specific activity of ornithine transcarbamylase which produces citrulline, (5) an increased ability to form aerial mycelium, and (6) an increased resistance to its own antibiotic. We propose that (1), (2) and (4) may be responsible for the high yields of bicozamycin and, possibly, of some other antibiotics produced by Streptomyces sp.

Changes in Nucleotide Pools during Sporulation of Streptomyces griseus in Submerged Culture

SUMMARY: Streptomyces griseus strain 13189 produced a large number of submerged spores in response to nitrogen-mediated nutritional shift-down. The pool size of ppGpp increased immediately after shift-down and was accompanied by a dramatic decrease in the GTP pool. In contrast, nutritional shift-down of a relaxed (rel) mutant derived from strain 13189 resulted in a slight increase in ppGpp pool size, a less extensive decrease in GTP pool size and spore titres 10-fold less than produced by the parental strain. Addition of decoyinine, a specific inhibitor of GMP synthetase, to the re1 mutant restored the sporulation frequency to the parental level. IMP dehydrogenase was competitively inhibited by ppGpp with a Ki of 0-05 mM, whereas other nucleotides were less effective inhibitors. The observed intracellular concentration of ppGpp during shift-down was sufficient to inhibit IMP dehydrogenase activity and to result in the decrease in the GTP pool. These results demonstrate that sporulation of S. griseus, like that of Bacillus subtilis, is nutrient-dependent and that the decrease in GTP content caused by the stringent response (ppGpp) is correlated with initiation of submerged spore formation due to amino acid starvation.

Mode of action of ribavirin: Effect of nucleotide pool alterations on influenza virus ribonucleoprotein synthesis

Ribavirin, a guanosine analogue, is a broad spectrum antiviral agent which is effective in the treatment of influenza. In this study, the effect of ribavirin on influenza virus ribonucleoprotein (RNP) synthesis and nucleotide pool sizes was simultaneously measured. Ribavirin (100 μM) reduced viral RNP synthesis 94% as measured by UTP incorporation. Intracellular GTP pools, measured by high performance liquid chromatography, were reduced approximately 45% in ribavirin treated cells, while other nucleotides remained near control values. Attempts to reverse ribavirin's inhibitory effects on viral RNP synthesis by addition of exogenous guanosine (50 μM) resulted in only a partial restoration of viral RNP synthesis, despite full restoration of the GTP pool. Dose-response experiments indicated that the GTP pool was significantly reducted (65% of control) at 25 μM ribavirin, and increasing concentrations of the drug caused only a small further reduction in the GTP pool (5–10% at 100 μM). In contrast, RNP synthesis was inhibited by 50% at 25 μM ribavirin and was further decreased to 5% of control at 100 μM ribavirin. Thus, ribavirin's antiviral activity may result from a reduction of the GTP pool size combined with direct effects on viral replicative enzymes.

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.

Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli

A new technique for measuring the rate of turnover and quantity of unstable RNA in exponentially growing cells has been developed which escapes the interpretive difficulties of earlier methods. It depends upon the fact that an unstable RNA fraction has easily measurable effects upon the kinetics of labelling of GTP; before the GTP pool can become fully labelled, unlabelled nucleotides present as GTP or in unstable RNA must be washed into stable RNA. (There is no equilibration with guanine in the medium.)Consequently, if the uptake of label into GTP is rapid, with only the delay predicted from the experimentally measured GTP pool size, then it is possible to conclude that there is little or no unstable RNA. In Bacillus subtilis, however, the GTP pool is labelled very slowly which, with other data, indicates a large amount of unstable RNA (9% of the guanine residues of the cell in unstable RNA). Similar measurements of the rapidity of GTP labelling in Escherichia coli indicate that there is much less unstable RNA (about 3%) in this organism.The curve expressing GTP specific activity as a function of time is shown to be the sum of two exponential terms with very different time constants. From the relative sizes of the two exponential terms, it is possible to estimate the rate of turnover of the unstable RNA. The result is weighted most heavily for the slow components of the decaying messenger, and thus it sets an upper limit to the true average decay time for the unstable RNA. This decay time (upper limit) was three minutes for the unstable RNA in B. subtilis grown at 37 °C with a doubling time of 115 minutes and six minutes when the cells were grown at 30 °C with a doubling time of 225 minutes. For E. coli (growing at 30 °C with a doubling time of 66 minutes) the decay time measured in this way was about four minutes. Thus the decay times as measured in these experiments are the same order of magnitude as have been observed by measuring the decay of pulse-labelled RNA and the decay of protein synthetic capacity after the addition of actinomycin. The various estimates of the decay rate of messenger RNA in bacteria are discussed in terms of the possible theoretical ambiguities in the interpretation of each.